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Sample records for human hematopoietic progenitor

  1. Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

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    Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

    2015-07-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

  2. Dendritic Cell Lineage Potential in Human Early Hematopoietic Progenitors

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    Julie Helft

    2017-07-01

    Full Text Available Conventional dendritic cells (cDCs are thought to descend from a DC precursor downstream of the common myeloid progenitor (CMP. However, a mouse lymphoid-primed multipotent progenitor has been shown to generate cDCs following a DC-specific developmental pathway independent of monocyte and granulocyte poiesis. Similarly, here we show that, in humans, a large fraction of multipotent lymphoid early progenitors (MLPs gives rise to cDCs, in particular the subset known as cDC1, identified by co-expression of DNGR-1 (CLEC9A and CD141 (BDCA-3. Single-cell analysis indicates that over one-third of MLPs have the potential to efficiently generate cDCs. cDC1s generated from CMPs or MLPs do not exhibit differences in transcriptome or phenotype. These results demonstrate an early imprinting of the cDC lineage in human hematopoiesis and highlight the plasticity of developmental pathways giving rise to human DCs.

  3. A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

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    Akira Niwa

    Full Text Available Elucidating the in vitro differentiation of human embryonic stem (ES and induced pluripotent stem (iPS cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

  4. Different Motile Behaviors of Human Hematopoietic Stem versus Progenitor Cells at the Osteoblastic Niche

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    Katie Foster

    2015-11-01

    Full Text Available Despite advances in our understanding of interactions between mouse hematopoietic stem cells (HSCs and their niche, little is known about communication between human HSCs and the microenvironment. Using a xenotransplantation model and intravital imaging, we demonstrate that human HSCs display distinct motile behaviors to their hematopoietic progenitor cell (HPC counterparts, and the same pattern can be found between mouse HSCs and HPCs. HSCs become significantly less motile after transplantation, while progenitor cells remain motile. We show that human HSCs take longer to find their niche than previously expected and suggest that the niche be defined as the position where HSCs stop moving. Intravital imaging is the only technique to determine where in the bone marrow stem cells stop moving, and future analyses should focus on the environment surrounding the HSC at this point.

  5. Novel pathways to erythropoiesis induced by dimerization of intracellular C-Mpl in human hematopoietic progenitors.

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    Parekh, Chintan; Sahaghian, Arineh; Kim, William; Scholes, Jessica; Ge, Shundi; Zhu, Yuhua; Asgharzadeh, Shahab; Hollis, Roger; Kohn, Donald; Ji, Lingyun; Malvar, Jemily; Wang, Xiaoyan; Crooks, Gay

    2012-04-01

    The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl. Copyright © 2012 AlphaMed Press.

  6. Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

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    Cashman, J.; Eaves, A.C.; Eaves, C.J.

    1985-01-01

    We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3 H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation

  7. Low antigenicity of hematopoietic progenitor cells derived from human ES cells

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    Eun-Mi Kim

    2010-02-01

    Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

  8. Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

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    Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

    1996-07-15

    Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

  9. Role of reactive oxygen species in the radiation response of human hematopoietic stem/progenitor cells.

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    Masaru Yamaguchi

    Full Text Available Hematopoietic stem/progenitor cells (HSPCs, which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS. The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34(+ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34(+ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34(+ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34(+ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34(+ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34(+ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.

  10. Direct evaluation of radiation damage in human hematopoietic progenitor cells in vivo

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    Kyoizumi, Seishi; McCune, J.M.; Namikawa, Reiko

    1994-01-01

    We have developed techniques by which normal functional elements of human bone marrow can be implanted into immunodeficient C.B-17 scid/scid (SCID) mice. Afterward, long-term multilineage human hematopoiesis is sustained in vivo. We evaluated the effect of irradiation on the function of human bone marrow with this in vivo model. After whole-body X irradiation of the engrafted animals, it was determined that the D 0 value of human committed progenitor cells within the human marrow was 1.00 ± 0.09 (SEM) Gy for granulocyte-macrophage colony-forming units (CFU-GM) and 0.74 ± 0.12 Gy for erythroidburst-forming units (BFU-E). The effects of irradiation on the hematopoietic elements were reduced when the radioprotective agent WR-2721 was administered prior to irradiation. After low-dose irradiation, recovery of human granulocyte colony-stimulating factor (G-CSF). This small animal model may prove amenable for the analysis of the risk of the exposure of humans to irradiation as well as for the development of new modalities for the prevention and treatment of radiation-induced hematopoietic damage. 41 refs., 5 figs., 1 tab

  11. Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects.

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    Mikirova, Nina A; Jackson, James A; Hunninghake, Ron; Kenyon, Julian; Chan, Kyle W H; Swindlehurst, Cathy A; Minev, Boris; Patel, Amit N; Murphy, Michael P; Smith, Leonard; Ramos, Famela; Ichim, Thomas E; Riordan, Neil H

    2010-04-08

    The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

  12. Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects

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    Minev Boris

    2010-04-01

    Full Text Available Abstract The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

  13. Individual differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

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    Oriya, Asami; Takahashi, Kenji; Kashiwakura, Ikuo; Inanami, Osamu; Kuwabara, Mikinori; Miura, Toshiaki; Abe, Yoshinao

    2008-01-01

    The aim of this study is to evaluate the individual differences in radiosensitivity of lineage-committed myeloid hematopoietic progenitors, colony-forming cells (CFC), detected in steady-state human peripheral blood (PB). Mononuclear cells were prepared from the buffy-coat of 30 individuals PB, and were assayed for CFC by semi-solid culture supplemented with cytokines. X irradiation was performed in the range of 0.5-4 Gy at a dose rate of about 80 cGy/min. The mean number of hematopoietic progenitor cells is 5866±3408 in 1 ml of buffy-coat, suggesting that the erythroid progenitor cells are the major population. The total CFC radiosensitivity parameter D 0 and n value are 1.18±0.24 and 1.89±0.98, respectively. Using a linear regression analysis, a statistically significant correlation is observed between the D 0 value and the surviving fraction at 4 Gy (r=0.611 p 0 parameter and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. The present study demonstrates that there are large individual differences in the radiosensitivity of hematopoietic progenitor cells as detected in steady-state human PB. These differences demonstrate almost no correlation with plasma or intracellular antioxidants. The prediction of individual differences in radiosensitivity of CFC can only be measured by 4 Gy irradiation. (author)

  14. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

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    Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

    2017-05-01

    Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

  15. Staurosporine Increases Lentiviral Vector Transduction Efficiency of Human Hematopoietic Stem and Progenitor Cells

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    Gretchen Lewis

    2018-06-01

    Full Text Available Lentiviral vector (LVV-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN approximately 2-fold when added to mobilized peripheral blood (mPB CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction, and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2 is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products. Keywords: lentiviral, HSPC, transduction

  16. PDGFRα and CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion.

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    Pinho, Sandra; Lacombe, Julie; Hanoun, Maher; Mizoguchi, Toshihide; Bruns, Ingmar; Kunisaki, Yuya; Frenette, Paul S

    2013-07-01

    The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51 ⁺subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.

  17. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

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    Tuoen Liu

    Full Text Available Myelodysplastic syndromes (MDS are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q compared to non-del(5q MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q-associated MDS.

  18. Engineered matrix coatings to modulate the adhesion of CD133+ human hematopoietic progenitor cells.

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    Franke, Katja; Pompe, Tilo; Bornhäuser, Martin; Werner, Carsten

    2007-02-01

    Interactions of hematopoietic progenitor cells (HPC) with their local microenvironments in the bone marrow are thought to control homing, differentiation, and self-renewal of the cells. To dissect the role of extracellular matrix (ECM) components of the niche microenvironment, a set of well-defined ECM coatings including fibronectin, heparin, heparan sulphate, hyaluronic acid, tropocollagen I, and co-fibrils of collagen I with heparin or hyaluronic acid was prepared and analysed with respect to the attachment of human CD133+ HPC in vitro. The extension of the adhesion areas of individual cells as well as the fraction of adherent cells were assessed by reflection interference contrast microscopy (RICM). Intense cell-matrix interactions were found on surfaces coated with fibronectin, heparin, heparan sulphate, and on the collagen I based co-fibrils. Insignificant adhesion was found for tropocollagen I and hyaluronic acid. The strongest adhesion of HPC was observed on fibronectin with contact areas of about 7 microm(2). Interaction of HPC with coatings consisting of heparin, heparan sulphate, and co-fibrils result in small circular shaped contact zones of 3 microm(2) pointing to another, less efficient, adhesion mechanism. Analysing the specificity of cell-matrix interaction by antibody blocking experiments suggests an integrin(alpha(5)beta(1))-specific adhesion on fibronectin, while adhesion on heparin was shown to be mediated by selectins (CD62L). Taken together, our data provide a basis for the design of advanced culture carriers supporting site-specific proliferation or differentiation of HPC.

  19. PLAG1 and USF2 Co-regulate Expression of Musashi-2 in Human Hematopoietic Stem and Progenitor Cells

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    Muluken S. Belew

    2018-04-01

    Full Text Available Summary: MSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs, enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry. : MSI2 is an essential human hematopoietic stem and progenitor cell (HSPC regulator, but knowledge of the mechanisms ensuring its appropriate expression in this context are lacking. Here, Hope and colleagues map the MSI2 promoter functional in hematopoietic cells and identify USF2 and PLAG1 as essential, cooperative enforcers of endogenous MSI2 expression and stemness traits in human HSPCs. Keywords: human hematopoietic stem cells, self-renewal, promoter, transcriptional regulation, transcription factors, Musashi-2, genome-wide DNA binding site mapping, PLAG1, USF2

  20. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

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    Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

    2013-01-01

    Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

  1. Transmission of clonal chromosomal abnormalities in human hematopoietic stem and progenitor cells surviving radiation exposure

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    Kraft, Daniela, E-mail: d.kraft@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Ritter, Sylvia, E-mail: s.ritter@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Durante, Marco, E-mail: m.durante@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Condensed Matter Physics, Physics Department, Technical University Darmstadt, Hochschulstraße 6-8, 64289 Darmstadt (Germany); Seifried, Erhard, E-mail: e.seifried@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Fournier, Claudia, E-mail: c.fournier@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Tonn, Torsten, E-mail: t.tonn@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Technische Universität Dresden, Med. Fakultät Carl Gustav Carus, Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Blasewitzer Straße 68/70, 01307 Dresden (Germany)

    2015-07-15

    Highlights: • Radiation induced formation and transmission of chromosomal aberrations were assessed. • Cytogenetic analysis was performed in human CD34+ HSPC by mFISH. • We report transmission of stable aberrations in irradiated, clonally expanded HSPC. • Unstable aberrations in clonally expanded HSPC occur independently of irradiation. • Carbon ions and X-rays bear a similar risk for propagation of cytogenetic changes. - Abstract: In radiation-induced acute myeloid leukemia (rAML), clonal chromosomal abnormalities are often observed in bone marrow cells of patients, suggesting that their formation is crucial in the development of the disease. Since rAML is considered to originate from hematopoietic stem and progenitor cells (HSPC), we investigated the frequency and spectrum of radiation-induced chromosomal abnormalities in human CD34{sup +} cells. We then measured stable chromosomal abnormalities, a possible biomarker of leukemia risk, in clonally expanded cell populations which were grown for 14 days in a 3D-matrix (CFU-assay). We compared two radiation qualities used in radiotherapy, sparsely ionizing X-rays and densely ionizing carbon ions (29 and 60–85 keV/μm, doses between 0.5 and 4 Gy). Only a negligible number of de novo arising, unstable aberrations (≤0.05 aberrations/cell, 97% breaks) were measured in the descendants of irradiated HSPC. However, stable aberrations were detected in colonies formed by irradiated HSPC. All cells of the affected colonies exhibited one or more identical aberrations, indicating their clonal origin. The majority of the clonal rearrangements (92%) were simple exchanges such as translocations (77%) and pericentric inversions (15%), which are known to contribute to the development of rAML. Carbon ions were more efficient in inducing cell killing (maximum of ∼30–35% apoptotic cells for 2 Gy carbon ions compared to ∼25% for X-rays) and chromosomal aberrations in the first cell-cycle after exposure (∼70% and

  2. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

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    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  3. Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Audigé, Annette; Rochat, Mary-Aude; Li, Duo; Ivic, Sandra; Fahrny, Audrey; Muller, Christina K S; Gers-Huber, Gustavo; Myburgh, Renier; Bredl, Simon; Schlaepfer, Erika; Scherrer, Alexandra U; Kuster, Stefan P; Speck, Roberto F

    2017-05-30

    Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time. Overall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.

  4. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Emanuela Chiarella

    Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

  5. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Science.gov (United States)

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

  6. Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures

    NARCIS (Netherlands)

    L.E. Duinhouwer (Lucia); N. Tüysüz (Nesrin); E.J. Rombouts (Elwin); M.N.D. Ter Borg (Mariëtte N. D.); E. Mastrobattista; J. Spanholtz (Jan); J.J. Cornelissen (Jan); D. ten Berge (Derk); E. Braakman (Eric)

    2015-01-01

    textabstractAbstract Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in

  7. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

    Science.gov (United States)

    Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

    2015-05-01

    Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy. © 2015 AlphaMed Press.

  8. The effects of X-irradiation on ex vivo expansion of cryopreserved human hematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Hayashi, Naoki; Takahashi, Kenji; Kashiwakura, Ikuo

    2010-01-01

    In our previous study (Life Sciences 84: 598, 2009), we demonstrated that placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells have the effect to support the regeneration of freshly prepared X-irradiated hematopoietic stem/progenitor cells (HSPCs). Generally, HSPCs are supplied from companies, institutions, and cell banks that cryopreserve them for clinical and experimental use. In this study, the influence of cryopreservation on the responses of HSPCs to irradiation and co-culture with stromal cells is assessed. After cryopreservation with the optimal procedure, 2 Gy-irradiated HSPCs were cultured with or without stromal cells supplemented with combination of interleukin-3, stem cell factor, and thrombopoietin. The population of relatively immature CD34 + /CD38 - cells in cryopreserved cells was significantly higher than in fresh cells prior to cryopreservation; furthermore, the hematopoietic progenitor populations of CD34 + /CD45RA + cells and CD34 + /CD117 + cells in cryopreserved cells were significantly lower than that in fresh cells. However, the rate of expansion in the cryopreserved HSPCs was lower than in the fresh HSPCs. In the culture of cryopreserved cells irradiated with 2 Gy, the growth rates of CD34 + cells, CD34 + /CD38 - cells, and hematopoietic progenitors were greater than growth rates of their counter parts in the culture of fresh cells. Surprisingly, the effect to support the hematopoiesis in co-culture with stromal cells was never observed in the X-irradiated HSPCs after cryopreservation. The present results demonstrated that cryopreserving process increased the rate of immature and radio-resistant HSPCs but decreased the effects to support the hematopoiesis by stromal cells, thus suggesting that cryopreservation changes the character of HSPCs. (author)

  9. Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

    Science.gov (United States)

    Buchanan, Sandhya S; Pyatt, David W; Carpenter, John F

    2010-09-01

    Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls pGM (approximately 35%, relative to unprocessed controls, p<0.0001), 112+/-68 BFU-E (approximately 26%, p<0.0001), and 36+/-17 CFU-GEMM ( approximately 82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and

  10. Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

    Directory of Open Access Journals (Sweden)

    Sandhya S Buchanan

    2010-09-01

    Full Text Available Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls p<0.0001, 170+/-70 BFU-E (approximately 40%, p<0.0001, and 41+/-22 CFU-GEMM (approximately 82%, p = 0.4171, and cells reconstituted after 28 days at room temperature produced 513+/-170 CFU-GM (approximately 35%, relative to unprocessed controls, p<0

  11. Molecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cells

    KAUST Repository

    Abusamra, Dina

    2016-12-01

    The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E

  12. In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells.

    Science.gov (United States)

    Rodman, C; Almeida-Porada, G; George, S K; Moon, J; Soker, S; Pardee, T; Beaty, M; Guida, P; Sajuthi, S P; Langefeld, C D; Walker, S J; Wilson, P F; Porada, C D

    2017-06-01

    Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.

  13. The influence of gender- and age-related differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

    International Nuclear Information System (INIS)

    Kato, Kengo; Kashiwakura, Ikuo; Kuwabara, Mikinori

    2011-01-01

    To investigate the importance of gender and aging on the individual radiosensitivity of lineage-committed myeloid hematopoietic stem/progenitor cells (HSPCs) detected in mononuclear cells (MNCs) of steady-state human peripheral blood (PB), the clonogenic survival of HPCs, including colony-forming unit-granulocyte macrophage; burst-forming unit-erythroid; colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte cells derived from MNCs exposed to 0.5 Gy and 2 Gy X-irradiation were estimated. MNCs were prepared from the buffy-coats of 59 healthy individual blood donors. The results showed that large individual differences exist in the number of HSPCs, as well as in the surviving fraction of cells. Furthermore, the number of progenitor cells strongly correlated with their surviving fraction, suggesting that the radiosensitivity of hematopoietic progenitor cells decreases with the number of cells in the 10 5 cells population. A statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of an individual, however, none of these correlations were observed after 2 Gy irradiation. No statistically significant difference was observed in individual radiosensitivity between males and females at either radiation dose. The present results indicated a correlation between the individual responsiveness of HSPCs to ionizing irradiation, especially to low dose irradiation, and aging. (author)

  14. Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

    KAUST Repository

    Merzaban, Jasmeen; Burdick, Monica M.; Gadhoum, Samah; Dagia, Nilesh M.; Chu, Julia T.; Fuhlbrigge, Robert C.; Sackstein, Robert D.

    2011-01-01

    Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using

  15. Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures

    NARCIS (Netherlands)

    Duinhouwer, Lucia E.; Tüysüz, Nesrin; Rombouts, Elwin W J C; Ter Borg, Mariette N D; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J.; Berge, Derk Ten; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with

  16. Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from X-irradiated human peripheral blood hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

    2009-01-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34 + hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34 + cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34 + cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34 + cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34 + cells. (author)

  17. Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

    Science.gov (United States)

    Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

    1999-12-01

    Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

  18. Deletion of the LTR enhancer/promoter has no impact on the integration profile of MLV vectors in human hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Arianna Moiani

    Full Text Available Moloney murine leukemia virus (MLV-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR, and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34(+ human hematopoietic stem/progenitor cells (HSPCs. We sequenced 13,011 unique SIN-MLV integration sites and compared them to 32,574 previously generated MLV sites in human HSPCs. The SIN-MLV vector recapitulates the integration pattern observed for MLV, with the characteristic clustering of integrations around enhancer and promoter regions associated to H3K4me3 and H3K4me1 histone modifications, specialized chromatin configurations (presence of the H2A.Z histone variant and binding of RNA Pol II. SIN-MLV and MLV integration clusters and hot spots overlap in most cases and are generated at a comparable frequency, indicating that the reduced genotoxicity of SIN-MLV vectors in hematopoietic cells is not due to a modified integration profile.

  19. Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

    Science.gov (United States)

    Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

    2015-01-01

    During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

  20. Mobilization of hematopoietic stem and progenitor cells in mice

    NARCIS (Netherlands)

    Robinson, Simon N; van Os, Ronald P; Bunting, Kevin

    2008-01-01

    Animal models have added significantly to our understanding of the mechanism(s) of hematopoietic stem and progenitor cell (HSPC) mobilization. Such models suggest that changes in the interaction between the HSPC and the hematopoietic microenvironmental 'niche' (cellular and extracellular components)

  1. Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2017-12-27

    CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.

  2. Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

    KAUST Repository

    Abu Samra, Dina Bashir Kamil; Aleisa, Fajr A; Al-Amoodi, Asma S.; Jalal Ahmed, Heba M.; Chin, Chee Jia; AbuElela, Ayman; Bergam, Ptissam; Sougrat, Rachid; Merzaban, Jasmeen

    2017-01-01

    CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.

  3. PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Varagnolo, Linda; Lin, Qiong; Obier, Nadine; Plass, Christoph; Dietl, Johannes; Zenke, Martin; Claus, Rainer; Müller, Albrecht M

    2015-07-22

    Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for transplantation approaches. However, the amount of cells per donor is limited and culture expansion of CB-HSCs is accompanied by a loss of engraftment potential. In order to analyze the molecular mechanisms leading to this impaired potential we profiled global and local epigenotypes during the expansion of human CB hematopoietic stem and progenitor cells (HPSCs). Human CB-derived CD34+ cells were cultured in serum-free medium together with SCF, TPO, FGF, with or without Igfbp2 and Angptl5 (STF/STFIA cocktails). As compared to the STF cocktail, the STFIA cocktail maintains in vivo repopulation capacity of cultured CD34+ cells. Upon expansion, CD34+ cells genome-wide remodel their epigenotype and depending on the cytokine cocktail, cells show different H3K4me3 and H3K27me3 levels. Expanding cells without Igfbp2 and Angptl5 leads to higher global H3K27me3 levels. ChIPseq analyses reveal a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Inhibition of the PRC2 component EZH2 counteracts the culture-associated loss of NOD scid gamma (NSG) engraftment potential. Collectively, our data reveal chromatin dynamics that underlie the culture-associated loss of engraftment potential. We identify PRC2 component EZH2 as being involved in the loss of engraftment potential during the in vitro expansion of HPSCs.

  4. Ionizing radiation induces apoptosis in hematopoietic stem and progenitor cells

    International Nuclear Information System (INIS)

    Meng, A.; Zhou, D.; Geiger, H.; Zant, G.V.

    2003-01-01

    The aims of this study was to determine if ionizing radiation (IR) induces apoptosis in hematopoietic stem (HSC) and progenitor cells. Lin-cells were isolated from mouse bone marrow (BM) and pretreated with vehicle or 100 μM z-VAD 1 h prior to exposure to 4 Gy IR. The apoptotic and/or necrotic responses of these cells to IR were analyzed by measuring the annexin V and/or 7-AAD staining in HSC and progenitor populations using flow cytometry, and hematopoietic function of these cells was determined by CAFC assay. Exposure of Lin-cells to IR selectively decreased the numbers of HSC and progenitors in association with an increase in apoptosis in a time-dependent manner. Pretreatment of Lin- cells with z-VAD significantly inhibited IR-induced apoptosis and the decrease in the numbers of HSC and progenitors. However, IR alone or in combination with z-VAD did not lead to a significant increase in necrotic cell death in either HSC or progenitors. In addition, pretreatment of BM cells with z-VAD significantly attenuated IR-induced reduction in the frequencies of day-7, -28 and -35 CAFC. Exposure of HSC and progenitors to IR induces apoptosis. The induction of HSC and progenitor apoptosis contributes to IR-induced suppression of their hematopoietic function

  5. Mobilization of primitive and committed hematopoietic progenitors in nonhuman primates treated with defibrotide and recombinant human granulocyte colony-stimulating factor.

    Science.gov (United States)

    Carlo-Stella, Carmelo; Di Nicola, Massimo; Longoni, Paolo; Milani, Raffaella; Milanesi, Marco; Guidetti, Anna; Haanstra, Krista; Jonker, Margaret; Cleris, Loredana; Magni, Michele; Formelli, Franca; Gianni, Alesssandro M

    2004-01-01

    The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys. Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 microg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, pdefibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, pdefibrotide enhances rhG-CSF-elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

  6. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-te...

  7. Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

    KAUST Repository

    Merzaban, Jasmeen S.

    2011-06-09

    Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

  8. Environmental and chemotherapeutic agents induce breakage at genes involved in leukemia-causing gene rearrangements in human hematopoietic stem/progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Thys, Ryan G., E-mail: rthys@wakehealth.edu [Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016 (United States); Lehman, Christine E., E-mail: clehman@wakehealth.edu [Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016 (United States); Pierce, Levi C.T., E-mail: Levipierce@gmail.com [Human Longevity, Inc., San Diego, California 92121 (United States); Wang, Yuh-Hwa, E-mail: yw4b@virginia.edu [Department of Biochemistry and Molecular Genetics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0733 (United States)

    2015-09-15

    Highlights: • Environmental/chemotherapeutic agents cause DNA breakage in MLL and CBFB in HSPCs. • Diethylnitrosamine-induced DNA breakage at MLL and CBFB shown for the first time. • Chemical-induced DNA breakage occurs at topoisomerase II cleavage sites. • Chemical-induced DNA breaks display a pattern similar to those in leukemia patients. • Long-term exposures suggested to generate DNA breakage at leukemia-related genes. - Abstract: Hematopoietic stem and progenitor cells (HSPCs) give rise to all of the cells that make up the hematopoietic system in the human body, making their stability and resilience especially important. Damage to these cells can severely impact cell development and has the potential to cause diseases, such as leukemia. Leukemia-causing chromosomal rearrangements have largely been studied in the context of radiation exposure and are formed by a multi-step process, including an initial DNA breakage and fusion of the free DNA ends. However, the mechanism for DNA breakage in patients without previous radiation exposure is unclear. Here, we investigate the role of non-cytotoxic levels of environmental factors, benzene, and diethylnitrosamine (DEN), and chemotherapeutic agents, etoposide, and doxorubicin, in generating DNA breakage at the patient breakpoint hotspots of the MLL and CBFB genes in human HSPCs. These conditions represent exposure to chemicals encountered daily or residual doses from chemotherapeutic drugs. Exposure of HSPCs to non-cytotoxic levels of environmental chemicals or chemotherapeutic agents causes DNA breakage at preferential sites in the human genome, including the leukemia-related genes MLL and CBFB. Though benzene, etoposide, and doxorubicin have previously been linked to leukemia formation, this is the first study to demonstrate a role for DEN in the generation of DNA breakage at leukemia-specific sites. These chemical-induced DNA breakpoints coincide with sites of predicted topoisomerase II cleavage. The

  9. Environmental and chemotherapeutic agents induce breakage at genes involved in leukemia-causing gene rearrangements in human hematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Thys, Ryan G.; Lehman, Christine E.; Pierce, Levi C.T.; Wang, Yuh-Hwa

    2015-01-01

    Highlights: • Environmental/chemotherapeutic agents cause DNA breakage in MLL and CBFB in HSPCs. • Diethylnitrosamine-induced DNA breakage at MLL and CBFB shown for the first time. • Chemical-induced DNA breakage occurs at topoisomerase II cleavage sites. • Chemical-induced DNA breaks display a pattern similar to those in leukemia patients. • Long-term exposures suggested to generate DNA breakage at leukemia-related genes. - Abstract: Hematopoietic stem and progenitor cells (HSPCs) give rise to all of the cells that make up the hematopoietic system in the human body, making their stability and resilience especially important. Damage to these cells can severely impact cell development and has the potential to cause diseases, such as leukemia. Leukemia-causing chromosomal rearrangements have largely been studied in the context of radiation exposure and are formed by a multi-step process, including an initial DNA breakage and fusion of the free DNA ends. However, the mechanism for DNA breakage in patients without previous radiation exposure is unclear. Here, we investigate the role of non-cytotoxic levels of environmental factors, benzene, and diethylnitrosamine (DEN), and chemotherapeutic agents, etoposide, and doxorubicin, in generating DNA breakage at the patient breakpoint hotspots of the MLL and CBFB genes in human HSPCs. These conditions represent exposure to chemicals encountered daily or residual doses from chemotherapeutic drugs. Exposure of HSPCs to non-cytotoxic levels of environmental chemicals or chemotherapeutic agents causes DNA breakage at preferential sites in the human genome, including the leukemia-related genes MLL and CBFB. Though benzene, etoposide, and doxorubicin have previously been linked to leukemia formation, this is the first study to demonstrate a role for DEN in the generation of DNA breakage at leukemia-specific sites. These chemical-induced DNA breakpoints coincide with sites of predicted topoisomerase II cleavage. The

  10. Quercetin alters the DNA damage response in human hematopoietic stem and progenitor cells via TopoII- and PI3K-dependent mechanisms synergizing in leukemogenic rearrangements.

    Science.gov (United States)

    Biechonski, Shahar; Gourevich, Dana; Rall, Melanie; Aqaqe, Nasma; Yassin, Muhammad; Zipin-Roitman, Adi; Trakhtenbrot, Luba; Olender, Leonid; Raz, Yael; Jaffa, Ariel J; Grisaru, Dan; Wiesmuller, Lisa; Elad, David; Milyavsky, Michael

    2017-02-15

    Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values. © 2016 UICC.

  11. Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle

    Directory of Open Access Journals (Sweden)

    Taponen Juhani

    2007-11-01

    Full Text Available Abstract Background Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization. Results Bull-derived CD34+ cells were detected in the liver of the female sibling (freemartin at 60 days gestation. The level of bull-derived CD34+ cells was lower in the freemartin than in its male siblings. Bull (Y+ and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y+CD34+ cells, Y+CD34- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation. Conclusion The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.

  12. Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Michelle Millington

    2009-07-01

    Full Text Available Hematopoietic stem cells (HSC, in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34(+ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.Using commercially available G-CSF mobilized peripheral blood (PB CD34(+ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI, transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV carrying enhanced green fluorescent protein (GFP was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+ cells.

  13. Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

    Directory of Open Access Journals (Sweden)

    Gene-Errol E Ringpis

    Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

  14. Molecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cells

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2016-01-01

    The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E-selectin was confirmed using some static and flow-based assays. E-selectin binds to CD34 with an affinity comparable to the well-described E-selLs CD44/HCELL and PSGL-1. CD34 knockdown resulted in faster-rolling velocities compared to control cells especially at and above three dyne/cm2. CD34 is the first selectin ligand since PSGL-1 reported to bind E-/P-/L-selectins and likely plays a key role in directing the migration of human HSPCs to the bone marrow.

  15. Synergistic actions of hematopoietic and mesenchymal stem/progenitor cells in vascularizing bioengineered tissues.

    Directory of Open Access Journals (Sweden)

    Eduardo K Moioli

    Full Text Available Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs and mesenchymal stem/progenitor cells (MSCs were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP scaffolds, followed by infusion of gel-suspended CD34(+ hematopoietic cells. Co-transplantation of CD34(+ HSCs and CD34(- MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromised mice yielded vascularized tissue. The average vascular number of co-transplanted CD34(+ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34(+ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34(+ cells. Based on additional in vitro results of endothelial differentiation of CD34(+ cells by vascular endothelial growth factor (VEGF, we adsorbed VEGF with co-transplanted CD34(+ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34(+ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone

  16. Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

    Science.gov (United States)

    Zonari, Erika; Desantis, Giacomo; Petrillo, Carolina; Boccalatte, Francesco E; Lidonnici, Maria Rosa; Kajaste-Rudnitski, Anna; Aiuti, Alessandro; Ferrari, Giuliana; Naldini, Luigi; Gentner, Bernhard

    2017-04-11

    Ex vivo gene therapy based on CD34 + hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34 + cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E 2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34 + CD38 - cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Effects of hematopoietic growth factors on purified bone marrow progenitor cells

    NARCIS (Netherlands)

    F.J. Bot (Freek)

    1992-01-01

    textabstractWe have used highly enriched hematopoietic progenitor cells and in-vitro culture to examine the following questions: 1. The effects of recombinant lL-3 and GM-CSF on proliferation and differentiation of enriched hematopoietic progenitor cells have not been clearly defined: - how do IL~3

  18. Cell cycle regulation of hematopoietic stem or progenitor cells.

    Science.gov (United States)

    Hao, Sha; Chen, Chen; Cheng, Tao

    2016-05-01

    The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

  19. Involvement of placental/umbilical cord blood acid-base status and gas values on the radiosensitivity of human fetal/neonatal hematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Yamaguchi, Masaru; Ebina, Satoko; Kashiwakura, Ikuo

    2013-01-01

    Arterial cord blood (CB) acid-base status and gas values, such as pH, PCO 2 , PO 2 , HCO 3 - and base excess, provide useful information on the fetal and neonatal condition. However, it remains unknown whether these values affect the radiosensitivity of fetal/neonatal hematopoiesis. The present study evaluated the relationship between arterial CB acid-base status, gas values, and the radiosensitivity of CB hematopoietic stem/progenitor cells (HSPCs). A total of 25 CB units were collected. The arterial CB acid-base status and gas values were measured within 30 min of delivery. The CD34 + HSPCs obtained from CB were exposed to 2 Gy X-irradiation, and then assayed for colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte erythroid, macrophage and megakaryocyte cells. Acid-base status and gas values for PCO 2 and HCO 3 - showed a statistically significant negative correlation with the surviving fraction of BFU-E. In addition, a significant positive correlation was observed between gestational age and PCO 2 . Moreover, the surviving fraction of BFU-E showed a significant negative correlation with gestational age. Thus, HSPCs obtained from CB with high PCO 2 /HCO 3 - levels were sensitive to X-irradiation, which suggests that the status of arterial PCO 2 /HCO 3 - influences the radiosensitivity of fetal/neonatal hematopoiesis, especially erythropoiesis. (author)

  20. In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice.

    Science.gov (United States)

    Río, Paula; Segovia, José Carlos; Hanenberg, Helmut; Casado, José Antonio; Martínez, Jesús; Göttsche, Kerstin; Cheng, Ngan Ching; Van de Vrugt, Henri J; Arwert, Fré; Joenje, Hans; Bueren, Juan A

    2002-09-15

    Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca(-/-) mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect of Fanca(-/-) progenitors, either when total BM or when purified Lin(-)Sca-1(+) cells were subjected to in vitro stimulation. Liquid cultures of Fanca(-/-) BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca(-/-) progenitors, purified Lin(-)Sca-1(+) cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCA genes. Lin(-)Sca-1(+) cells from Fanca(-/-) mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors of Fanca(-/-) mice.

  1. Functional evaluation of circulating hematopoietic progenitors in Noonan syndrome

    Science.gov (United States)

    TIMEUS, FABIO; CRESCENZIO, NICOLETTA; BALDASSARRE, GIUSEPPINA; DORIA, ALESSANDRA; VALLERO, STEFANO; FOGLIA, LUISELDA; PAGLIANO, SARA; ROSSI, CESARE; SILENGO, MARGHERITA CIRILLO; RAMENGHI, UGO; FAGIOLI, FRANCA; DI MONTEZEMOLO, LUCA CORDERO; FERRERO, GIOVANNI BATTISTA

    2013-01-01

    Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, multiple dysmorphisms and congenital heart defects. A myeloproliferative disorder (NS/MPD), resembling juvenile myelomonocytic leukemia (JMML), is occasionally diagnosed in infants with NS. In the present study, we performed a functional evaluation of the circulating hematopoietic progenitors in a series of NS, NS/MPD and JMML patients. The different functional patterns were compared with the aim to identify a possible NS subgroup worthy of stringent hematological follow-up for an increased risk of MPD development. We studied 27 NS and 5 JMML patients fulfilling EWOG-MDS criteria. The more frequent molecular defects observed in NS were mutations in the PTPN11 and SOS genes. The absolute count of monocytes, circulating CD34+ hematopoietic progenitors, their apoptotic rate and the number of circulating CFU-GMs cultured in the presence of decreasing concentrations or in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated. All JMML patients showed monocytosis >1,000/μl. Ten out of the 27 NS patients showed monocytosis >1,000/μl, which included the 3 NS/MPD patients. In JMML patients, circulating CD34+ cells were significantly increased (median, 109.8/μl; range, 44–232) with a low rate of apoptosis (median, 2.1%; range, 0.4–12.1%), and circulating CFU-GMs were hyper-responsive to GM-CSF. NS/MPD patients showed the same flow cytometric pattern as the JMML patients (median, CD34+ cells/μl, 205.7; range, 58–1374; median apoptotic rate, 1.4%; range, 0.2–2.4%) and their circulating CFU-GMs were hyper-responsive to GM-CSF. These functional alterations appeared 10 months before the typical clinical manifestations in 1 NS/MPD patient. In NS, the CD34+ absolute cell count and circulating CFU-GMs showed a normal pattern (median CD34+ cells/μl, 4.9; range, 1.3–17.5), whereas the CD34+ cell apoptotic rate was significantly decreased in

  2. Pre-malignant lymphoid cells arise from hematopoietic stem/progenitor cells in chronic lymphocytic leukemia.

    Science.gov (United States)

    Kikushige, Yoshikane; Miyamoto, Toshihiro

    2015-11-01

    Human malignancies progress through a multistep process that includes the development of critical somatic mutations over the clinical course. Recent novel findings have indicated that hematopoietic stem cells (HSCs), which have the potential to self-renew and differentiate into multilineage hematopoietic cells, are an important cellular target for the accumulation of critical somatic mutations in hematological malignancies and play a central role in myeloid malignancy development. In contrast to myeloid malignancies, mature lymphoid malignancies, such as chronic lymphocytic leukemia (CLL), are thought to originate directly from differentiated mature lymphocytes; however, recent compelling data have shown that primitive HSCs and hematopoietic progenitor cells contribute to the pathogenesis of mature lymphoid malignancies. Several representative mutations of hematological malignancies have been identified within the HSCs of CLL and lymphoma patients, indicating that the self-renewing long-lived fraction of HSCs can serve as a reservoir for the development of oncogenic events. Novel mice models have been established as human mature lymphoma models, in which specific oncogenic events target the HSCs and immature progenitor cells. These data collectively suggest that HSCs can be the cellular target involved in the accumulation of oncogenic events in the pathogenesis of mature lymphoid and myeloid malignancies.

  3. Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition

    Directory of Open Access Journals (Sweden)

    Aimin Yang

    2015-01-01

    Full Text Available Abnormal activation of the mammalian target of rapamycin (mTOR signaling pathway has been observed in a variety of human cancers. Therefore, targeting of the mTOR pathway is an attractive strategy for cancer treatment and several mTOR inhibitors, including AZD8055 (AZD, a novel dual mTORC1/2 inhibitor, are currently in clinical trials. Although bone marrow (BM suppression is one of the primary side effects of anticancer drugs, it is not known if pharmacological inhibition of dual mTORC1/2 affects BM hematopoietic stem and progenitor cells (HSPCs function and plasticity. Here we report that dual inhibition of mTORC1/2 by AZD or its analogue (KU-63794 depletes mouse BM Lin−Sca-1+c-Kit+ cells in cultures via the induction of apoptotic cell death. Subsequent colony-forming unit (CFU assays revealed that inhibition of mTORC1/2 suppresses the clonogenic function of hematopoietic progenitor cells (HPCs in a dose-dependent manner. Surprisingly, we found that dual inhibition of mTORC1/2 markedly inhibits the growth of day-14 cobblestone area-forming cells (CAFCs but enhances the generation of day-35 CAFCs. Given the fact that day-14 and day-35 CAFCs are functional surrogates of HPCs and hematopoietic stem cells (HSCs, respectively, these results suggest that dual inhibition of mTORC1/2 may have distinct effects on HPCs versus HSCs.

  4. Contribution of endothelial progenitors and proangiogenic hematopoietic cells to vascularization of tumor and ischemic tissue

    Science.gov (United States)

    Kopp, Hans-Georg; Ramos, Carlos A.; Rafii, Shahin

    2010-01-01

    Purpose of review During the last several years, a substantial amount of evidence from animal as well as human studies has advanced our knowledge of how bone marrow derived cells contribute to neoangiogenesis. In the light of recent findings, we may have to redefine our thinking of endothelial cells as well as of perivascular mural cells. Recent findings Inflammatory hematopoietic cells, such as macrophages, have been shown to promote neoangiogenesis during tumor growth and wound healing. Dendritic cells, B lymphocytes, monocytes, and other immune cells have also been found to be recruited to neoangiogenic niches and to support neovessel formation. These findings have led to the concept that subsets of hematopoietic cells comprise proangiogenic cells that drive adult revascularization processes. While evidence of the importance of endothelial progenitor cells in adult vasculogenesis increased further, the role of these comobilized hematopoietic cells has been intensely studied in the last few years. Summary Angiogenic factors promote mobilization of vascular endothelial growth factor receptor 1-positive hematopoietic cells through matrix metalloproteinase-9 mediated release of soluble kit-ligand and recruit these proangiogenic cells to areas of hypoxia, where perivascular mural cells present stromal-derived factor 1 (CXCL-12) as an important retention signal. The same factors are possibly involved in mobilization of vascular endothelial growth factor receptor 2-positive endothelial precursors that may participate in neovessel formation. The complete characterization of mechanisms, mediators and signaling pathways involved in these processes will provide novel targets for both anti and proangiogenic therapeutic strategies. PMID:16567962

  5. Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease

    International Nuclear Information System (INIS)

    Fink, J.K.; Correll, P.H.; Perry, L.K.; Brady, R.O.; Karlsson, S.

    1990-01-01

    Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, the authors developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py + /Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hemaptopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py + /Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py + /Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity has been normalized

  6. Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease

    Energy Technology Data Exchange (ETDEWEB)

    Fink, J.K.; Correll, P.H.; Perry, L.K.; Brady, R.O.; Karlsson, S. (National Institutes of Health, Bethesda, MD (USA))

    1990-03-01

    Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, the authors developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py{sup +}/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hemaptopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py{sup +}/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py{sup +}/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity has been normalized.

  7. Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

    Science.gov (United States)

    Xie, Ying; Koch, Mia Lee; Zhang, Xin; Hamblen, Melanie J; Godinho, Frank J; Fujiwara, Yuko; Xie, Huafeng; Klusmann, Jan-Henning; Orkin, Stuart H; Li, Zhe

    2017-07-01

    ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Erg kd ) in which Erg expression can be conditionally restored by Cre recombinase. Erg kd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Erg kd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin - Sca-1 + c-Kit + (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Erg kd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785. © 2017 AlphaMed Press.

  8. An update on ABO incompatible hematopoietic progenitor cell transplantation.

    Science.gov (United States)

    Staley, Elizabeth M; Schwartz, Joseph; Pham, Huy P

    2016-06-01

    Hematopoietic progenitor cell (HPC) transplantation has long been established as the optimal treatment for many hematologic malignancies. In the setting of allogenic HLA matched HPC transplantation, greater than 50% of unrelated donors and 30% of related donors demonstrate some degree of ABO incompatibility (ABOi), which is classified in one of three ways: major, minor, or bidirectional. Major ABOi refers to the presence of recipient isoagglutinins against the donor's A and/or B antigen. Minor ABOi occurs when the HPC product contains the isoagglutinins targeting the recipient's A and/or B antigen. Bidirectional refers to the presence of both major and minor ABOi. Major adverse events associated with ABOi HPC transplantation includes acute and delayed hemolysis, pure red cell aplasia, and delayed engraftment. ABOi HPC transplantation poses a unique challenge to the clinical transplantation unit, the HPC processing lab, and the transfusion medicine service. Therefore, it is essential that these services actively communicate with one another to ensure patient safety. This review will attempt to globally address the challenges related to ABOi HPC transplantation, with an increased focus on aspects related to the laboratory and transfusion medicine services. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Hematopoietic Stem and Progenitor Cells as Effectors in Innate Immunity

    Directory of Open Access Journals (Sweden)

    Jennifer L. Granick

    2012-01-01

    Full Text Available Recent research has shed light on novel functions of hematopoietic stem and progenitor cells (HSPC. While they are critical for maintenance and replenishment of blood cells in the bone marrow, these cells are not limited to the bone marrow compartment and function beyond their role in hematopoiesis. HSPC can leave bone marrow and circulate in peripheral blood and lymph, a process often manipulated therapeutically for the purpose of transplantation. Additionally, these cells preferentially home to extramedullary sites of inflammation where they can differentiate to more mature effector cells. HSPC are susceptible to various pathogens, though they may participate in the innate immune response without being directly infected. They express pattern recognition receptors for detection of endogenous and exogenous danger-associated molecular patterns and respond not only by the formation of daughter cells but can themselves secrete powerful cytokines. This paper summarizes the functional and phenotypic characterization of HSPC, their niche within and outside of the bone marrow, and what is known regarding their role in the innate immune response.

  10. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

    Science.gov (United States)

    Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

    2015-01-01

    Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

  11. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    Energy Technology Data Exchange (ETDEWEB)

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Schulz, Robert A., E-mail: rschulz@nd.edu

    2014-10-24

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche.

  12. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    International Nuclear Information System (INIS)

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Schulz, Robert A.

    2014-01-01

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche

  13. Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

    Science.gov (United States)

    Rissone, Alberto; Weinacht, Katja Gabriele; la Marca, Giancarlo; Bishop, Kevin; Giocaliere, Elisa; Jagadeesh, Jayashree; Felgentreff, Kerstin; Dobbs, Kerry; Al-Herz, Waleed; Jones, Marypat; Chandrasekharappa, Settara; Kirby, Martha; Wincovitch, Stephen; Simon, Karen Lyn; Itan, Yuval; DeVine, Alex; Schlaeger, Thorsten; Schambach, Axel; Sood, Raman

    2015-01-01

    Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD. PMID:26150473

  14. CXC chemokine receptor 3 expression on CD34(+) hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor

    DEFF Research Database (Denmark)

    Jinquan, T; Quan, S; Jacobi, H H

    2000-01-01

    -induced CD34(+) progenitor chemotaxis. These chemotactic attracted CD34(+) progenitors are colony-forming units-granulocyte-macrophage. gamma IP-10 and Mig also induced GM-CSF-stimulated CD34(+) progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 m......Ab blocked these functions of gammaIP-10 and Mig but not of chemokine stromal cell-derived factor 1 alpha. gamma IP-10-induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF-stimulated CD34(+) progenitors. Moreover, gamma IP-10 and Mig...... stimulated CXCR3 redistribution and cellular polarization in GM-CSF-stimulated CD34(+) progenitors. These results indicate that CXCR3-gamma IP-10 and CXCR3-Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment...

  15. Exercise increases the frequency of circulating hematopoietic progenitor cells, but reduces hematopoietic colony-forming capacity.

    Science.gov (United States)

    Kroepfl, Julia Maria; Pekovits, Karin; Stelzer, Ingeborg; Fuchs, Robert; Zelzer, Sieglinde; Hofmann, Peter; Sedlmayr, Peter; Dohr, Gottfried; Wallner-Liebmann, Sandra; Domej, Wolfgang; Mueller, Wolfram

    2012-11-01

    Circulating hematopoietic progenitor cells (CPCs) may be triggered by physical exercise and/or normobaric hypoxia from the bone marrow. The aim of the study was to investigate the influence of physical exercise and normobaric hypoxia on CPC number and functionality in the peripheral blood as well as the involvement of oxidative stress parameters as possibly active agents. Ten healthy male subjects (25.3±4.4 years) underwent a standardized cycle incremental exercise test protocol (40 W+20 W/min) under either normoxic (FiO2 ∼0.21) or hypoxic conditions (FiO2exercise. The number of CPCs in the peripheral blood was analyzed by flow cytometry (CD34/CD45-positive cells). The functionality of cells present was addressed by secondary colony-forming unit-granulocyte macrophage (CFU-GM) assays. To determine a possible correlation between the mobilization of CPCs and reactive oxygen species, parameters for oxidative stress such as malondialdehyde (MDA) and myeloperoxidase (MPO) were obtained. Data showed a significant increase of CPC release under normoxic as well as hypoxic conditions after 10 min of recovery (Pexercise (Pexercise, possibly due to the influence of increased oxidative stress levels.

  16. Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors

    Directory of Open Access Journals (Sweden)

    Dongjun Lee

    2014-11-01

    Full Text Available Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1 or are upstream and regulatory of it (mTORC2. Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2 negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.

  17. The hematopoietic transcription factor PU.1 regulates RANK gene expression in myeloid progenitors

    International Nuclear Information System (INIS)

    Kwon, Oh Hyung; Lee, Chong-Kil; Lee, Young Ik; Paik, Sang-Gi; Lee, Hyun-Jun

    2005-01-01

    Osteoclasts are bone resorbing cells of hematopoietic origin. The hematopoietic transcription factor PU.1 is critical for osteoclastogenesis; however, the molecular mechanisms of PU.1-regulated osteoclastogenesis have not been explored. Here, we present evidence that the receptor activator of nuclear factor κB (RANK) gene that has been shown to be crucial for osteoclastogenesis is a transcriptional target of PU.1. The PU.1 -/- progenitor cells failed to express the RANK gene and reconstitution of PU.1 in these cells induced RANK expression. Treatment of the PU.1 reconstituted cells with M-CSF and RANKL further augmented the RANK gene expression. To explore the regulatory mechanism of the RANK gene expression by PU.1, we have cloned the human RANK promoter. Transient transfection assays have revealed that the 2.2-kb RANK promoter was functional in a monocyte line RAW264.7, whereas co-transfection of PU.1 transactivated the RANK promoter in HeLa cells. Taken together, these results suggest that PU.1 regulates the RANK gene transcription and this may represent one of the key roles of PU.1 in osteoclast differentiation

  18. The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation

    NARCIS (Netherlands)

    Walasek, Marta A.; Bystrykh, Leonid; van den Boom, Vincent; Olthof, Sandra; Ausema, Albertina; Ritsema, Martha; Huls, Gerwin; de Haan, Gerald; van Os, Ronald

    2012-01-01

    Despite increasing knowledge on the regulation of hematopoietic stem/progenitor cell (HSPC) self-renewal and differentiation, in vitro control of stem cell fate decisions has been difficult. The ability to inhibit HSPC commitment in culture may be of benefit to cell therapy protocols. Small

  19. Regulatory Systems in Bone Marrow for Hematopoietic Stem/Progenitor Cells Mobilization and Homing

    Directory of Open Access Journals (Sweden)

    P. Alvarez

    2013-01-01

    Full Text Available Regulation of hematopoietic stem cell release, migration, and homing from the bone marrow (BM and of the mobilization pathway involves a complex interaction among adhesion molecules, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells. The identification of new mechanisms that regulate the trafficking of hematopoietic stem/progenitor cells (HSPCs cells has important implications, not only for hematopoietic transplantation but also for cell therapies in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and stroke, among others. This paper reviews the regulation mechanisms underlying the homing and mobilization of BM hematopoietic stem/progenitor cells, investigating the following issues: (a the role of different factors, such as stromal cell derived factor-1 (SDF-1, granulocyte colony-stimulating factor (G-CSF, and vascular cell adhesion molecule-1 (VCAM-1, among other ligands; (b the stem cell count in peripheral blood and BM and influential factors; (c the therapeutic utilization of this phenomenon in lesions in different tissues, examining the agents involved in HSPCs mobilization, such as the different forms of G-CSF, plerixafor, and natalizumab; and (d the effects of this mobilization on BM-derived stem/progenitor cells in clinical trials of patients with different diseases.

  20. Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1

    Science.gov (United States)

    Zhou, Bo O; Ding, Lei; Morrison, Sean J

    2015-01-01

    Hematopoietic stem cells (HSCs) are maintained by a perivascular niche in bone marrow but it is unclear whether the niche is reciprocally regulated by HSCs. Here, we systematically assessed the expression and function of Angiopoietin-1 (Angpt1) in bone marrow. Angpt1 was not expressed by osteoblasts. Angpt1 was most highly expressed by HSCs, and at lower levels by c-kit+ hematopoietic progenitors, megakaryocytes, and Leptin Receptor+ (LepR+) stromal cells. Global conditional deletion of Angpt1, or deletion from osteoblasts, LepR+ cells, Nes-cre-expressing cells, megakaryocytes, endothelial cells or hematopoietic cells in normal mice did not affect hematopoiesis, HSC maintenance, or HSC quiescence. Deletion of Angpt1 from hematopoietic cells and LepR+ cells had little effect on vasculature or HSC frequency under steady-state conditions but accelerated vascular and hematopoietic recovery after irradiation while increasing vascular leakiness. Hematopoietic stem/progenitor cells and LepR+ stromal cells regulate niche regeneration by secreting Angpt1, reducing vascular leakiness but slowing niche recovery. DOI: http://dx.doi.org/10.7554/eLife.05521.001 PMID:25821987

  1. Expression and function of PML-RARA in the hematopoietic progenitor cells of Ctsg-PML-RARA mice.

    Directory of Open Access Journals (Sweden)

    Lukas D Wartman

    Full Text Available Because PML-RARA-induced acute promyelocytic leukemia (APL is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte versus an hematopoietic stem/progenitor cell (HSPC. We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice, are expressed in the purified KLS cells of these mice (KLS = Kit(+Lin(-Sca(+, which are highly enriched for HSPCs, and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs and granulocyte/monocyte progenitors (GMPs], which have a distinct gene expression signature compared to wild-type (WT mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.

  2. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

    Directory of Open Access Journals (Sweden)

    Giorgia Salvagiotto

    2011-03-01

    Full Text Available Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.

  3. PRDM11 is dispensable for the maintenance and function of hematopoietic stem and progenitor cells

    DEFF Research Database (Denmark)

    Thoren, Lina A; Fog, Cathrine K; Jensen, Klaus T

    2013-01-01

    Hematopoietic stem cells (HSC)(1) supply organisms with life-long output of mature blood cells. To do so, the HSC pool size has to be maintained by HSC self-renewing divisions. PRDM3 and PRDM16 have been documented to regulate HSC self-renewal, maintenance and function. We found Prdm11 to have...... similar expression patterns in the hematopoietic stem and progenitor cell (HSPC) compartments as Prdm3 and Prdm16. Therefore, we undertook experiments to test if PRDM11 regulates HSC self-renewal, maintenance and function by investigating the Prdm11(-/-) mice. Our data shows that phenotypic HSPCs...

  4. Cadmium modulates hematopoietic stem and progenitor cells and skews toward myelopoiesis in mice

    International Nuclear Information System (INIS)

    Zhang, Yandong; Yu, Xinchun; Sun, Shuhui; Li, Qian; Xie, Yunli; Li, Qiang; Zhao, Yifan; Pei, Jianfeng; Zhang, Wenmin; Xue, Peng; Zhou, Zhijun; Zhang, Yubin

    2016-01-01

    The heavy metal cadmium (Cd) is known to modulate immunity and cause osteoporosis. However, how Cd influences on hematopoiesis remain largely unknown. Herein, we show that wild-type C57BL/6 (B6) mice exposed to Cd for 3 months had expanded bone marrow (BM) populations of long-term hematopoietic stem cells (LT-HSCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), while having reduced populations of multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). A competitive mixed BM transplantation assay indicates that BM from Cd-treated mice had impaired LT-HSC ability to differentiate into mature cells. In accordance with increased myeloid progenitors and decreased lymphoid progenitors, the BM and spleens of Cd-treated mice had more monocytes and/or neutrophils and fewer B cells and T cells. Cd impaired the ability of the non-hematopoietic system to support LT-HSCs, in that lethally irradiated Cd-treated recipients transplanted with normal BM cells had reduced LT-HSCs after the hematopoietic system was fully reconstituted. This is consistent with reduced osteoblasts, a known critical component for HSC niche, observed in Cd-treated mice. Conversely, lethally irradiated control recipients transplanted with BM cells from Cd-treated mice had normal LT-HSC reconstitution. Furthermore, both control mice and Cd-treated mice that received Alendronate, a clinical drug used for treating osteoporosis, had BM increases of LT-HSCs. Thus, the results suggest Cd increase of LT-HSCs is due to effects on HSCs and not on osteoblasts, although, Cd causes osteoblast reduction and impaired niche function for maintaining HSCs. Furthermore, Cd skews HSCs toward myelopoiesis. - Highlights: • Cd increases the number of LT-HSCs but impairs their development. • Cd-treated hosts have compromised ability to support LT-HSCs. • Cd promotes myelopoiesis at the expense of lymphopoiesis at the MPP level.

  5. Cadmium modulates hematopoietic stem and progenitor cells and skews toward myelopoiesis in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yandong; Yu, Xinchun [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China); Sun, Shuhui [Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Li, Qian [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China); Xie, Yunli [Insititute of Brain Sciences, Fudan University, Shanghai 200032 (China); Li, Qiang [Putuo District Center for Disease Control and Prevention, Shanghai 200062 (China); Zhao, Yifan; Pei, Jianfeng; Zhang, Wenmin; Xue, Peng; Zhou, Zhijun [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China); Zhang, Yubin, E-mail: yz001@fudan.edu.cn [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China)

    2016-12-15

    The heavy metal cadmium (Cd) is known to modulate immunity and cause osteoporosis. However, how Cd influences on hematopoiesis remain largely unknown. Herein, we show that wild-type C57BL/6 (B6) mice exposed to Cd for 3 months had expanded bone marrow (BM) populations of long-term hematopoietic stem cells (LT-HSCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), while having reduced populations of multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). A competitive mixed BM transplantation assay indicates that BM from Cd-treated mice had impaired LT-HSC ability to differentiate into mature cells. In accordance with increased myeloid progenitors and decreased lymphoid progenitors, the BM and spleens of Cd-treated mice had more monocytes and/or neutrophils and fewer B cells and T cells. Cd impaired the ability of the non-hematopoietic system to support LT-HSCs, in that lethally irradiated Cd-treated recipients transplanted with normal BM cells had reduced LT-HSCs after the hematopoietic system was fully reconstituted. This is consistent with reduced osteoblasts, a known critical component for HSC niche, observed in Cd-treated mice. Conversely, lethally irradiated control recipients transplanted with BM cells from Cd-treated mice had normal LT-HSC reconstitution. Furthermore, both control mice and Cd-treated mice that received Alendronate, a clinical drug used for treating osteoporosis, had BM increases of LT-HSCs. Thus, the results suggest Cd increase of LT-HSCs is due to effects on HSCs and not on osteoblasts, although, Cd causes osteoblast reduction and impaired niche function for maintaining HSCs. Furthermore, Cd skews HSCs toward myelopoiesis. - Highlights: • Cd increases the number of LT-HSCs but impairs their development. • Cd-treated hosts have compromised ability to support LT-HSCs. • Cd promotes myelopoiesis at the expense of lymphopoiesis at the MPP level.

  6. Nicaraven attenuates radiation-induced injury in hematopoietic stem/progenitor cells in mice.

    Directory of Open Access Journals (Sweden)

    Miho Kawakatsu

    Full Text Available Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in hematopoietic stem/progenitor cells, which is the conmen complication of radiotherapy and one of the major causes of death in sub-acute phase after accidental exposure to high dose radiation. C57BL/6 mice were exposed to 1 Gy γ-ray radiation daily for 5 days in succession (a total of 5 Gy, and given nicaraven or a placebo after each exposure. The mice were sacrificed 2 days after the last radiation treatment, and the protective effects and relevant mechanisms of nicaraven in hematopoietic stem/progenitor cells with radiation-induced damage were investigated by ex vivo examination. We found that post-radiation administration of nicaraven significantly increased the number, improved the colony-forming capacity, and decreased the DNA damage of hematopoietic stem/progenitor cells. The urinary levels of 8-oxo-2'-deoxyguanosine, a marker of DNA oxidation, were significantly lower in mice that were given nicaraven compared with those that received a placebo treatment, although the levels of intracellular and mitochondrial reactive oxygen species in the bone marrow cells did not differ significantly between the two groups. Interestingly, compared with the placebo treatment, the administration of nicaraven significantly decreased the levels of the inflammatory cytokines IL-6 and TNF-α in the plasma of mice. Our data suggest that nicaraven effectively diminished the effects of radiation-induced injury in hematopoietic stem/progenitor cells, which is likely associated with the anti-oxidative and anti-inflammatory properties of this compound.

  7. OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Xiaoli Chen

    2015-09-01

    Full Text Available Generating engraftable hematopoietic stem cells (HSCs from pluripotent stem cells (PSCs is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2 accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo.

  8. Changing patterns of radiosensitivity of hematopoietic progenitors from chronically irradiated dogs prone either to aplastic anemia or to myeloproliferative disease

    International Nuclear Information System (INIS)

    Seed, T.M.; Kaspar, L.V.

    1990-01-01

    Hematopoietic patterns have been assessed in chronic 60 Co gamma irradiated dogs during preclinical phases of evolving aplastic anemia (AA) or myeloproliferative disease (MPD), principally myeloid leukemia. The results support the concept that acquired radioresistance of vital granulocyte/monocyte lineage-committed hematopoietic progenitors is temporally, perhaps causally, linked to the processes mediating hematopoietic recovery and accommodation under chronic irradiation, and in turn to preclinical events of evolving MPD. In addition, the marked differential responses of progenitors to gamma and neutron irradiation in vitro might suggest differences in the nature of cellular lesions elicited by chronic gamma irradiation, in vivo. (author)

  9. Changing patterns of radiosensitivity of hematopoietic progenitors from chronically irradiated dogs prone either to aplastic anemia or to myeloproliferative disease

    Energy Technology Data Exchange (ETDEWEB)

    Seed, T.M.; Kaspar, L.V. (Oak Ridge National Lab., TN (USA))

    1990-01-01

    Hematopoietic patterns have been assessed in chronic {sup 60}Co gamma irradiated dogs during preclinical phases of evolving aplastic anemia (AA) or myeloproliferative disease (MPD), principally myeloid leukemia. The results support the concept that acquired radioresistance of vital granulocyte/monocyte lineage-committed hematopoietic progenitors is temporally, perhaps causally, linked to the processes mediating hematopoietic recovery and accommodation under chronic irradiation, and in turn to preclinical events of evolving MPD. In addition, the marked differential responses of progenitors to gamma and neutron irradiation in vitro might suggest differences in the nature of cellular lesions elicited by chronic gamma irradiation, in vivo. (author).

  10. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

    Science.gov (United States)

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-Ping; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

    2016-05-01

    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI.

    Science.gov (United States)

    Jin, Hao; Sood, Raman; Xu, Jin; Zhen, Fenghua; English, Milton A; Liu, P Paul; Wen, Zilong

    2009-02-01

    One unique feature of vertebrate definitive hematopoiesis is the ontogenic switching of hematopoietic stem cells from one anatomical compartment or niche to another. In mice, hematopoietic stem cells are believed to originate in the aorta-gonad-mesonephros (AGM), subsequently migrate to the fetal liver (FL) and finally colonize the bone marrow (BM). Yet, the differentiation potential of hematopoietic stem cells within early niches such as the AGM and FL remains incompletely defined. Here, we present in vivo analysis to delineate the differentiation potential of definitive hematopoietic stem/progenitor cells (HSPCs) in the zebrafish AGM and FL analogies, namely the ventral wall of dorsal aorta (VDA) and the posterior blood island (PBI), respectively. Cell fate mapping and analysis of zebrafish runx1(w84x) and vlad tepes (vlt(m651)) mutants revealed that HSPCs in the PBI gave rise to both erythroid and myeloid lineages. However, we surprisingly found that HSPCs in the VDA were not quiescent but were uniquely adapted to generate myeloid but not erythroid lineage cells. We further showed that such distinct differentiation output of HSPCs was, at least in part, ascribed to the different micro-environments present in these two niches. Our results highlight the importance of niche in shaping the differentiation output of developing HSPCs.

  12. Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression.

    Science.gov (United States)

    Guo, Peipei; Poulos, Michael G; Palikuqi, Brisa; Badwe, Chaitanya R; Lis, Raphael; Kunar, Balvir; Ding, Bi-Sen; Rabbany, Sina Y; Shido, Koji; Butler, Jason M; Rafii, Shahin

    2017-12-01

    Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

  13. Comparative study of hematopoietic stem and progenitor cells between sexes in mice under physiological conditions along time.

    Science.gov (United States)

    Gasco, Samanta; Rando, Amaya; Zaragoza, Pilar; García-Redondo, Alberto; Calvo, Ana Cristina; Osta, Rosario

    2017-12-01

    Hematopoietic stem and progenitor cells (HSPCs) are attractive targets in regenerative medicine, although the differences in their homeostatic maintenance between sexes along time are still under debate. We accurately monitored hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs) frequencies by flow cytometry, by performing serial peripheral blood extractions from male and female B6SJL wild-type mice and found no significant differences. Only modest differences were found in the gene expression profile of Slamf1 and Gata2. Our findings suggest that both sexes could be used indistinctly to perform descriptive studies in the murine hematopoietic system, especially for flow cytometry studies in peripheral blood. This would allow diminishing the number of animals needed for the experimental procedures. In addition, the use of serial extractions in the same animals drastically decreases the number of animals needed. © 2017 International Federation for Cell Biology.

  14. Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

    Science.gov (United States)

    Doulatov, Sergei; Vo, Linda T.; Macari, Elizabeth R.; Wahlster, Lara; Kinney, Melissa A.; Taylor, Alison M.; Barragan, Jessica; Gupta, Manav; McGrath, Katherine; Lee, Hsiang-Ying; Humphries, Jessica M.; DeVine, Alex; Narla, Anupama; Alter, Blanche P.; Beggs, Alan H.; Agarwal, Suneet; Ebert, Benjamin L.; Gazda, Hanna T.; Lodish, Harvey F.; Sieff, Colin A.; Schlaeger, Thorsten M.; Zon, Leonard I.; Daley, George Q.

    2017-01-01

    Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. PMID:28179501

  15. Oxidative stress due to radiation in CD34+ hematopoietic progenitor cells. Protection by IGF-1

    International Nuclear Information System (INIS)

    Floratou, K.; Karakantza, M.; Giannopoulou, E.; Antonacopoulou, A.; Adonakis, G.; Kardamakis, D.; Matsouka, P.

    2012-01-01

    Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34 + irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34 + from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis. (author)

  16. Imaging Macrophage and Hematopoietic Progenitor Proliferation in Atherosclerosis

    DEFF Research Database (Denmark)

    Ye, Yu-Xiang; Calcagno, Claudia; Binderup, Tina

    2015-01-01

    tomography-computed tomography imaging of cell proliferation in atherosclerosis. METHODS AND RESULTS: (18)F-FLT positron emission tomography-computed tomography was performed in mice, rabbits, and humans with atherosclerosis. In apolipoprotein E knock out mice, increased (18)F-FLT signal was observed...

  17. Molecular regulation of human hematopoietic stem cells

    NARCIS (Netherlands)

    van Galen, P.L.J.

    2014-01-01

    Peter van Galen focuses on understanding the determinants that maintain the stem cell state. Using human hematopoietic stem cells (HSCs) as a model, processes that govern self-renewal and tissue regeneration were investigated. Specifically, a role for microRNAs in balancing the human HSC

  18. Subsequent donation requests among 2472 unrelated hematopoietic progenitor cell donors are associated with bone marrow harvest

    Science.gov (United States)

    Lown, Robert N.; Tulpule, Sameer; Russell, Nigel H.; Craddock, Charles F.; Roest, Rochelle; Madrigal, J. Alejandro; Shaw, Bronwen E.

    2013-01-01

    Approximately 1 in 20 unrelated donors are asked to make a second donation of hematopoietic progenitor cells, the majority for the same patient. Anthony Nolan undertook a study of subsequent hematopoietic progenitor cell donations made by its donors from 2005 to 2011, with the aims of predicting those donors more likely to be called for a second donation, assessing rates of serious adverse reactions and examining harvest yields. This was not a study of factors predictive of second allografts. During the study period 2591 donations were made, of which 120 (4.6%) were subsequent donations. The median time between donations was 179 days (range, 21–4016). Indications for a second allogeneic transplant included primary graft failure (11.7%), secondary graft failure (53.2%), relapse (30.6%) and others (1.8%). On multivariate analysis, bone marrow harvest at first donation was associated with subsequent donation requests (odds ratio 2.00, P=0.001). The rate of serious adverse reactions in donors making a subsequent donation appeared greater than the rate in those making a first donation (relative risk=3.29, P=0.005). Harvest yields per kilogram recipient body weight were equivalent between donations, although females appeared to have a lower yield at the subsequent donation. Knowledge of these factors will help unrelated donor registries to counsel their donors. PMID:23812935

  19. SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Mina N.F. Morcos

    2017-06-01

    Full Text Available Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs express the surface molecule Stem cell antigen 1 (SCA-1/LY6A. Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage−SCA-1+KIT+ (LSK population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN. We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

  20. Transient loading of CD34+ hematopoietic progenitor cells with polystyrene nanoparticles

    Directory of Open Access Journals (Sweden)

    Deville S

    2017-01-01

    Full Text Available Sarah Deville,1,2 Wahyu Wijaya Hadiwikarta,1 Nick Smisdom,1,2 Bart Wathiong,1,3 Marcel Ameloot,2 Inge Nelissen,1 Jef Hooyberghs1,3 1VITO, Flemish Institute for Technological Research, Mol, Belgium; 2Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 3Theoretical Physics, Hasselt University, Diepenbeek, Belgium Abstract: CD34+ hematopoietic progenitor cells (HPCs offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon. Keywords: nanoparticles, hematopoietic progenitor cells, dendritic cells, uptake, release

  1. Catalase inhibits ionizing radiation-induced apoptosis in hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Xiao, Xia; Luo, Hongmei; Vanek, Kenneth N; LaRue, Amanda C; Schulte, Bradley A; Wang, Gavin Y

    2015-06-01

    Hematologic toxicity is a major cause of mortality in radiation emergency scenarios and a primary side effect concern in patients undergoing chemo-radiotherapy. Therefore, there is a critical need for the development of novel and more effective approaches to manage this side effect. Catalase is a potent antioxidant enzyme that coverts hydrogen peroxide into hydrogen and water. In this study, we evaluated the efficacy of catalase as a protectant against ionizing radiation (IR)-induced toxicity in hematopoietic stem and progenitor cells (HSPCs). The results revealed that catalase treatment markedly inhibits IR-induced apoptosis in murine hematopoietic stem cells and hematopoietic progenitor cells. Subsequent colony-forming cell and cobble-stone area-forming cell assays showed that catalase-treated HSPCs can not only survive irradiation-induced apoptosis but also have higher clonogenic capacity, compared with vehicle-treated cells. Moreover, transplantation of catalase-treated irradiated HSPCs results in high levels of multi-lineage and long-term engraftments, whereas vehicle-treated irradiated HSPCs exhibit very limited hematopoiesis reconstituting capacity. Mechanistically, catalase treatment attenuates IR-induced DNA double-strand breaks and inhibits reactive oxygen species. Unexpectedly, we found that the radioprotective effect of catalase is associated with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway and pharmacological inhibition of STAT3 abolishes the protective activity of catalase, suggesting that catalase may protect HSPCs against IR-induced toxicity via promoting STAT3 activation. Collectively, these results demonstrate a previously unrecognized mechanism by which catalase inhibits IR-induced DNA damage and apoptosis in HSPCs.

  2. Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation.

    Science.gov (United States)

    Li, Wei; Wang, Guanjun; Cui, Jiuwei; Xue, Lu; Cai, Lu

    2004-11-01

    The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a

  3. Proliferative status of primitive hematopoietic progenitors from patients with acute myelogenous leukemia (AML).

    Science.gov (United States)

    Guan, Y; Hogge, D E

    2000-12-01

    One possible explanation for the competitive advantage that malignant cells in patients with acute myelogenous leukemia (AML) appear to have over normal hematopoietic elements is that leukemic progenitors proliferate more rapidly than their normal progenitor cell counterparts. To test this hypothesis, an overnight 3H-thymidine (3H-Tdr) suicide assay was used to analyze the proliferative status of malignant progenitors detected in both colony-forming cell (CFC) and long-term culture initiating cell (LTC-IC) assays from the peripheral blood of nine patients with newly diagnosed AML. Culture of AML cells in serum-free medium with 100 ng/ml Steel factor (SF), 20 ng/ml interleukin 3 (IL-3) and 20 ng/ml granulocyte colony-stimulating factor (G-CSF) for 16-24 h maintained the number of AML-CFC and LTC-IC at near input values (mean % input +/- s.d. for CFC and LTC-IC were 78 +/- 33 and 126 +/- 53, respectively). The addition of 20 muCi/ml high specific activity 3H-Tdr to these cultures reduced the numbers of both progenitor cell types from most of the patient samples substantially: mean % kill +/- s.d. for AML-CFC and LTC-IC were 64 +/- 27 and 82 +/- 16, respectively, indicating that a large proportion of both progenitor populations were actively cycling. FISH analysis of colonies from CFC and LTC-IC assays confirmed that most cytogenetically abnormal CFC and LTC-IC were actively cycling (mean % kill +/- s.d.: 68 +/- 26 and 85 +/- 13, respectively). Interestingly, in six patient samples where a significant number of cytogenetically normal LTC-ICs were detected, the % kill of these cells (74 +/- 20) was similar to that of the abnormal progenitors. These data contrast with the predominantly quiescent cell cycle status of CFC and LTC-IC previously observed in steady-state peripheral blood from normal individuals but also provide evidence that a significant proportion of primitive malignant progenitors from AML patients are quiescent and therefore may be resistant to standard

  4. Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

    NARCIS (Netherlands)

    Braakman, E.; Schuurhuis, G.J.; Preijers, F.W.M.B.; Voermans, C.; Theunissen, K.; Riet, I. van; Fibbe, W.E.; Slaper-Cortenbach, I.C.M.

    2008-01-01

    BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure

  5. Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products

    NARCIS (Netherlands)

    Braakman, E.; Schuurhuis, G. J.; Preijers, F. W. M. B.; Voermans, C.; Theunissen, K.; van Riet, I.; Fibbe, W. E.; Slaper-Cortenbach, I.

    2008-01-01

    BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure

  6. Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products

    NARCIS (Netherlands)

    E. Braakman (Eric); G.J. Schuurhuis (Gerrit Jan); F.W.M.B. Preijers (Frank); C. Voermans; K. Theunissen; I. van Riet; W.E. Fibbe (Willem); I. Slaper-Cortenbach (Ineke)

    2008-01-01

    textabstractBackground: Immunomagnetic selection of CD34+hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+cells recommended by the manufacturer for processing in a single

  7. Comparative Analysis of the Hematopoietic Progenitor Cells from Placenta, Cord Blood, and Fetal Liver, Based on Their Immunophenotype

    Directory of Open Access Journals (Sweden)

    Maria D. Kuchma

    2015-01-01

    Full Text Available We have investigated the characteristics of human hematopoietic progenitor cells (HPCs with the CD34+CD45lowSSClow phenotype from full-term placental tissue (FTPT as compared to cord blood (CB and fetal liver (FL cells. We demonstrated the presence of cell subpopulations at various stages of the differentiation with such immunophenotypes as CD34+/lowCD45low/-, CD34++CD45low/-, CD34+++CD45low/-, CD34+/lowCD45hi, and CD34++CD45hi in both first trimester placental tissue (FiTPT and FTPT which implies their higher phenotypic heterogeneity compared to CB. HPCs of the FTPT origin expressed the CD90 antigen at a higher level compared to its expression by the CB HPCs and the CD133 antigen expression being at the same level in both cases. The HPCs compartment of FTPT versus CB contained higher number of myeloid and erythroid committed cells but lower number of myeloid and lymphoid ones compared to FL HPCs. HPCs of the FTPT and CB origin possess similar potentials for the multilineage differentiation in vitro and similar ratios of myeloid and erythroid progenitors among the committed cells. This observation suggests that the active hematopoiesis occurs in the FTPT. We obtained viable HPCs from cryopreserved placental tissue fragments allowing us to develop procedures for banking and testing of placenta-derived HPCs for clinical use.

  8. Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

    Science.gov (United States)

    Podestà, Marina; Bruschettini, Matteo; Cossu, Claudia; Sabatini, Federica; Dagnino, Monica; Romantsik, Olga; Spaggiari, Grazia Maria; Ramenghi, Luca Antonio; Frassoni, Francesco

    2015-01-01

    Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15-4.8) vs 0.3% (0.032-2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18-4.8) and 0.36% (0.15-3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.

  9. Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

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    Marina Podestà

    Full Text Available Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups.Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15-4.8 vs 0.3% (0.032-2.23 p = 0.0001 and in neonates <32 weeks of gestational age (GA compared to those ≥32 wks GA: 0.95% (range 0.18-4.8 and 0.36% (0.15-3.2 respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL resulted higher compared to term (p = 0.004 and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017. The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168.We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.

  10. Comparison of cryopreservation bags for hematopoietic progenitor cells using a WBC-enriched product.

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    Dijkstra-Tiekstra, Margriet J; Hazelaar, Sandra; Gkoumassi, Effimia; Weggemans, Margienus; de Wildt-Eggen, Janny

    2015-04-01

    Hematopoietic progenitor cells (HPC) are stored in cryopreservation bags that are resistant to liquid nitrogen. Since Cryocyte bags of Baxter (B-bags) are no longer available, an alternative bag was sought. Also, the influence of freezing volume was studied. Miltenyi Biotec (MB)- and MacoPharma (MP)-bags passed the integrity tests without failure. Comparing MB- and MP-bags with B-bags, no difference in WBC recovery or viability was found when using a WBC-enriched product as a "dummy" HPC product. Further, a freezing volume of 30 mL resulted in better WBC recovery and viability than 60 mL. Additonal studies using real HPC might be necessary. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors.

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    Mega, Tiziana; Lupia, Michela; Amodio, Nicola; Horton, Sarah J; Mesuraca, Maria; Pelaggi, Daniela; Agosti, Valter; Grieco, Michele; Chiarella, Emanuela; Spina, Raffaella; Moore, Malcolm A S; Schuringa, Jan Jacob; Bond, Heather M; Morrone, Giovanni

    2011-07-01

    Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.

  12. The role of tumor suppressor p15Ink4b in the regulation of hematopoietic progenitor cell fate

    International Nuclear Information System (INIS)

    Humeniuk, R; Rosu-Myles, M; Fares, J; Koller, R; Bies, J; Wolff, L

    2013-01-01

    Epigenetic silencing of the tumor suppressor gene p15Ink4b (CDKN2B) is a frequent event in blood disorders like acute myeloid leukemia and myelodysplastic syndromes. The molecular function of p15Ink4b in hematopoietic differentiation still remains to be elucidated. Our previous study demonstrated that loss of p15Ink4b in mice results in skewing of the differentiation pattern of the common myeloid progenitor towards the myeloid lineage. Here, we investigated a function of p15Ink4b tumor suppressor gene in driving erythroid lineage commitment in hematopoietic progenitors. It was found that p15Ink4b is expressed more highly in committed megakaryocyte–erythroid progenitors than granulocyte–macrophage progenitors. More importantly, mice lacking p15Ink4b have lower numbers of primitive red cell progenitors and a severely impaired response to 5-fluorouracil- and phenylhydrazine-induced hematopoietic stress. Introduction of p15Ink4b into multipotential progenitors produced changes at the molecular level, including activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling, increase GATA-1, erythropoietin receptor (EpoR) and decrease Pu1, GATA-2 expression. These changes rendered cells more permissive to erythroid commitment and less permissive to myeloid commitment, as demonstrated by an increase in early burst-forming unit-erythroid formation with concomitant decrease in myeloid colonies. Our results indicate that p15Ink4b functions in hematopoiesis, by maintaining proper lineage commitment of progenitors and assisting in rapid red blood cells replenishment following stress

  13. Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

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    Young, Kira; Borikar, Sneha; Bell, Rebecca; Kuffler, Lauren; Philip, Vivek; Trowbridge, Jennifer J

    2016-10-17

    Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging. © 2016 Young et al.

  14. Endothelial and circulating progenitor cells in hematological diseases and allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Ruggeri, Annalisa; Paviglianiti, Annalisa; Volt, Fernanda; Kenzey, Chantal; Rafii, Hanadi; Rocha, Vanderson; Gluckman, Eliane

    2017-10-12

    Circulating endothelial cells (CECs), originated form endothelial progenitors (EPCs) are mature cells which are not associated with vessel walls, and that are detached from the endothelium. Normally, they are present in insignificant amounts in the peripheral blood of healthy individuals. On the other hand, elevated CECs and EPCs levels have been reported in the peripheral blood of patients with different types of cancers and some other diseases. Consequently, CECs and EPCs represent a potential biomarker in several clinical conditions involving endothelial turnover and remodeling, such as hematological diseases. These cells may be involved in disease progression and the neoplastic angiogenesis process. Moreover, CESs and EPCs are probably involved in endothelial damage that is a marker of several complications following allogeneic hematopoietic stem cell transplantation. This review aims to provide an overview on the characterization of CECs and EPCs, describe isolation methods and to identify the potential role of these cells in hematological diseases and hematopoietic stem cell transplantation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Reciprocal upregulation of Notch signaling molecules in hematopoietic progenitor and mesenchymal stromal cells

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    Kikuchi Y

    2011-01-01

    Full Text Available Although mesenchymal stem cells (MSCs play pivotal supportive roles in hematopoiesis, how they interact with hematopoietic stem cells (HSCs is not well understood. We investigated the interaction between HSCs and surrogate MSCs (C3H10T1/2 stromal cells, focusing on the molecular events induced by cell contact of these bipartite populations. C3H10T1/2 is a mesenchymal stromal cell line that can be induced to differentiate into preadipocytes (A54 and myoblasts (M1601. The stromal cell derivatives were cocultured with murine HSCs (Lineage-Sca1+, and gene expression profiles in stromal cells and HSCs were compared before and after the coculture. HSCs gave rise to cobblestone areas only on A54 cells, with ninefold more progenitors than on M1601 or undifferentiated C3H10T1/2 cells. Microarray-based screening and a quantitative reverse transcriptase directed-polymerase chain reaction showed that the levels of Notch ligands (Jagged1 and Delta-like 3 were increased in A54 cells upon interaction with HSCs. On the other hand, the expression of Notch1 and Hes1 was upregulated in the HSCs cocultured with A54 cells. A transwell assay revealed that the reciprocal upregulation was dependent on cell-to-cell contact. The result suggested that in the hematopoietic niche, HSCs help MSCs to produce Notch ligands, and in turn, MSCs help HSCs to express Notch receptor. Such a reciprocal upregulation would reinforce the downstream signaling to determine the fate of hematopoietic cell lineage. Clarification of the initiating events on cell contact should lead to the identification of specific molecular targets to facilitate HSC engraftment in transplantation therapy.

  16. Probing altered hematopoietic progenitors of preleukemic dogs with JANUS fission neutrons

    International Nuclear Information System (INIS)

    Seed, T.M.; Kaspar, L.V.

    1990-01-01

    Toward the goal of developing basic insights to mechanisms of radiation leukemogenesis, the authors have developed a canine model that responds to protracted courses of low-daily-dose gamma irradiation with high incidences of myeloproliferative disease (MPD), principally myeloid leukemia. Using this model system, the authors have identified and partially characterized a four-phase preclinical sequence in the induction of MPD, including (1) suppression, (2) recovery, (3) accommodation, and (4) preleukemic transition. Further, they have identified within this sequence, a critical early hematopoietic target cell event that appears to promote progression of the initial preclinical phase to the second preclinical phase. This key target cell event is characterized by the acquisition of increased radioresistance to low-LET gamma rays by granulocyte/monocyte-committed progenitors (CFU-GM). In order to gain further insight into the basis of this critical event, the acquired survival responses of preleukemic progenitors have been probed in vitro with high-LET fission neutrons. 23 refs., 4 figs., 1 tab

  17. Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

    International Nuclear Information System (INIS)

    Corcelle, V.; Stieger, B.; Gjinovci, A.; Wollheim, C.B.; Gauthier, B.R.

    2006-01-01

    Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl 4 . Livers were removed 9 to 13 days post-CCl 4 treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b + cells fail to propagate while c-kit + -HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit + -HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion

  18. Haemopoietic progenitor cells in human peripheral blood

    International Nuclear Information System (INIS)

    Zwaan, F.E.

    1980-01-01

    The purpose of the investigation reported is to purify haemopoietic progenitor cells from human peripheral blood using density gradient centrifugation in order to isolate a progenitor cell fraction without immunocompetent cells. The purification technique of peripheral blood flow colony forming unit culture (CFU-c) by means of density gradient centrifugation and a combined depletion of various rosettes is described. The results of several 'in vitro' characteristics of purified CFU-c suspensions and of the plasma clot diffusion chamber culture technique are presented. Irradiation studies revealed that for both human bone marrow and peripheral blood the CFU-c were less radioresistant than clusters. Elimination of monocytes (and granulocytes) from the test suspensions induced an alteration in radiosensitivity pararmeters. The results obtained with the different techniques are described by analysing peripheral progenitor cell activity in myeloproliferative disorders. (Auth.)

  19. Generation of induced pluripotent stem cells from peripheral blood CD34+ hematopoietic progenitors of a 31 year old healthy woman

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    Amornrat Tangprasittipap

    2017-04-01

    Full Text Available The MUi019-A human induced pluripotent stem cell line was generated from peripheral blood CD34+ hematopoietic progenitors of a healthy woman using a non-integrative reprogramming method. Episomal vectors carrying reprogramming factors OCT4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 and EBNA-1 were delivered using electroporation. The iPSC line can be used as a control in studying disease mechanisms. Furthermore, gene editing approaches can be used to introduce specific mutations into the MUi019-A to model disease while the cell type affected by the disease is inaccessible.

  20. Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency

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    Andrea Z. Tuckett

    2017-05-01

    Full Text Available Abstract Background Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγnull mice is feasible and facilitates the generation of functional T cells conferring protective immunity. Methods Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase+, CD45.1+ and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγnull recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγnull mice was assessed in a B cell lymphoma model. Results Despite the small size of the thymic remnant in NOD-scid IL2rγnull mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγnull mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγnull mice, suggesting that immune dysregulation is not a major concern

  1. HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

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    Parisa Imanirad

    2014-01-01

    Full Text Available Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α, a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver, and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.

  2. Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

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    Pascariello Caterina

    2008-05-01

    Full Text Available Abstract Background Aldehyde dehydrogenase (ALDH is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007. The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively. As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a. Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA. Conclusion Our study, comparing surface antigen expression of

  3. Hematopoietic Stem and Progenitor Cell Migration After Hypofractionated Radiation Therapy in a Murine Model

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    Kane, Jonathan [Department of Biological Sciences, Oakland University, Rochester, Michigan (United States); Radiation Oncology, William Beaumont Health System, Royal Oak, Michigan (United States); Krueger, Sarah A.; Dilworth, Joshua T.; Torma, John T.; Wilson, George D.; Marples, Brian [Radiation Oncology, William Beaumont Health System, Royal Oak, Michigan (United States); Madlambayan, Gerard J., E-mail: madlamba@oakland.edu [Department of Biological Sciences, Oakland University, Rochester, Michigan (United States)

    2013-12-01

    Purpose: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. Methods and Materials: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. Results: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133{sup +} HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b{sup +} counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. Conclusions: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

  4. Hematopoietic Stem and Progenitor Cell Migration After Hypofractionated Radiation Therapy in a Murine Model

    International Nuclear Information System (INIS)

    Kane, Jonathan; Krueger, Sarah A.; Dilworth, Joshua T.; Torma, John T.; Wilson, George D.; Marples, Brian; Madlambayan, Gerard J.

    2013-01-01

    Purpose: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. Methods and Materials: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. Results: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133 + HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b + counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. Conclusions: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors

  5. In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

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    P.B. Soares

    2013-01-01

    Full Text Available Imatinib mesylate (IM is used to treat chronic myeloid leukemia (CML because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM. The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM, using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells increased. At higher concentrations (15 µM, the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control. Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

  6. Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients.

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    Diez, Begoña; Genovese, Pietro; Roman-Rodriguez, Francisco J; Alvarez, Lara; Schiroli, Giulia; Ugalde, Laura; Rodriguez-Perales, Sandra; Sevilla, Julian; Diaz de Heredia, Cristina; Holmes, Michael C; Lombardo, Angelo; Naldini, Luigi; Bueren, Juan Antonio; Rio, Paula

    2017-11-01

    Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  7. Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide

    International Nuclear Information System (INIS)

    Winton, E.F.; Colenda, K.W.

    1987-01-01

    The continued retrieval of progenitor cells (CFU-GEMM, BFU-E, CFU-E, CFU-GM) from human long-term marrow cultures (LTMC) is not uncommonly used as evidence that proliferation and differentiation are occurring in more primitive hematopoietic stem cells (HSC) in these cultures. Alternatively, the continued presence of progenitors in LTMC could be the result of survival and/or limited self-renewal of progenitor cells present when the culture was initiated, and such progenitors would have little relevance to the parent HSC. The following studies were designed to determine the relative contributions of precursors of progenitor cells to the total progenitor cells present in LTMC using a two-stage regeneration model. The adherent layer in LTMC was established over 3 weeks, irradiated (875 rad) to permanently eliminate resident hematopoietic cells, and recharged with autologous cryo-preserved marrow that was either treated or not treated (control) with 4-hydroperoxycyclophosphamide (4-HC, 100 micrograms/ml for 30 min). The 4-HC-treated marrow contained no progenitor cells, yet based on clinical autologous bone marrow transplant experience, has intact HSC. Within 1-3 weeks, progenitor cells reappeared in the irradiated LTMC recharged with 4-HC-treated marrow, and were preferentially located in the adherent layer. By 2-6 weeks, the number of progenitor cells in the adherent layer of LTMC recharged with 4-HC marrow was equivalent to control LTMC. The progenitors regenerating in the irradiated LTMC recharged with 4-HC-treated marrow appear to originate from precursors of progenitor cells, perhaps HSC. We propose this model may be useful in elucidating cellular and molecular correlates of progenitor cell regeneration from precursors

  8. Sonic Hedgehog Signaling Regulates Hematopoietic Stem/Progenitor Cell Activation during the Granulopoietic Response to Systemic Bacterial Infection.

    Science.gov (United States)

    Shi, Xin; Wei, Shengcai; Simms, Kevin J; Cumpston, Devan N; Ewing, Thomas J; Zhang, Ping

    2018-01-01

    Activation and reprogramming of hematopoietic stem/progenitor cells play a critical role in the granulopoietic response to bacterial infection. Our current study determined the significance of Sonic hedgehog (SHH) signaling in the regulation of hematopoietic precursor cell activity during the host defense response to systemic bacterial infection. Bacteremia was induced in male Balb/c mice via intravenous injection (i.v.) of Escherichia coli (5 × 10 7 CFUs/mouse). Control mice received i.v. saline. SHH protein level in bone marrow cell (BMC) lysates was markedly increased at both 24 and 48 h of bacteremia. By contrast, the amount of soluble SHH ligand in marrow elutes was significantly reduced. These contrasting alterations suggested that SHH ligand release from BMCs was reduced and/or binding of soluble SHH ligand to BMCs was enhanced. At both 12 and 24 h of bacteremia, SHH mRNA expression by BMCs was significantly upregulated. This upregulation of SHH mRNA expression was followed by a marked increase in SHH protein expression in BMCs. Activation of the ERK1/2-SP1 pathway was involved in mediating the upregulation of SHH gene expression. The major cell type showing the enhancement of SHH expression in the bone marrow was lineage positive cells. Gli1 positioned downstream of the SHH receptor activation serves as a key component of the hedgehog (HH) pathway. Primitive hematopoietic precursor cells exhibited the highest level of baseline Gli1 expression, suggesting that they were active cells responding to SHH ligand stimulation. Along with the increased expression of SHH in the bone marrow, expression of Gli1 by marrow cells was significantly upregulated at both mRNA and protein levels following bacteremia. This enhancement of Gli1 expression was correlated with activation of hematopoietic stem/progenitor cell proliferation. Mice with Gli1 gene deletion showed attenuation in activation of marrow hematopoietic stem/progenitor cell proliferation and inhibition

  9. Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes

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    Moufida Ben Nasr

    2018-06-01

    Full Text Available Hematopoietic stem and progenitor cells (HSPCs are multipotent stem cells that have been harnessed as a curative therapy for patients with hematological malignancies. Notably, the discovery that HSPCs are endowed with immunoregulatory properties suggests that HSPC-based therapeutic approaches may be used to treat autoimmune diseases. Indeed, infusion with HSPCs has shown promising results in the treatment of type 1 diabetes (T1D and remains the only “experimental therapy” that has achieved a satisfactory rate of remission (nearly 60% in T1D. Patients with newly diagnosed T1D have been successfully reverted to normoglycemia by administration of autologous HSPCs in association with a non-myeloablative immunosuppressive regimen. However, this approach is hampered by a high incidence of adverse effects linked to immunosuppression. Herein, we report that while the use of autologous HSPCs is capable of improving C-peptide production in patients with T1D, ex vivo modulation of HSPCs with prostaglandins (PGs increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. Surprisingly, CXCR4 was upregulated as well, which could enhance HSPC trafficking toward the inflamed pancreatic zone. When tested in murine and human in vitro autoimmune assays, PG-modulated HSPCs were shown to abrogate the autoreactive T cell response. The use of PG-modulated HSPCs may thus provide an attractive and novel treatment of autoimmune diabetes.

  10. Enhanced genetic modification of adult growth factor mobilized peripheral blood hematopoietic stem and progenitor cells with rapamycin.

    Science.gov (United States)

    Li, Lijing; Torres-Coronado, Mónica; Gu, Angel; Rao, Anitha; Gardner, Agnes M; Epps, Elizabeth W; Gonzalez, Nancy; Tran, Chy-Anh; Wu, Xiwei; Wang, Jin-Hui; DiGiusto, David L

    2014-10-01

    Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long-term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene-modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene-modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell-based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene-modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene-modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene-modified autologous HSPCs for our HIV gene therapy clinical trials. ©AlphaMed Press.

  11. Two Ellagic Acids Isolated from Roots of Sanguisorba officinalis L. Promote Hematopoietic Progenitor Cell Proliferation and Megakaryocyte Differentiation

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    Xiaoping Gao

    2014-04-01

    Full Text Available Using a bioassay-directed chromatographic separation, two ellagic acids were obtained from the ethyl acetate extract of the roots of Sanguisorba officinalis L. On the basis of chemical and spectroscopic methods, the two ellagic acids were identified as 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside and 3,3',4-tri-O-methylellagic acid. Stimulation of cell proliferation was assayed in hematopoietic progenitor cells using the Cell Counting kit-8 method. The megakaryocyte differentiation was determined in human erythroleukemia (HEL cells using Giemsa staining and flow cytometry analysis. The ellagic acids significantly stimulated the proliferation of Baf3/Mpl cells. Morphology analysis and megakaryocyte specific-marker CD41 staining confirmed that the ellagic acids induced megakaryocyte differentiation in HEL cells. This is the first time that 3,3',4-tri-O-methylellagic acid or 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside are reported to induce megakaryopoiesis, suggesting a class of small molecules which differ from others non-peptidyl, and appears to have potential for clinical development as a therapeutic agent for patients with blood platelet disorders.

  12. Long-active granulocyte colony-stimulating factor for peripheral blood hematopoietic progenitor cell mobilization.

    Science.gov (United States)

    Martino, Massimo; Laszlo, Daniele; Lanza, Francesco

    2014-06-01

    Peg-filgrastim (PEG-FIL), a polyethylene glycol-conjugated form of granulocyte colony-stimulating factor (G-CSF), has been introduced in clinical practice and is effective in shortening the time of neutropenia after cytotoxic chemotherapy. G-CSF has emerged as the preferred cytokine for hematopoietic progenitor cells' (HPC) mobilization. Nevertheless, data on the ability of PEG-FIL in this field have been published. We review publications in the field with the goal of providing an overview of this approach. PEG-FIL may be able to mobilize CD34(+) cells in a more timely fashion than G-CSF, with the advantages of only a single-dose administration, an earlier start and a reduction in the number of apheresis procedures. The main controversies concern the dosage of the drug and the optimal dose. In the context of chemo-mobilization, a single dose of 6 mg PEG-FIL seems effective in terms of HPC's mobilization and there is no increase in this effect if the dose is doubled to 12 mg. Steady-state mobilization requires higher doses of PEG-FIL and this approach is not cost-effective when compared with G-CSF. The experiences with PEG-FIL in the healthy donor setting are very limited.

  13. Hypercholesterolemia Tunes Hematopoietic Stem/Progenitor Cells for Inflammation and Atherosclerosis.

    Science.gov (United States)

    Ma, Xiaojuan; Feng, Yingmei

    2016-07-19

    As the pathological basis of cardiovascular disease (CVD), atherosclerosis is featured as a chronic inflammation. Hypercholesterolemia is an independent risk factor for CVD. Accumulated studies have shown that hypercholesterolemia is associated with myeloid cell expansion, which stimulates innate and adaptive immune responses, strengthens inflammation, and accelerates atherosclerosis progression. Hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) expresses a panel of lipoprotein receptors to control cholesterol homeostasis. Deficiency of these receptors abrogates cellular cholesterol efflux, resulting in HSPC proliferation and differentiation in hypercholesterolemic mice. Reduction of the cholesterol level in the lipid rafts by infusion of reconstituted high-density lipoprotein (HDL) or its major apolipoprotein, apoA-I, reverses hypercholesterolemia-induced HSPC expansion. Apart from impaired cholesterol metabolism, inhibition of reactive oxygen species production suppresses HSPC activation and leukocytosis. These data indicate that the mechanisms underlying the effects of hypercholesterolemia on HSPC proliferation and differentiation could be multifaceted. Furthermore, dyslipidemia also regulates HSPC-neighboring cells, resulting in HSPC mobilization. In the article, we review how hypercholesterolemia evokes HSPC activation and mobilization directly or via its modification of BM microenvironment. We hope this review will bring light to finding key molecules to control HSPC expansion, inflammation, and atherosclerosis for the treatment of CVD.

  14. Ryk receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice

    Science.gov (United States)

    Povinelli, Benjamin J.; Srivastava, Pragya; Nemeth, Michael J.

    2017-01-01

    Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Ryk. Expression of Ryk on HSPCs in vivo is associated with a lower rate of proliferation and following treatment with fluorouracil (5-FU), the percentage of Ryk+/high HSPCs increased while the percent of Ryk−/low HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling in vivo resulted in increased HSC proliferation and decreased HSC function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury. PMID:25461251

  15. Transient loading of CD34+ hematopoietic progenitor cells with polystyrene nanoparticles.

    Science.gov (United States)

    Deville, Sarah; Hadiwikarta, Wahyu Wijaya; Smisdom, Nick; Wathiong, Bart; Ameloot, Marcel; Nelissen, Inge; Hooyberghs, Jef

    2017-01-01

    CD34 + hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon.

  16. Open the gates: vascular neurocrine signaling mobilizes hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Itkin, Tomer; Gómez-Salinero, Jesús María; Rafii, Shahin

    2017-12-01

    Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) into the peripheral blood is a complex process that is enhanced dramatically under stress-induced conditions. A better understanding of how the mobilization process is regulated will likely facilitate the development of improved clinical protocols for stem cell harvesting and transplantation. In this issue of the JCI, Singh et al. (1) showed that the truncated cleaved form of neurotransmitter neuropeptide Y (NPY) actively promotes a breach of BM vascular sinusoidal portals, thereby augmenting HSPC trafficking to the circulation. The authors report a previously unrecognized axis, in which expression of the enzyme dipeptidylpeptidase-4 (DPP4)/CD26 by endothelial cells activates NPY-mediated signaling by increasing the bioavailability of the truncated form of NPY. These findings underscore the importance of and urgency to develop pharmacological therapies that target the vasculature and regulate diverse aspects of hematopoiesis, such as HSPC trafficking, in steady-state and stress-induced conditions.

  17. Reconstruction of hematopoietic inductive microenvironment after transplantation of VCAM-1-modified human umbilical cord blood stromal cells.

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    Yao Liu

    Full Text Available The hematopoietic inductive microenvironment (HIM is where hematopoietic stem/progenitor cells grow and develop. Hematopoietic stromal cells were the key components of the HIM. In our previous study, we had successfully cultured and isolated human cord blood-derived stromal cells (HUCBSCs and demonstrated that they could secret hemopoietic growth factors such as GM-CSF, TPO, and SCF. However, it is still controversial whether HUCBSCs can be used for reconstruction of HIM. In this study, we first established a co-culture system of HUCBSCs and cord blood CD34(+ cells and then determined that using HUCBSCs as the adherent layer had significantly more newly formed colonies of each hematopoietic lineage than the control group, indicating that HUCBSCs had the ability to promote the proliferation of hematopoietic stem cells/progenitor cells. Furthermore, the number of colonies was significantly higher in vascular cell adhesion molecule-1 (VCAM-1-modified HUCBSCs, suggesting that the ability of HUCBSCs in promoting the proliferation of hematopoietic stem cells/progenitor cells was further enhanced after having been modified with VCAM-1. Next, HUCBSCs were infused into a radiation-damaged animal model, in which the recovery of hematopoiesis was observed. The results demonstrate that the transplanted HUCBSCs were "homed in" to bone marrow and played roles in promoting the recovery of irradiation-induced hematopoietic damage and repairing HIM. Compared with the control group, the HUCBSC group had significantly superior effectiveness in terms of the recovery time for hemogram and myelogram, CFU-F, CFU-GM, BFU-E, and CFU-Meg. Such differences were even more significant in VCAM-1-modified HUCBSCs group. We suggest that HUCBSCs are able to restore the functions of HIM and promote the recovery of radiation-induced hematopoietic damage. VCAM-1 plays an important role in supporting the repair of HIM damage.

  18. Combining G-CSF with a blockade of adhesion strongly improves the reconstitutive capacity of mobilized hematopoietic progenitor cells.

    Science.gov (United States)

    Christ, O; Kronenwett, R; Haas, R; Zöller, M

    2001-03-01

    Mobilization of hematopoietic progenitor cells is achieved mainly by application of growth factors and, more recently, by blockade of adhesion. In this report, we describe the advantages of a combined treatment with granulocyte colony-stimulating factor (G-CSF) and anti-VLA4 (CD49d)/anti-CD44 as compared to treatment with the individual components. Mobilization by intravenous injection of anti-CD44, anti-VLA4, or G-CSF was controlled in spleen and bone marrow with regard to frequencies of multipotential colony-forming unit (C-CFU), marrow repopulating ability, long-term reconstitution, recovery of myelopoiesis, and regain of immunocompetence. Mobilization by anti-CD44 had a strong effect on expansion of early progenitor cells in the bone marrow, while the recovery in the spleen was poor. In anti-CD49d-mobilized noncommitted and committed progenitors, progenitor expansion was less pronounced, but settlement in the spleen was quite efficient. Thus, anti-CD44 and anti-CD49d differently influenced mobilization. Accordingly, mobilization and recovery after transfer were improved by combining anti-CD44 with anti-CD49d treatment. Mobilization by G-CSF was most efficient with respect to recovery of progenitor cells in the spleen. However, when transferring G-CSF-mobilized cells, regain of immunocompetence was strongly delayed. This disadvantage could be overridden when progenitor cells were mobilized via blockade of adhesion and when expansion of these mobilized progenitor cells was supported by low-dose G-CSF only during the last 24 hours before transfer. Mobilization of pluripotent progenitor cells via antibody blockade of CD44 or CD49d or via G-CSF relies on distinct mechanisms. Therefore, the reconstitutive capacity of a transplant can be significantly improved by mobilization regimens combining antibody with low-dose G-CSF treatment.

  19. Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Chow, Paik Wah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Chan, Kok Meng [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Inayat-Hussain, Salmaan Hussain [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Rajab, Nor Fadilah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia)

    2015-04-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and

  20. Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

    International Nuclear Information System (INIS)

    Chow, Paik Wah; Abdul Hamid, Zariyantey; Chan, Kok Meng; Inayat-Hussain, Salmaan Hussain; Rajab, Nor Fadilah

    2015-01-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e + cells but reduced the total counts of Sca-1 + , CD11b + , Gr-1 + , and CD45 + cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and progenitors. • 1,4-BQ

  1. Cell-Cycle-Specific Function of p53 in Fanconi Anemia Hematopoietic Stem and Progenitor Cell Proliferation

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    Xiaoli Li

    2018-02-01

    Full Text Available Summary: Overactive p53 has been proposed as an important pathophysiological factor for bone marrow failure syndromes, including Fanconi anemia (FA. Here, we report a p53-dependent effect on hematopoietic stem and progenitor cell (HSPC proliferation in mice deficient for the FA gene Fanca. Deletion of p53 in Fanca−/− mice leads to replicative exhaustion of the hematopoietic stem cell (HSC in transplant recipients. Using Fanca−/− HSCs expressing the separation-of-function mutant p53515C transgene, which selectively impairs the p53 function in apoptosis but keeps its cell-cycle checkpoint activities intact, we show that the p53 cell-cycle function is specifically required for the regulation of Fanca−/− HSC proliferation. Our results demonstrate that p53 plays a compensatory role in preventing FA HSCs from replicative exhaustion and suggest a cautious approach to manipulating p53 signaling as a therapeutic utility in FA. : In this article, Pang and colleagues demonstrate a p53-dependent HSPC proliferation regulation in mice deficient for the Fanca gene in the Fanconi anemia (FA pathway. They show that the p53 cell-cycle function is specifically required for the regulation of FA HSC proliferation. These results suggest that overactive p53 may represent a compensatory checkpoint mechanism for FA HSC proliferation. Keywords: p53, bone marrow failure, Fanconi anemia, hematopoietic stem and progenitor cells, apoptosis, cell cycle, proliferation

  2. A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.

    Science.gov (United States)

    Ward, Maureen; Sattler, Rose; Grossman, I Robert; Bell, Anthony J; Skerrett, Donna; Baxi, Laxmi; Bank, Arthur

    2003-11-01

    Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines. However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC. The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34+ cells using transient packaging systems and to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained. We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34+ cells. Viral particles from this cell line were concentrated up to 100-fold (up to 10(7) viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+ cells were efficiently transduced with a Neo(R)-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+ cells and 51% of progenitors from sickle cell CD34+ cells expressed the NeoR gene. We also show transfer of a human beta-globin gene into progenitor cells from CD34+ cells from sickle cell patients with this new RD114 stable packaging system. The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy.

  3. Sources of Hematopoietic Stem and Progenitor Cells and Methods to Optimize Yields for Clinical Cell Therapy.

    Science.gov (United States)

    Panch, Sandhya R; Szymanski, James; Savani, Bipin N; Stroncek, David F

    2017-08-01

    Bone marrow (BM) aspirates, mobilized peripheral blood, and umbilical cord blood (UCB) have developed as graft sources for hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation and other cellular therapeutics. Individualized techniques are necessary to enhance graft HSPC yields and cell quality from each graft source. BM aspirates yield adequate CD34 + cells but can result in relative delays in engraftment. Granulocyte colony-stimulating factor (G-CSF)-primed BM HSPCs may facilitate faster engraftment while minimizing graft-versus-host disease in certain patient subsets. The levels of circulating HSPCs are enhanced using mobilizing agents, such as G-CSF and/or plerixafor, which act via the stromal cell-derived factor 1/C-X-C chemokine receptor type 4 axis. Alternate niche pathway mediators, including very late antigen-4/vascular cell adhesion molecule-1, heparan sulfate proteoglycans, parathyroid hormone, and coagulation cascade intermediates, may offer promising alternatives for graft enhancement. UCB grafts have been expanded ex vivo with cytokines, notch-ligand, or mesenchymal stromal cells, and most studies demonstrated greater quantities of CD34 + cells ex vivo and improved short-term engraftment. No significant changes were observed in long-term repopulating potential or in patient survival. Early phase clinical trials using nicotinamide and StemReginin1 may offer improved short- and long-term repopulating ability. Breakthroughs in genome editing and stem cell reprogramming technologies may hasten the generation of pooled, third-party HSPC grafts. This review elucidates past, present, and potential future approaches to HSPC graft optimization. Published by Elsevier Inc.

  4. Standard sub-thermoneutral caging temperature influences radiosensitivity of hematopoietic stem and progenitor cells.

    Directory of Open Access Journals (Sweden)

    Benjamin J Povinelli

    Full Text Available The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs. To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI. Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.

  5. Standard sub-thermoneutral caging temperature influences radiosensitivity of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Povinelli, Benjamin J; Kokolus, Kathleen M; Eng, Jason W-L; Dougher, Christopher W; Curtin, Leslie; Capitano, Maegan L; Sailsbury-Ruf, Christi T; Repasky, Elizabeth A; Nemeth, Michael J

    2015-01-01

    The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.

  6. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    Science.gov (United States)

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

  7. Phenotypic and Functional Changes Induced in Hematopoietic Stem/Progenitor Cells After Gamma-Ray Radiation Exposure

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    Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D. [Institute of Emerging Diseases and Innovative Therapies, Functional Bioengineering Laboratory, Commissariat a l' Energie Atomique (CEA), Evry (France); Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D. [Institut National de la Sante et de la Recherche Medicale (INSERM) U733 (Unite Mixte de Recherche) - UMR INSERM CEA Paris XI (France); Vaigot, P. [Institute of Cellular and Molecular Radiation Biology, Department of Genetic Instability, Recombination and Repair, Commissariat a l' Energie Atomique, Fontenay-aux-Roses (France); Vaigot, P. [UMR 217, UMR-CEA-Centre National de la Recherche Scientifique (France); Barroca, V. [Laboratory of Gametogenesis, Apoptosis, Genotoxicity, Institute of Cellular and Molecular Radiation Biology, Commissariat a l' Energie Atomique, Fontenay-aux-Roses (France); Barroca, V. [Institut National de la Sante et de la Recherche Medicale U566 - UMR INSERM-CEA-PARIS VII (France); Leboulch, Ph. [Genetics Division, Brigham and Women' s Hospital and Harvard Medical School, Boston, Massachusetts (US)

    2009-07-01

    Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding 'side population (SP)' early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SPSK cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit{sup +}Sca-1{sup +}Lin{sup -/low} (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150{sup +}Flk2{sup -} and CD150{sup -}Flk2{sup +} cells, respectively. CD150{sup +} KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not age matched controls, allowed engraftment and significant hematopoietic contribution from transplanted con-genic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice. (authors)

  8. Phenotypic and Functional Changes Induced in Hematopoietic Stem/Progenitor Cells After Gamma-Ray Radiation Exposure

    International Nuclear Information System (INIS)

    Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D.; Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D.; Vaigot, P.; Vaigot, P.; Barroca, V.; Barroca, V.; Leboulch, Ph.

    2009-01-01

    Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding 'side population (SP)' early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SPSK cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit + Sca-1 + Lin -/low (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150 + Flk2 - and CD150 - Flk2 + cells, respectively. CD150 + KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not age matched controls, allowed engraftment and significant hematopoietic contribution from transplanted con-genic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice. (authors)

  9. Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

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    2005-08-01

    Full Text Available Although several reports have characterized the hematopoietic stem cell (HSC transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+(CD33-(CD38-Rho(lo(c-kit+ cells, enriched for hematopoietic stem/progenitor cells with (CD34+(CD33-(CD38-Rho(hi cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23% of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global

  10. Proliferative compensation of residual radiation damage in the compartment of hematopoietic early progenitor cells of the mouse

    International Nuclear Information System (INIS)

    Huebner, G.E.; Wangenheim, K.H. von; Feinendegen, L.E.

    1984-01-01

    The rate of cell entry from the compartment of hematopoietic early progenitor cells into differentiation was determined in sublethally irradiated mice. By use of the criterion of repopulating ability, transplantation of 5-( 125 I) iodo-2'-deoxyuridine labeled bone marrow cells into fatally irradiated syngeneic recipients allows to measure the relative number of early progenitor cells lodging in the spleen and the turnover of these cells in the donors. Following 450 rad the relative number of transplantable early progenitor cells in S-phase recovers to normal within 2 weeks and stabilizes after 5 weeks. At this time, the labeled progenitors turn over with a half-time of 1.4-2.2 days; the respective times for unirradiated mice are 1.5-1.8 days. This, quantitative and qualitative residual radiation damage that is known to exist in the compartment of CFU-S, is disguised within 2-5 weeks after irradiation by proliferative compensation in the entirety of early hemopoietic precursor cells which are here defined by their capacity of selfrenewal and delivery of differentiated cells and of seeding to spleens of lethally irradiated recipients. (orig.)

  11. Circulating hematopoietic progenitors and CD34+ cells predicted successful hematopoietic stem cell harvest in myeloma and lymphoma patients: experiences from a single institution

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    Yu JT

    2016-02-01

    Full Text Available Jui-Ting Yu,1,2,* Shao-Bin Cheng,3,* Youngsen Yang,1 Kuang-Hsi Chang,4 Wen-Li Hwang,1 Chieh-Lin Jerry Teng,1,5,6 1Division of Hematology/Medical Oncology, Department of Medicine, Taichung Veterans General Hospital, 2Division of Hematology/Medical Oncology, Tungs' Taichung MetroHarbor Hospital, 3Division of General Surgery, Department of Surgery, 4Department of Medical Research and Education, Taichung Veterans General Hospital, 5Department of Life Science, Tunghai University, 6School of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China *These authors contributed equally to this work Background: Previous studies have shown that the numbers of both circulating hematopoietic progenitor cell (HPC and CD34+ cell are positively correlated with CD34+ cell harvest yield. However, the minimal numbers of both circulating HPCs and CD34+ cells required for performing an efficient hematopoietic stem cell (HSC harvest in lymphoma and myeloma patients have not been defined in our institution. Patients and methods: Medical records of 50 lymphoma and myeloma patients undergoing peripheral blood HSC harvest in our institution were retrospectively reviewed. The minimal and optimal HSC harvest yield required for the treatment was considered to be ≥2×106 CD34+ cells/kg and ≥5×106 CD34+ cells/kg, respectively. Results: The minimally required or optimal HSC yield obtained was not influenced by age (≥60 years, sex, underlying malignancies, disease status, multiple rounds of chemotherapy, or history of radiotherapy. The numbers of both circulating HPC and CD34+ cell were higher in patients with minimally required HSC yields (P=0.000 for HPC and P=0.000 for CD34+ cell and also in patients with optimal HSC yields (P=0.011 for HPC and P=0.006 for CD34+ cell. The cell count cutoff for obtaining minimally required HSC harvest was determined to be 20/mm3 for HPCs and 10/mm3 for CD34+ cells. Furthermore, the cell count cutoff for obtaining

  12. Assessment of benzene-induced hematotoxicity using a human-like hematopoietic lineage in NOD/Shi-scid/IL-2Rγnull mice.

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    Masayuki Takahashi

    Full Text Available Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγ(null (NOG mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice. Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice. A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.

  13. The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells

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    Prasuna Paluru

    2014-03-01

    Full Text Available The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41 + CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP. Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

  14. Analysis and manipulation of hematopoietic progenitor and stem cells from murine embryonic tissues

    NARCIS (Netherlands)

    A. Medvinsky (Alexander); S. Taoudi (Samir); S.C. Mendes (Sandra); E.A. Dzierzak (Elaine)

    2008-01-01

    textabstractHematopoietic development begins in several locations in the mammalian embryo: yolk sac, aorta-gonad-mesonephros region (AGM), and the chorio-allantoic placenta. Generation of the most potent cells, adult definitive hematopoietic stem cells (HSCs), occurs within the body of the mouse

  15. Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

    Science.gov (United States)

    Magnusson, Mattias; Sierra, Maria I; Sasidharan, Rajkumar; Prashad, Sacha L; Romero, Melissa; Saarikoski, Pamela; Van Handel, Ben; Huang, Andy; Li, Xinmin; Mikkola, Hanna K A

    2013-01-01

    Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

  16. Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

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    Mattias Magnusson

    Full Text Available Lack of HLA-matched hematopoietic stem cells (HSC limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC stroma that protects human hematopoietic stem/progenitor cells (HSPC from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+ characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

  17. A Method to Study the Epigenetic Chromatin States of Rare Hematopoietic Stem and Progenitor Cells; MiniChIP–Chip

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    Weishaupt Holger

    2010-01-01

    Full Text Available Abstract Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP–chip, we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions. Additional file 1 Click here for file

  18. Mesenchymal stromal cells from patients with acute myeloid leukemia have altered capacity to expand differentiated hematopoietic progenitors.

    Science.gov (United States)

    Chandran, Priya; Le, Yevgeniya; Li, Yuhua; Sabloff, Mitchell; Mehic, Jelica; Rosu-Myles, Michael; Allan, David S

    2015-04-01

    The bone marrow microenvironment may be permissive to the emergence and progression of acute myeloid leukemia (AML). Studying interactions between the microenvironment and leukemia cells should provide new insight for therapeutic advances. Mesenchymal stromal cells (MSCs) are central to the maintenance of the hematopoietic niche. Here we compared the functions and gene expression patterns of MSCs derived from bone marrow aspirates of healthy donors and patients with AML. MSCs expanded from AML patients had heterogeneous morphology and displayed a wide range of proliferation capacity compared to MSCs from healthy controls. The ability of AML-MSCs to support the expansion of committed hematopoietic progenitors from umbilical cord blood-derived CD34+ cells may be impaired while the expression of genes associated with maintaining hematopoietic quiescence appeared to be increased in AML-MSCs compared to healthy donors. These results highlight important potential differences in the biologic profile of MSCs from AML patients compared to healthy donors that may contribute to the emergence or progression of leukemia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Related-to-receptor tyrosine kinase receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice.

    Science.gov (United States)

    Povinelli, Benjamin J; Srivastava, Pragya; Nemeth, Michael J

    2015-03-01

    Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a noncanonical Wnt ligand receptor termed 'related-to-receptor tyrosine kinase' (Ryk). Expression of Ryk on HSPCs in vivo is associated with a lower rate of proliferation, and, following treatment with fluorouracil (5-FU), the percentage of Ryk(+/high) HSPCs increased and the percentage of Ryk(-/low) HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling in vivo resulted in increased hematopoietic-stem-cell proliferation and decreased hematopoietic-stem-cell function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  20. MicroRNAs and Metabolites in Serum Change after Chemotherapy: Impact on Hematopoietic Stem and Progenitor Cells.

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    Thomas Walenda

    Full Text Available Hematopoietic regeneration after high dose chemotherapy necessitates activation of the stem cell pool. There is evidence that serum taken after chemotherapy comprises factors stimulating proliferation and self-renewal of CD34(+ hematopoietic stem and progenitor cells (HSPCs--however, the nature of these feedback signals is yet unclear. Here, we addressed the question if specific microRNAs (miRNAs or metabolites are affected after high dose chemotherapy. Serum taken from the same patients before and after chemotherapy was supplemented for in vitro cultivation of HSPCs. Serum taken after chemotherapy significantly enhanced HSPC proliferation, better maintained a CD34(+ immunophenotype, and stimulated colony forming units. Microarray analysis revealed that 23 miRNAs changed in serum after chemotherapy--particularly, miRNA-320c and miRNA-1275 were down-regulated whereas miRNA-3663-3p was up-regulated. miRNA-320c was exemplarily inhibited by an antagomiR, which seemed to increase proliferation. Metabolomic profiling demonstrated that 44 metabolites were less abundant, whereas three (including 2-hydroxybutyrate and taurocholenate sulphate increased in serum upon chemotherapy. Nine of these metabolites were subsequently tested for effects on HSPCs in vitro, but none of them exerted a clear concentration dependent effect on proliferation, immunophenotype and colony forming unit formation. Taken together, serum profiles of miRNAs and metabolites changed after chemotherapy. Rather than individually, these factors may act in concert to recruit HSPCs into action for hematopoietic regeneration.

  1. FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage.

    Science.gov (United States)

    Bigarella, Carolina L; Li, Jianfeng; Rimmelé, Pauline; Liang, Raymond; Sobol, Robert W; Ghaffari, Saghi

    2017-02-17

    Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age. Despite this, the regulatory pathways implicated in the HSC DNA damage response have not been fully elucidated. One of the sources of DNA damage is reactive oxygen species (ROS) generated by both exogenous and endogenous insults. Balancing ROS levels in HSC requires FOXO3, which is an essential transcription factor for HSC maintenance implicated in HSC aging. Elevated ROS levels result in defective Foxo3 -/- HSC cycling, among many other deficiencies. Here, we show that loss of FOXO3 leads to the accumulation of DNA damage in primitive hematopoietic stem and progenitor cells (HSPC), associated specifically with reduced expression of genes implicated in the repair of oxidative DNA damage. We provide further evidence that Foxo3 -/- HSPC are defective in DNA damage repair. Specifically, we show that the base excision repair pathway, the main pathway utilized for the repair of oxidative DNA damage, is compromised in Foxo3 -/- primitive hematopoietic cells. Treating mice in vivo with N -acetylcysteine reduces ROS levels, rescues HSC cycling defects, and partially mitigates HSPC DNA damage. These results indicate that DNA damage accrued as a result of elevated ROS in Foxo3 -/- mutant HSPC is at least partially reversible. Collectively, our findings suggest that FOXO3 serves as a protector of HSC genomic stability and health. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Loss of Pcgf5 Affects Global H2A Monoubiquitination but Not the Function of Hematopoietic Stem and Progenitor Cells.

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    Sha Si

    Full Text Available Polycomb-group RING finger proteins (Pcgf1-Pcgf6 are components of Polycomb repressive complex 1 (PRC1-related complexes that catalyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub1, an epigenetic mark associated with repression of genes. Pcgf5 has been characterized as a component of PRC1.5, one of the non-canonical PRC1, consisting of Ring1a/b, Rybp/Yaf2 and Auts2. However, the biological functions of Pcgf5 have not yet been identified. Here we analyzed the impact of the deletion of Pcgf5 specifically in hematopoietic stem and progenitor cells (HSPCs. Pcgf5 is expressed preferentially in hematopoietic stem cells (HSCs and multipotent progenitors (MPPs compared with committed myeloid progenitors and differentiated cells. We transplanted bone marrow (BM cells from Rosa::Cre-ERT control and Cre-ERT;Pcgf5fl/fl mice into lethally irradiated recipient mice. At 4 weeks post-transplantation, we deleted Pcgf5 by injecting tamoxifen, however, no obvious changes in hematopoiesis were detected including the number of HSPCs during a long-term observation period following the deletion. Competitive BM repopulating assays revealed normal repopulating capacity of Pcgf5-deficient HSCs. Nevertheless, Pcgf5-deficient HSPCs showed a significant reduction in H2AK119ub1 levels compared with the control. ChIP-sequence analysis confirmed the reduction in H2AK119ub1 levels, but revealed no significant association of changes in H2AK119ub1 levels with gene expression levels. Our findings demonstrate that Pcgf5-containing PRC1 functions as a histone modifier in vivo, but its role in HSPCs is limited and can be compensated by other PRC1-related complexes in HSPCs.

  3. Gene expression profiling in the inductive human hematopoietic microenvironment

    International Nuclear Information System (INIS)

    Zhao Yongjun; Chen, Edwin; Li Liheng; Gong Baiwei; Xie Wei; Nanji, Shaherose; Dube, Ian D.; Hough, Margaret R.

    2004-01-01

    Human hematopoietic stem cells (HSCs) and their progenitors can be maintained in vitro in long-term bone marrow cultures (LTBMCs) in which constituent HSCs can persist within the adherent layers for up to 2 months. Media replenishment of LTBMCs has been shown to induce transition of HSCs from a quiescent state to an active cycling state. We hypothesize that the media replenishment of the LTBMCs leads to the activation of important regulatory genes uniquely involved in HSC proliferation and differentiation. To profile the gene expression changes associated with HSC activation, we performed suppression subtractive hybridization (SSH) on day 14 human LTBMCs following 1-h media replenishment and on unmanipulated controls. The generated SSH library contained 191 differentially up-regulated expressed sequence tags (ESTs), the majority corresponding to known genes related to various intracellular processes, including signal transduction pathways, protein synthesis, and cell cycle regulation. Nineteen ESTs represented previously undescribed sequences encoding proteins of unknown function. Differential up-regulation of representative genes, including IL-8, IL-1, putative cytokine 21/HC21, MAD3, and a novel EST was confirmed by semi-quantitative RT-PCR. Levels of fibronectin, G-CSF, and stem cell factor also increased in the conditioned media of LTBMCs as assessed by ELISA, indicating increased synthesis and secretion of these factors. Analysis of our library provides insights into some of the immediate early gene changes underlying the mechanisms by which the stromal elements within the LTBMCs contribute to the induction of HSC activation and provides the opportunity to identify as yet unrecognized factors regulating HSC activation in the LTBMC milieu

  4. Whole body proton irradiation causes acute damage to bone marrow hematopoietic progenitor and stem cells in mice.

    Science.gov (United States)

    Chang, Jianhui; Wang, Yingying; Pathak, Rupak; Sridharan, Vijayalakshmi; Jones, Tamako; Mao, Xiao Wen; Nelson, Gregory; Boerma, Marjan; Hauer-Jensen, Martin; Zhou, Daohong; Shao, Lijian

    2017-12-01

    Exposure to proton irradiation during missions in deep space can lead to bone marrow injury. The acute effects of proton irradiation on hematopoietic stem and progenitor cells remain undefined and thus were investigated. We exposed male C57BL/6 mice to 0.5 and 1.0 Gy proton total body irradiation (proton-TBI, 150 MeV) and examined changes in peripheral blood cells and bone marrow (BM) progenitors and LSK cells 2 weeks after exposure. 1.0 Gy proton-TBI significantly reduced the numbers of peripheral blood cells compared to 0.5 Gy proton-TBI and unirradiated animals, while the numbers of peripheral blood cell counts were comparable between 0.5 Gy proton-TBI and unirradiated mice. The frequencies and numbers of LSK cells and CMPs in BM of 0.5 and 1.0 Gy irradiated mice were decreased in comparison to those of normal controls. LSK cells and CMPs and their progeny exhibited a radiation-induced impairment in clonogenic function. Exposure to 1.0 Gy increased cellular apoptosis but not the production of reactive oxygen species (ROS) in CMPs two weeks after irradiation. LSK cells from irradiated mice exhibited an increase in ROS production and apoptosis. Exposure to proton-TBI can induce acute damage to BM progenitors and LSK cells.

  5. Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic

  6. Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia

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    Miguel Quijada-Álamo

    2017-04-01

    Full Text Available Abstract Background Chronic lymphocytic leukemia (CLL is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored. Methods Amplicon-based deep next-generation sequencing (NGS studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56 to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities. Results NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9 and XPO1 (4/4 mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2, FBXW7 (2/2, and SF3B1 (3/4 showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7. Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2 in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of

  7. The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

    Energy Technology Data Exchange (ETDEWEB)

    Ali, Haytham [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Department of Medical Physiology and Cell Biology, Qena Faculty of Medicine, South Valley University (Egypt); Galal, Omima [Department of Medical Physiology and Cell Biology, Qena Faculty of Medicine, South Valley University (Egypt); Urata, Yoshishige; Goto, Shinji; Guo, Chang-Ying; Luo, Lan [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Abdelrahim, Eman [Department of Medical Histology, Qena Faculty of Medicine, South Valley University (Egypt); Ono, Yusuke [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Mostafa, Emtethal [Department of Medical Physiology and Cell Biology, Qena Faculty of Medicine, South Valley University (Egypt); Li, Tao-Sheng, E-mail: litaoshe@nagasaki-u.ac.jp [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2014-09-26

    Highlights: • Nicaraven mitigated the radiation-induced reduction of c-kit{sup +} stem cells. • Nicaraven enhanced the function of hematopoietic stem/progenitor cells. • Complex mechanisms involved in the protection of nicaraven to radiation injury. - Abstract: Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days in sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit{sup +} stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60–90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit{sup +} stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms.

  8. The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

    International Nuclear Information System (INIS)

    Ali, Haytham; Galal, Omima; Urata, Yoshishige; Goto, Shinji; Guo, Chang-Ying; Luo, Lan; Abdelrahim, Eman; Ono, Yusuke; Mostafa, Emtethal; Li, Tao-Sheng

    2014-01-01

    Highlights: • Nicaraven mitigated the radiation-induced reduction of c-kit + stem cells. • Nicaraven enhanced the function of hematopoietic stem/progenitor cells. • Complex mechanisms involved in the protection of nicaraven to radiation injury. - Abstract: Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days in sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit + stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60–90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit + stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms

  9. Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin.

    Directory of Open Access Journals (Sweden)

    Xiaoxia Jin

    Full Text Available Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A, hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

  10. Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

    Science.gov (United States)

    Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

    2012-01-01

    Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self

  11. Frontline Science: Wnt/β-catenin pathway promotes early engraftment of fetal hematopoietic stem/progenitor cells.

    Science.gov (United States)

    Kwarteng, Edward O; Hétu-Arbour, Roxann; Heinonen, Krista M

    2018-03-01

    The switch from fetal to adult hematopoietic stem/progenitor cells (HSPCs) is associated with profound changes in several genetic programs. Although HSPC ageing corresponds to alterations in Wnt signaling, relatively little is known about the relative roles of different Wnt signaling pathways in HSPC ontogeny. We hypothesized that proliferating fetal HSPCs would be more dependent on canonical β-catenin-dependent Wnt signaling when compared to quiescent adult bone marrow HSPCs. We have compared here Wnt signaling activities in murine fetal and adult HSPCs and demonstrate a shift from Wnt/β-catenin-dependent signaling in fetal liver HSPCs to more predominantly noncanonical Wnt/polarity signaling in adult HSPCs. β-Catenin was selectively required for fetal HSPC competitiveness shortly after transplant, and protected cells from oxidative stress. Our results emphasize the complexity of Wnt signaling dynamics in HSPC maintenance and function. ©2018 Society for Leukocyte Biology.

  12. Longitudinal Analysis of DNA Methylation in CD34+ Hematopoietic Progenitors in Myelodysplastic Syndrome

    DEFF Research Database (Denmark)

    Wong, Yan Fung; Micklem, Chris N; Taguchi, Masataka

    2014-01-01

    Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease...... regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population...... is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS...

  13. Circulating hematopoietic progenitors and CD34(+) cells predicted successful hematopoietic stem cell harvest in myeloma and lymphoma patients: experiences from a single institution.

    Science.gov (United States)

    Yu, Jui-Ting; Cheng, Shao-Bin; Yang, Youngsen; Chang, Kuang-Hsi; Hwang, Wen-Li; Teng, Chieh-Lin Jerry

    2016-01-01

    Previous studies have shown that the numbers of both circulating hematopoietic progenitor cell (HPC) and CD34(+) cell are positively correlated with CD34(+) cell harvest yield. However, the minimal numbers of both circulating HPCs and CD34(+) cells required for performing an efficient hematopoietic stem cell (HSC) harvest in lymphoma and myeloma patients have not been defined in our institution. Medical records of 50 lymphoma and myeloma patients undergoing peripheral blood HSC harvest in our institution were retrospectively reviewed. The minimal and optimal HSC harvest yield required for the treatment was considered to be ≥2×10(6) CD34(+) cells/kg and ≥5×10(6) CD34(+) cells/kg, respectively. The minimally required or optimal HSC yield obtained was not influenced by age (≥60 years), sex, underlying malignancies, disease status, multiple rounds of chemotherapy, or history of radiotherapy. The numbers of both circulating HPC and CD34(+) cell were higher in patients with minimally required HSC yields (P=0.000 for HPC and P=0.000 for CD34(+) cell) and also in patients with optimal HSC yields (P=0.011 for HPC and P=0.006 for CD34(+) cell). The cell count cutoff for obtaining minimally required HSC harvest was determined to be 20/mm(3) for HPCs and 10/mm(3) for CD34(+) cells. Furthermore, the cell count cutoff for obtaining optimal HSC harvest was determined to be 60/mm(3) for HPCs and 35/mm(3) for CD34(+) cells. A total of 60/mm(3) of HPCs and 35/mm(3) of CD34(+) cells in peripheral blood predicted optimal HSC harvest in lymphoma and myeloma patients.

  14. Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Chenxi [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Chen, Mouying [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); You, Huihui [Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Qiu, Feng [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); Wen, Huaxiao; Yuan, Junlin [Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Xiang, Shuanglin, E-mail: xshlin@hunnu.edu.cn [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); Yang, Xu, E-mail: yangxu@mail.ccnu.edu.cn [Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China)

    2017-06-01

    Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3 mg/m{sup 3} FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk. - Highlights: • Nucleated bone marrow cell count recovered after 7 days post-FA and/or BZ exposure. • CFU-GM showed an increase in colonies and 8-OHdG after 7 days post-FA + BZ exposure. • Levels of ROS in CFU-GM and BFU-E were increased by FA or FA + BZ during recovery. • Levels of

  15. Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

    International Nuclear Information System (INIS)

    Wei, Chenxi; Chen, Mouying; You, Huihui; Qiu, Feng; Wen, Huaxiao; Yuan, Junlin; Xiang, Shuanglin; Yang, Xu

    2017-01-01

    Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3 mg/m 3 FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk. - Highlights: • Nucleated bone marrow cell count recovered after 7 days post-FA and/or BZ exposure. • CFU-GM showed an increase in colonies and 8-OHdG after 7 days post-FA + BZ exposure. • Levels of ROS in CFU-GM and BFU-E were increased by FA or FA + BZ during recovery. • Levels of GM

  16. Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

    Science.gov (United States)

    Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

    2017-05-04

    Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

  17. M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation

    Science.gov (United States)

    Sarrazin, Sandrine; Redelberger, David

    2016-01-01

    Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation. PMID:27811055

  18. Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

    Science.gov (United States)

    Gori, Jennifer L; Butler, Jason M; Kunar, Balvir; Poulos, Michael G; Ginsberg, Michael; Nolan, Daniel J; Norgaard, Zachary K; Adair, Jennifer E; Rafii, Shahin; Kiem, Hans-Peter

    2017-03-01

    Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 + cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 + C38 - HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 + cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 + cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  19. Endothelial Cells Promote Expansion of Long‐Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

    Science.gov (United States)

    Gori, Jennifer L.; Butler, Jason M.; Kunar, Balvir; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Norgaard, Zachary K.; Adair, Jennifer E.; Rafii, Shahin

    2016-01-01

    Abstract Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self‐renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self‐renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17‐fold, with a significant increase in hematopoietic colony‐forming activity compared with cells cultured with cytokines alone (colony‐forming unit‐granulocyte‐erythroid‐macrophage‐monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long‐term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864–876 PMID:28297579

  20. Harmine stimulates proliferation of human neural progenitors

    Directory of Open Access Journals (Sweden)

    Vanja Dakic

    2016-12-01

    Full Text Available Harmine is the β-carboline alkaloid with the highest concentration in the psychotropic plant decoction Ayahuasca. In rodents, classical antidepressants reverse the symptoms of depression by stimulating neuronal proliferation. It has been shown that Ayahuasca presents antidepressant effects in patients with depressive disorder. In the present study, we investigated the effects of harmine in cell cultures containing human neural progenitor cells (hNPCs, 97% nestin-positive derived from pluripotent stem cells. After 4 days of treatment, the pool of proliferating hNPCs increased by 71.5%. Harmine has been reported as a potent inhibitor of the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK1A, which regulates cell proliferation and brain development. We tested the effect of analogs of harmine, an inhibitor of DYRK1A (INDY, and an irreversible selective inhibitor of monoamine oxidase (MAO but not DYRK1A (pargyline. INDY but not pargyline induced proliferation of hNPCs similarly to harmine, suggesting that inhibition of DYRK1A is a possible mechanism to explain harmine effects upon the proliferation of hNPCs. Our findings show that harmine enhances proliferation of hNPCs and suggest that inhibition of DYRK1A may explain its effects upon proliferation in vitro and antidepressant effects in vivo.

  1. Lhx2 expression promotes self-renewal of a distinct multipotential hematopoietic progenitor cell in embryonic stem cell-derived embryoid bodies.

    Directory of Open Access Journals (Sweden)

    Lina Dahl

    Full Text Available The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2(-/- mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES cell-derived embryoid bodies (EBs can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

  2. Infusion of megakaryocytic progenitor products generated from cord blood hematopoietic stem/progenitor cells: results of the phase 1 study.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available BACKGROUND: Currently, a constant shortage in the supply of platelets has become an important medical and society challenge, especially in developing country, and the in vitro production of megakaryocytic progenitor cells (MPs from cord blood could represent an effective platelet substitute. In the present study, our objective was to determine the safety and feasibility of ex vivo generated MPs in patients. METHODS AND FINDINGS: MPs were produced and characterized from cord blood mononuclear cells under a serum free medium with cytokines. We investigated the feasibility of expansion and infusion of cord blood-derived MPs in 24 patients with advanced hematological malignancies. The primary end point was the safety and tolerability of the infusion of cord blood-derived MPs. No adverse effects were observed in patients who received ex vivo-generated cells at concentrations of up to a median value of 5.45 × 10(6cells/kg of body weight. With one year follow-up, acute and chronic GVHD had not been observed among patients who received MPs infusion, even without ABO blood group and HLA typing matching. CONCLUSIONS: These initial results in patients are very encouraging. They suggest that infusion of cord blood-derived MPs appears safe and feasible for treatment of thrombocytopenia.

  3. HEMATOPOIETIC PROGENITOR CELL CONTENT OF VERTEBRAL BODY MARROW USED FOR COMBINED SOLID ORGAN AND BONE MARROW TRANSPLANTATION

    Science.gov (United States)

    Rybka, Witold B.; Fontes, Paulo A.; Rao, Abdul S.; Winkelstein, Alan; Ricordi, Camillo; Ball, Edward D.; Starzl, Thomas E.

    2010-01-01

    While cadaveric vertebral bodies (VB) have long been proposed as a suitable source of bone marrow (BM) for transplantation (BMT), they have rarely been used for this purpose. We have infused VB BM immediately following whole organ (WO) transplantation to augment donor cell chimerism. We quantified the hematopoietic progenitor cell (HPC) content of VB BM as well as BM obtained from the iliac crests (IC) of normal allogeneic donors (ALLO) and from patients with malignancy undergoing autologous marrow harvest (AUTO). Patients undergoing WOIBM transplantation also had AUTO BM harvested in the event that subsequent lymphohematopoietic reconstitution was required. Twenty-four VB BM, 24 IC BM-ALLO, 31 IC AUTO, and 24 IC WO-AUTO were harvested. VB BM was tested 12 to 72 hr after procurement and infused after completion ofWO grafting. IC BM was tested and then used or cryopreserved immediately. HPC were quantified by clonal assay measuring CFU-GM, BFU-E, and CFU-GEMM, and by flow cytometry for CD34+ progenitor cells. On an average, 9 VB were processed during each harvest, and despite an extended processing time the number of viable nucleated cells obtained was significantly higher than that from IC. Furthermore, by HPC content, VB BM was equivalent to IC BM, which is routinely used for BMT. We conclude that VB BM is a clinically valuable source of BM for allogeneic transplantation. PMID:7701582

  4. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    Directory of Open Access Journals (Sweden)

    David M Harris

    Full Text Available Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza and the growth factors (GF granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

  5. Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

    Energy Technology Data Exchange (ETDEWEB)

    Holbrook, S.T.; Ohls, R.K.; Schibler, K.R.; Yang, Y.C.; Christensen, R.D. (Univ. of Utah School of Medicine, Salt Lake City (USA))

    1991-05-15

    We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6.

  6. Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

    International Nuclear Information System (INIS)

    Holbrook, S.T.; Ohls, R.K.; Schibler, K.R.; Yang, Y.C.; Christensen, R.D.

    1991-01-01

    We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6

  7. Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

    Science.gov (United States)

    Markina, Elena; Andreeva, Elena; Andrianova, Irina; Sotnezova, Elena; Buravkova, Ludmila

    2018-05-02

    Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45 + MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

  8. Expression of cytochrome P450 genes in CD34(+) hematopoietic stem and progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Souček, P.; Anzenbacher, P.; Skoumalová, I.; Dvořák, Michal

    2005-01-01

    Roč. 23, č. 9 (2005), s. 1417-1422 ISSN 1066-5099 Institutional research plan: CEZ:AV0Z50520514 Keywords : CD34+ stem/progenitor cells * cytochrome P450 isoforms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.094, year: 2005

  9. Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors.

    Science.gov (United States)

    Hwang, Yongsung; Broxmeyer, Hal E; Lee, Man Ryul

    2017-07-01

    Hematopoietic cell transplantation (HCT) is a successful treatment modality for patients with malignant and nonmalignant disorders, usually when no other treatment option is available. The cells supporting long-term reconstitution after HCT are the hematopoietic stem cells (HSCs), which can be limited in numbers. Moreover, finding an appropriate human leukocyte antigen-matched donor can be problematic. If HSCs can be stably produced in large numbers from autologous or allogeneic cell sources, it would benefit HCT. Induced pluripotent stem cells (iPSCs) established from patients' own somatic cells can be differentiated into hematopoietic cells in vitro. This review will highlight recent methods for regulating human (h) iPSC production of HSCs and more mature blood cells. Advancements in transcription factor-mediated regulation of the developmental stages of in-vivo hematopoietic lineage commitment have begun to provide an understanding of the molecular mechanism of hematopoiesis. Such studies involve not only directed differentiation in which transcription factors, specifically expressed in hematopoietic lineage-specific cells, are overexpressed in iPSCs, but also direct conversion in which transcription factors are introduced into patient-derived somatic cells which are dedifferentiated to hematopoietic cells. As iPSCs derived from patients suffering from genetically mutated diseases would express the same mutated genetic information, CRISPR-Cas9 gene editing has been utilized to differentiate genetically corrected iPSCs into normal hematopoietic cells. IPSCs provide a model for molecular understanding of disease, and also may function as a cell population for therapy. Efficient differentiation of patient-specific iPSCs into HSCs and progenitor cells is a potential means to overcome limitations of such cells for HCT, as well as for providing in-vitro drug screening templates as tissue-on-a-chip models.

  10. Metabolic regulation of hematopoietic and leukemic stem/progenitor cells under homeostatic and stress conditions.

    Science.gov (United States)

    Karigane, Daiki; Takubo, Keiyo

    2017-07-01

    Hematopoietic stem cells (HSCs) exhibit multilineage differentiation and self-renewal activities that maintain the entire hematopoietic system during an organism's lifetime. These abilities are sustained by intrinsic transcriptional programs and extrinsic cues from the microenvironment or niche. Recent studies using metabolomics technologies reveal that metabolic regulation plays an essential role in HSC maintenance. Metabolic pathways provide energy and building blocks for other factors functioning at steady state and in stress. Here we review recent advances in our understanding of metabolic regulation in HSCs relevant to cell cycle quiescence, symmetric/asymmetric division, and proliferation following stress and lineage commitment, and discuss the therapeutic potential of targeting metabolic factors or pathways to treat hematological malignancies.

  11. Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance

    OpenAIRE

    Campbell, Kirsteen J.; Bath, Mary L.; Turner, Marian L.; Vandenberg, Cassandra J.; Bouillet, Philippe; Metcalf, Donald; Scott, Clare L.; Cory, Suzanne

    2010-01-01

    Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular...

  12. Cardiac Progenitor Cell Extraction from Human Auricles

    KAUST Repository

    Di Nardo, Paolo; Pagliari, Francesca

    2017-01-01

    by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient

  13. Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

    Science.gov (United States)

    Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

    2010-04-29

    Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

  14. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

    International Nuclear Information System (INIS)

    Di Gioacchino, Mario; Petrarca, Claudia; Perrone, Angela; Farina, Massimo; Sabbioni, Enrico; Hartung, Thomas; Martino, Simone; Esposito, Diana L.; Lotti, Lavinia Vittoria; Mariani-Costantini, Renato

    2008-01-01

    Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 μM and 10 μM Cr(VI) or Cd. Cultures treated with 10 μM Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 μM Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure

  15. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Di Gioacchino, Mario [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Medicine and Science of Ageing University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy)], E-mail: m.digioacchino@unich.it; Petrarca, Claudia; Perrone, Angela [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Medicine and Science of Ageing University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy); Farina, Massimo; Sabbioni, Enrico; Hartung, Thomas [Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy); Martino, Simone [Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome (Italy); Esposito, Diana L. [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy); Lotti, Lavinia Vittoria [Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome (Italy); Mariani-Costantini, Renato [Aging Research Center, ' G. d' Annunzio' University Foundation, Via Colle dell' Ara, 66100 Chieti (Italy); Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti (Italy)

    2008-03-15

    Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 {mu}M and 10 {mu}M Cr(VI) or Cd. Cultures treated with 10 {mu}M Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 {mu}M Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure.

  16. Human endothelial progenitor cells internalize high-density lipoprotein.

    Directory of Open Access Journals (Sweden)

    Kaemisa Srisen

    Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal

  17. Hematopoietic stem/progenitor cell proliferation and differentiation is differentially regulated by high-density and low-density lipoproteins in mice.

    Directory of Open Access Journals (Sweden)

    Yingmei Feng

    Full Text Available RATIONALE: Hematopoietic stem/progenitor cells (HSPC are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation. OBJECTIVES: We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells. METHODS AND RESULTS: HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr(-/- mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB. In addition, an increased proportion of BM HSPC was in G(2M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP increased in BM of LDLr(-/- mice. When BM Lin-Sca-1+cKit+ (i.e. "LSK" cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin- cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL or reconstituted HDL (rHDL, the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro. CONCLUSION: Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.

  18. Phenotypic and Functional Alterations of Hematopoietic Stem and Progenitor Cells in an In Vitro Leukemia-Induced Microenvironment

    Directory of Open Access Journals (Sweden)

    Jean-Paul Vernot

    2017-02-01

    Full Text Available An understanding of the cell interactions occurring in the leukemic microenvironment and their functional consequences for the different cell players has therapeutic relevance. By co-culturing mesenchymal stem cells (MSC with the REH acute lymphocytic leukemia (ALL cell line, we have established an in vitro leukemic niche for the functional evaluation of hematopoietic stem/progenitor cells (HSPC, CD34+ cells. We showed that the normal homeostatic control exerted by the MSC over the HSPC is considerably lost in this leukemic microenvironment: HSPC increased their proliferation rate and adhesion to MSC. The adhesion molecules CD54 and CD44 were consequently upregulated in HSPC from the leukemic niche. Consequently, with this adhesive phenotype, HSPC showed less Stromal derived factor-1 (SDF-1-directed migration. Interestingly, multipotency was severely affected with an important reduction in the absolute count and the percentage of primitive progenitor colonies. It was possible to simulate most of these HSPC alterations by incubation of MSC with a REH-conditioned medium, suggesting that REH soluble factors and their effect on MSC are important for the observed changes. Of note, these HSPC alterations were reproduced when primary leukemic cells from an ALL type B (ALL-B patient were used to set up the leukemic niche. These results suggest that a general response is induced in the leukemic niche to the detriment of HSPC function and in favor of leukemic cell support. This in vitro leukemic niche could be a valuable tool for the understanding of the molecular events responsible for HSPC functional failure and a useful scenario for therapeutic evaluation.

  19. Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

    Science.gov (United States)

    Kent, David G; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C; Silber, Yvonne; Hamilton, Tina L; Sneade, Rachel; Simons, Benjamin D; Green, Anthony R

    2013-01-01

    Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F

  20. HEMATOPOIETIC PROGENITOR CELLS AS A PREDICTIVE OF CD34+ ENUMERATION PRIOR TO PERIPHERAL BLOOD STEM CELLS HARVESTING

    Directory of Open Access Journals (Sweden)

    Z. Zulkafli

    2014-09-01

    Full Text Available Background: To date, the CD34+ cell enumeration has relied predominantly on flow cytometry technique. However, flow cytometry is time consuming and operator dependent. The application of the hematopoietic progenitor cells (HPCs channel in Sysmex XE-2100, a fully automated hematology analyzer offers an alternative approach, which is with minimal sample manipulation and less operator dependent. This study evaluates the utility of HPC counts as a predictive of CD34+ enumeration prior to peripheral blood stem cells harvesting. Materials and methods: HPC, CD34+, white blood cell (WBC, reticulocytes (retic, immature platelet fraction (IPF and immature reticulocyte fraction (IRF were determined in 61 samples from 19 patients with hematological malignancies (15 lymphoma and 4 multiple myeloma patients at Hospital Universiti Sains Malaysia (Hospital USM who had received granulocyte-colony stimulating factor (G-CSF and planned for autologous transplantation. Results: CD34+ count showed strong and significant correlation with HPC. The receiver operating characteristics (ROC curve analysis revealed that HPC count > 21.5 x 106 / L can predicts a pre harvest CD34+ count of >20 x 106 / L with sensitivity of 77%, specificity of 64% and area under the curve (AUC of 0.802. Conclusion: We concluded that HPC count can be a useful potential parameter in optimizing timing for CD34+ enumeration prior to leukapheresis.

  1. Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2.

    Science.gov (United States)

    Ting, Stephen B; Deneault, Eric; Hope, Kristin; Cellot, Sonia; Chagraoui, Jalila; Mayotte, Nadine; Dorn, Jonas F; Laverdure, Jean-Philippe; Harvey, Michael; Hawkins, Edwin D; Russell, Sarah M; Maddox, Paul S; Iscove, Norman N; Sauvageau, Guy

    2012-03-15

    The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.

  2. Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature

    Directory of Open Access Journals (Sweden)

    Zúñiga-Pflücker Juan

    2011-03-01

    Full Text Available Abstract Background T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB hematopoietic stem cells (HSCs in coculture with OP9-DL1 cells. Results HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38, secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule. Conclusion Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.

  3. Deregulation of vital mitotic kinase-phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia.

    Science.gov (United States)

    Chatterjee, Ritam; Chattopadhyay, Sukalpa; Law, Sujata

    2016-11-01

    Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3β, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3β axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

  4. Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera.

    Science.gov (United States)

    Kawahara, Masahiro; Chen, Jianhong; Sogo, Takahiro; Teng, Jinying; Otsu, Makoto; Onodera, Masafumi; Nakauchi, Hiromitsu; Ueda, Hiroshi; Nagamune, Teruyuki

    2011-09-01

    Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Cardiac Progenitor Cell Extraction from Human Auricles

    KAUST Repository

    Di Nardo, Paolo

    2017-02-22

    For many years, myocardial tissue has been considered terminally differentiated and, thus, incapable of regenerating. Recent studies have shown, instead, that cardiomyocytes, at least in part, are slowly substituted by new cells originating by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient. The protocol here reported describes how from auricles a population of multipotent, cardiogenic cells can be isolated, cultured, and differentiated. Further studies are needed to fully exploit this cell population, but, sampling auricles, it could be possible to treat cardiac patients using their own cells circumventing rejection or organ shortage limitations.

  6. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    NARCIS (Netherlands)

    Guadix, Juan Antonio; Orlova, Valeria V.; Giacomelli, Elisa; Bellin, Milena; Ribeiro, Marcelo C.; Mummery, Christine L.; Pérez-Pomares, José M.; Passier, Robert

    2017-01-01

    Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA)

  7. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

    Directory of Open Access Journals (Sweden)

    Olga Schmal

    2016-01-01

    Full Text Available Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs with bone marrow-derived mesenchymal stromal cells (MSCs. When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

  8. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

    Science.gov (United States)

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E; Walter, Christina B; Aicher, Wilhelm K; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

  9. F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

    Science.gov (United States)

    Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

    2016-01-01

    Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

  10. Correlated miR-mRNA expression signatures of mouse hematopoietic stem and progenitor cell subsets predict "Stemness" and "Myeloid" interaction networks.

    Directory of Open Access Journals (Sweden)

    Diane Heiser

    Full Text Available Several individual miRNAs (miRs have been implicated as potent regulators of important processes during normal and malignant hematopoiesis. In addition, many miRs have been shown to fine-tune intricate molecular networks, in concert with other regulatory elements. In order to study hematopoietic networks as a whole, we first created a map of global miR expression during early murine hematopoiesis. Next, we determined the copy number per cell for each miR in each of the examined stem and progenitor cell types. As data is emerging indicating that miRs function robustly mainly when they are expressed above a certain threshold (∼100 copies per cell, our database provides a resource for determining which miRs are expressed at a potentially functional level in each cell type. Finally, we combine our miR expression map with matched mRNA expression data and external prediction algorithms, using a Bayesian modeling approach to create a global landscape of predicted miR-mRNA interactions within each of these hematopoietic stem and progenitor cell subsets. This approach implicates several interaction networks comprising a "stemness" signature in the most primitive hematopoietic stem cell (HSC populations, as well as "myeloid" patterns associated with two branches of myeloid development.

  11. Correlated miR-mRNA expression signatures of mouse hematopoietic stem and progenitor cell subsets predict "Stemness" and "Myeloid" interaction networks.

    Science.gov (United States)

    Heiser, Diane; Tan, Yee Sun; Kaplan, Ian; Godsey, Brian; Morisot, Sebastien; Cheng, Wen-Chih; Small, Donald; Civin, Curt I

    2014-01-01

    Several individual miRNAs (miRs) have been implicated as potent regulators of important processes during normal and malignant hematopoiesis. In addition, many miRs have been shown to fine-tune intricate molecular networks, in concert with other regulatory elements. In order to study hematopoietic networks as a whole, we first created a map of global miR expression during early murine hematopoiesis. Next, we determined the copy number per cell for each miR in each of the examined stem and progenitor cell types. As data is emerging indicating that miRs function robustly mainly when they are expressed above a certain threshold (∼100 copies per cell), our database provides a resource for determining which miRs are expressed at a potentially functional level in each cell type. Finally, we combine our miR expression map with matched mRNA expression data and external prediction algorithms, using a Bayesian modeling approach to create a global landscape of predicted miR-mRNA interactions within each of these hematopoietic stem and progenitor cell subsets. This approach implicates several interaction networks comprising a "stemness" signature in the most primitive hematopoietic stem cell (HSC) populations, as well as "myeloid" patterns associated with two branches of myeloid development.

  12. Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

    Science.gov (United States)

    Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L

    2017-10-05

    Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Juan Antonio Guadix

    2017-12-01

    Full Text Available Summary: Human pluripotent stem cells (hPSCs are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA and bone morphogenetic protein 4 (BMP4 promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (proepicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (proepicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (proepicardial-like cells displaying functional properties (adhesion and spreading over the myocardium of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (proepicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration. : The authors have shown that hPSCs can be instructed in vitro to differentiate into a specific cardiac embryonic progenitor cell population called the proepicardium. Proepicardial cells are required for normal formation of the heart during development and might contribute to the development of cell-based therapies for heart repair. Keywords: human pluripotent stem cells, proepicardium, progenitor cells, cardiovascular, differentiation

  14. Adrenaline administration promotes the efficiency of granulocyte colony stimulating factor-mediated hematopoietic stem and progenitor cell mobilization in mice.

    Science.gov (United States)

    Chen, Chong; Cao, Jiang; Song, Xuguang; Zeng, Lingyu; Li, Zhenyu; Li, Yong; Xu, Kailin

    2013-01-01

    A high dose of granulocyte colony stimulating factor (G-CSF) is widely used to mobilize hematopoietic stem and progenitor cells (HSPC), but G-CSF is relatively inefficient and may cause adverse effects. Recently, adrenaline has been found to play important roles in HSPC mobilization. In this study, we explored whether adrenaline combined with G-CSF could induce HSPC mobilization in a mouse model. Mice were treated with adrenaline and either a high or low dose of G-CSF alone or in combination. Peripheral blood HSPC counts were evaluated by flow cytometry. Levels of bone marrow SDF-1 were measured by ELISA, the transcription of CXCR4 and SDF-1 was measured by real-time RT-PCR, and CXCR4 protein was detected by Western blot. Our results showed that adrenaline alone fails to mobilize HSPCs into the peripheral blood; however, when G-CSF and adrenaline are combined, the WBC counts and percentages of HSPCs are significantly higher compared to those in mice that received G-CSF alone. The combined use of adrenaline and G-CSF not only accelerated HSPC mobilization, but also enabled the efficient mobilization of HSPCs into the peripheral blood at lower doses of G-CSF. Adrenaline/G-CSF treatment also extensively downregulated levels of SDF-1 and CXCR4 in mouse bone marrow. These results demonstrated that adrenaline combined with G-CSF can induce HSPC mobilization by down-regulating the CXCR4/SDF-1 axis, indicating that the use of adrenaline may enable the use of reduced dosages or durations of G-CSF treatment, minimizing G-CSF-associated complications.

  15. Expression of p210 BCR/ABl increases hematopoietic progenitor cell radiosensitivity

    International Nuclear Information System (INIS)

    Santucci, M.A.; Anklesaria, P.; Das, I.J.; Sakakeeny, M.A.; FitzGerald, T.J.; Greenberger, J.S.; Laneuville, P.

    1993-01-01

    The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. The authors tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D 0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 product induced a significant increase in radiosensitivity at the clinically relevant radiation therapy dose-rate. The increased radiosensitivity of p210-expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of p210 cDNA in the 32D-PC1 and 32D-LG7 subclones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. These data suggest a role for p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells. 43 refs., 3 figs., 1 tab

  16. Inactivation of the forkhead transcription factor FoxO3 is essential for PKB-mediated survival of hematopoietic progenitor cells by kit ligand

    DEFF Research Database (Denmark)

    Engström, Maria; Karlsson, Richard; Jönsson, Jan-Ingvar

    2003-01-01

    OBJECTIVE: Kit ligand (KL) is a major survival factor for hematopoietic stem cells. Although anti-apoptotic bcl-2 family members are expressed in these cells, the survival effects by KL appear to involve other mechanisms. Survival signals can also be elicited by the activation of phosphatidylinos......OBJECTIVE: Kit ligand (KL) is a major survival factor for hematopoietic stem cells. Although anti-apoptotic bcl-2 family members are expressed in these cells, the survival effects by KL appear to involve other mechanisms. Survival signals can also be elicited by the activation......, immunofluorescence, and subcellular fractionation, we analyzed the effects of KL on PKB and different forkhead family members in two factor-dependent cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow-derived Lin(-) progenitors. Forced overexpression of triple mutated form of FoxO3 by retroviral...

  17. Autologous hematopoietic progenitor cell mobilization and collection in adult patients presenting with multiple myeloma and lymphoma: A position-statement from the Turkish Society of Apheresis (TSA).

    Science.gov (United States)

    Tekgündüz, Emre; Arat, Mutlu; Göker, Hakan; Özdoğu, Hakan; Kaynar, Leylagül; Çağırgan, Seçkin; Erkurt, Mehmet Ali; Vural, Filiz; Kiki, İlhami; Altuntaş, Fevzi; Demirkan, Fatih

    2017-12-01

    Autologous hematopoietic cell transplantation (AHCT) is a routinely used procedure in the treatment of adult patients presenting with multiple myeloma (MM), Hodgkin lymphoma (HL) and various subtypes of non-Hodgkin lymphoma (NHL) in upfront and relapsed/refractory settings. Successful hematopoietic progenitor cell mobilization (HPCM) and collection are the rate limiting first steps for application of AHCT. In 2015, almost 1700 AHCT procedures have been performed for MM, HL and NHL in Turkey. Although there are recently published consensus guidelines addressing critical issues regarding autologous HPCM, there is a tremendous heterogeneity in terms of mobilization strategies of transplant centers across the world. In order to pave the way to a more standardized HPCM approach in Turkey, Turkish Society of Apheresis (TSA) assembled a working group consisting of experts in the field. Here we report the position statement of TSA regarding autologous HPCM mobilization strategies in adult patients presenting with MM and lymphoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

    Science.gov (United States)

    Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook; Kim, Jung-Hyun

    2017-03-01

    Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34 + CD43 + hematopoietic progenitor cells (HPCs) and CD34 + CD45 + HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro . In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

  19. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    International Nuclear Information System (INIS)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa; Nagai, Mami; Matsui, Keiji; Hasegawa, Natsumi; Roeder, Robert G.; Asano, Shigetaka; Ito, Mitsuhiro

    2013-01-01

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1 +/+ MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1 −/− MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1 +/+ and Med1 −/− MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells

  20. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Nagai, Mami [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Matsui, Keiji; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Asano, Shigetaka [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan)

    2013-10-11

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.

  1. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

    International Nuclear Information System (INIS)

    Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook; Kim, Sun Kyung; Jang, In Keun; Eom, Young-woo; Park, Joon Seong; Kim, Hugh C.; Song, Kye Yong; Park, Soon Cheol; Lim, Hwan Sub; Kim, Young Jin

    2007-01-01

    Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications

  2. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

    Directory of Open Access Journals (Sweden)

    Nadia T Sebastian

    2017-07-01

    Full Text Available Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  3. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

    Science.gov (United States)

    Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

    2017-07-01

    Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  4. Hematopoietic growth factors and human acute leukemia.

    Science.gov (United States)

    Löwenberg, B; Touw, I

    1988-10-22

    The study of myelopoietic maturation arrest in acute myeloblastic leukemia (AML) has been eased by availability of the human recombinant hemopoietic growth factors, macrophage colony stimulating factor (M-CSF), granulocyte-(G-CSF), granulocyte-macrophage-(GM-CSF) and multilineage stimulating factor (IL-3). Nonphysiological expansion of the leukemic population is not due to escape from control by these factors. Proliferation in vitro of AML cells is dependent on the presence of one or several factors in most cases. The pattern of factor-dependency does not correlate with morphological criteria in individual cases, and may thus offer a new tool for classification of AML. Overproduction of undifferentiated cells is not due to abnormal expression of receptors for the stimulating factors acting at an immature level. Rather, autocrine secretion of early acting lymphokines maintains proliferation of the leukemic clone. When looking at causes of leukemic dysregulation, yet undefined inhibitors of differentiation probably are of equal importance as dysequilibrated stimulation by lymphokines.

  5. Transmembrane inhibitor of RICTOR/mTORC2 in hematopoietic progenitors

    NARCIS (Netherlands)

    D. Lee (Dongjun); S.M. Sykes (Stephen M.); D. Kalaitzidis (Demetrios); A.A. Lane (Andrew A.); Y. Kfoury (Youmna); M.H.G.P. Raaijmakers (Marc H.G.P.); Y.-H. Wang (Ying-Hua); S.A. Armstrong (Scott); D.T. Scadden (David T.)

    2014-01-01

    textabstractCentral to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of

  6. Molecular functions of the LIM-homeobox transcription factor Lhx2 in hematopoietic progenitor cells derived from mouse embryonic stem cells.

    Science.gov (United States)

    Kitajima, Kenji; Kawaguchi, Manami; Iacovino, Michelina; Kyba, Michael; Hara, Takahiko

    2013-12-01

    We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells. © AlphaMed Press.

  7. Efficacy and Safety of Human Retinal Progenitor Cells

    Science.gov (United States)

    Semo, Ma'ayan; Haamedi, Nasrin; Stevanato, Lara; Carter, David; Brooke, Gary; Young, Michael; Coffey, Peter; Sinden, John; Patel, Sara; Vugler, Anthony

    2016-01-01

    Purpose We assessed the long-term efficacy and safety of human retinal progenitor cells (hRPC) using established rodent models. Methods Efficacy of hRPC was tested initially in Royal College of Surgeons (RCS) dystrophic rats immunosuppressed with cyclosporine/dexamethasone. Due to adverse effects of dexamethasone, this drug was omitted from a subsequent dose-ranging study, where different hRPC doses were tested for their ability to preserve visual function (measured by optokinetic head tracking) and retinal structure in RCS rats at 3 to 6 months after grafting. Safety of hRPC was assessed by subretinal transplantation into wild type (WT) rats and NIH-III nude mice, with analysis at 3 to 6 and 9 months after grafting, respectively. Results The optimal dose of hRPC for preserving visual function/retinal structure in dystrophic rats was 50,000 to 100,000 cells. Human retinal progenitor cells integrated/survived in dystrophic and WT rat retina up to 6 months after grafting and expressed nestin, vimentin, GFAP, and βIII tubulin. Vision and retinal structure remained normal in WT rats injected with hRPC and there was no evidence of tumors. A comparison between dexamethasone-treated and untreated dystrophic rats at 3 months after grafting revealed an unexpected reduction in the baseline visual acuity of dexamethasone-treated animals. Conclusions Human retinal progenitor cells appear safe and efficacious in the preclinical models used here. Translational Relevance Human retinal progenitor cells could be deployed during early stages of retinal degeneration or in regions of intact retina, without adverse effects on visual function. The ability of dexamethasone to reduce baseline visual acuity in RCS dystrophic rats has important implications for the interpretation of preclinical and clinical cell transplant studies. PMID:27486556

  8. Using Proteomics to 1) Identify the Bone Marrow Homing Receptors Expressed on Human Hematopoietic Stem Cells and 2) Elucidate Critical Signaling Pathways Responsible for the Blockage of Hematopoietic Differentiation in Leukemia

    KAUST Repository

    Chin, Chee J.

    2011-01-01

    Successful hematopoiesis requires the trafficking of hematopoietic stem/progenitor cells (HSPCs) to their bone marrow (BM) niche, where they can differentiate to produce all blood lineages. Leukemia arises when there is a blockage of differentiation

  9. TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells

    DEFF Research Database (Denmark)

    Langlois, Thierry; da Costa Reis Monte Mor, Barbara; Lenglet, Gaëlle

    2014-01-01

    . Here, we show that TET2 expression is low in human embryonic stem (ES) cell lines and increases during hematopoietic differentiation. ShRNA-mediated TET2 knockdown had no effect on the pluripotency of various ES cells. However, it skewed their differentiation into neuroectoderm at the expense...... profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm...... and hematopoietic differentiation. Stem Cells 2014....

  10. Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues

    NARCIS (Netherlands)

    Jokubaitis, Vanta J.; Sinka, Lidia; Driessen, Rebecca; Whitty, Genevieve; Haylock, David N.; Bertoncello, Ivan; Smith, Ian; Peault, Bruno; Tavian, Manuela; Simmons, Paul J.

    2008-01-01

    Previous studies revealed that mAb BB9 reacts with a subset of CD34(+) human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map B89 expression throughout hernatopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and

  11. Modulation of hematopoietic progenitor cell fate in vitro by varying collagen oligomer matrix stiffness in the presence or absence of osteoblasts.

    Science.gov (United States)

    Chitteti, Brahmananda Reddy; Kacena, Melissa A; Voytik-Harbin, Sherry L; Srour, Edward F

    2015-10-01

    To recreate the in vivo hematopoietic cell microenvironment or niche and to study the impact of extracellular matrix (ECM) biophysical properties on hematopoietic progenitor cell (HPC) proliferation and function, mouse bone-marrow derived HPC (Lin-Sca1+cKit+/(LSK) were cultured within three-dimensional (3D) type I collagen oligomer matrices. To generate a more physiologic milieu, 3D cultures were established in both the presence and absence of calvariae-derived osteoblasts (OB). Collagen oligomers were polymerized at varying concentration to give rise to matrices of different fibril densities and therefore matrix stiffness (shear storage modulus, 50-800 Pa). Decreased proliferation and increased clonogenicity of LSK cells was associated with increase of matrix stiffness regardless of whether OB were present or absent from the 3D culture system. Also, regardless of whether OB were or were not added to the 3D co-culture system, LSK within 800 Pa collagen oligomer matrices maintained the highest percentage of Lin-Sca1+ cells as well as higher percentage of cells in quiescent state (G0/G1) compared to 50 Pa or 200Pa matrices. Collectively, these data illustrate that biophysical features of collagen oligomer matrices, specifically fibril density-induced modulation of matrix stiffness, provide important guidance cues in terms of LSK expansion and differentiation and therefore maintenance of progenitor cell function. Copyright © 2015. Published by Elsevier B.V.

  12. Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.

    Science.gov (United States)

    Suzuki, Naoya M; Niwa, Akira; Yabe, Miharu; Hira, Asuka; Okada, Chihiro; Amano, Naoki; Watanabe, Akira; Watanabe, Ken-Ichiro; Heike, Toshio; Takata, Minoru; Nakahata, Tatsutoshi; Saito, Megumu K

    2015-04-01

    Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF. ©AlphaMed Press.

  13. Endogenous DNA Damage Leads to p53-Independent Deficits in Replicative Fitness in Fetal Murine Fancd2−/− Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Young me Yoon

    2016-11-01

    Full Text Available Our mechanistic understanding of Fanconi anemia (FA pathway function in hematopoietic stem and progenitor cells (HSPCs owes much to their role in experimentally induced DNA crosslink lesion repair. In bone marrow HSPCs, unresolved stress confers p53-dependent apoptosis and progressive cell attrition. The role of FA proteins during hematopoietic development, in the face of physiological replicative demand, remains elusive. Here, we reveal a fetal HSPC pool in Fancd2−/− mice with compromised clonogenicity and repopulation. Without experimental manipulation, fetal Fancd2−/− HSPCs spontaneously accumulate DNA strand breaks and RAD51 foci, associated with a broad transcriptional DNA-damage response, and constitutive activation of ATM as well as p38 stress kinase. Remarkably, the unresolved stress during rapid HSPC pool expansion does not trigger p53 activation and apoptosis; rather, it constrains proliferation. Collectively our studies point to a role for the FA pathway during hematopoietic development and provide a new model for studying the physiological function of FA proteins.

  14. Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice

    Directory of Open Access Journals (Sweden)

    Cesare Lancini

    2016-03-01

    Full Text Available The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ. Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR (Jackson and Durocher, 2013 [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008 [2,3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014 [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article “Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells” (Lancini et al., 2014 [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002 [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495. With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis. Keywords: B

  15. Thymic B cell development is controlled by the B potential of progenitors via both hematopoietic-intrinsic and thymic microenvironment-intrinsic regulatory mechanisms.

    Directory of Open Access Journals (Sweden)

    Shiyun Xiao

    Full Text Available Hematopoietic stem cells (HSCs derived from birth through adult possess differing differentiation potential for T or B cell fate in the thymus; neonatal bone marrow (BM cells also have a higher potential for B cell production in BM compared to adult HSCs. We hypothesized that this hematopoietic-intrinsic B potential might also regulate B cell development in the thymus during ontogeny.Foxn1lacZ mutant mice are a model in which down regulation of a thymic epithelial cell (TEC specific transcription factor beginning one week postnatal causes a dramatic reduction of thymocytes production. In this study, we found that while T cells were decreased, the frequency of thymic B cells was greatly increased in these mutants in the perinatal period. We used this model to characterize the mechanisms in the thymus controlling B cell development.Foxn1lacZ mutants, T cell committed intrathymic progenitors (DN1a,b were progressively reduced beginning one week after birth, while thymic B cells peaked at 3-4 weeks with pre-B-II progenitor phenotype, and originated in the thymus. Heterochronic chimeras showed that the capacity for thymic B cell production was due to a combination of higher B potential of neonatal HSCs, combined with a thymic microenvironment deficiency including reduction of DL4 and increase of IL-7 that promoted B cell fate.Our findings indicate that the capacity and time course for thymic B-cell production are primarily controlled by the hematopoietic-intrinsic potential for B cells themselves during ontogeny, but that signals from TECs microenvironment also influence the frequency and differentiation potential of B cell development in the thymus.

  16. Identification of the homing molecules that escort pluripotent stem cells-derived hematopoietic stem cells to their niches and human activated T-cells to inflammatory sites.

    KAUST Repository

    Ali, Amal

    2017-12-01

    Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the

  17. Proliferation extent of CD34+ cells as a key parameter to maximize megakaryocytic differentiation of umbilical cord blood-derived hematopoietic stem/progenitor cells in a two-stage culture protocol

    Directory of Open Access Journals (Sweden)

    Javad Hatami

    2014-12-01

    Full Text Available Co-infusion of ex-vivo generated megakaryocytic progenitors with hematopoietic stem/progenitor cells (HSC/HPC may contribute to a faster platelet recovery upon umbilical cord blood (UCB transplantation. A two stage protocol containing cell expansion and megakaryocyte (Mk differentiation was established using human UCB CD34+-enriched cells. The expansion stage used a pre-established protocol supported by a human bone marrow mesenchymal stem cells (MSC feeder layer and the differentiation stage used TPO (100 ng/mL and IL-3 (10 ng/mL. 18% of culture-derived Mks had higher DNA content (>4 N and were able to produce platelet-like particles. The proliferation extent of CD34+ cells obtained in the expansion stage (FI-CD34+, rather than expansion duration, determined as a key parameter for efficient megakaryocytic differentiation. A maximum efficiency yield (EY of 48 ± 7.7 Mks/input CD34+ cells was obtained for a FI-CD34+ of 17 ± 2.5, where a higher FI-CD34+ of 42 ± 13 resulted in a less efficient megakaryocytic differentiation (EY of 22 ± 6.7 and 19 ± 4.6 %CD41.

  18. Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation.

    Science.gov (United States)

    Kahn, Joy; Byk, Tamara; Jansson-Sjostrand, Lottie; Petit, Isabelle; Shivtiel, Shoham; Nagler, Arnon; Hardan, Izhar; Deutsch, Varda; Gazit, Zulma; Gazit, Dan; Karlsson, Stefan; Lapidot, Tsvee

    2004-04-15

    A major limitation to clinical stem cell-mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34+ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34+ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34+/CD38(-/low) cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34+ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors.

  19. Aging impairs long-term hematopoietic regeneration after autologous stem cell transplantation

    NARCIS (Netherlands)

    Woolthuis, Carolien M; Mariani, Niccoló; Verkaik-Schakel, Rikst Nynke; Brouwers-Vos, Annet Z.; Schuringa, Jan Jacob; Vellenga, Edo; de Wolf, Joost T M; Huls, Gerwin

    Most of our knowledge of the effects of aging on the hematopoietic system comes from studies in animal models. In this study, to explore potential effects of aging on human hematopoietic stem and progenitor cells (HSPCs), we evaluated CD34(+) cells derived from young (<35 years) and old (>60 years)

  20. Effects of abdominal lavage fluid from rats with radiation injury and combined radiation-burn injury on growth of hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Su, Y.-P.; Cheng, T.-M.; Guo, C.-H.; Liu, X.-H.; Qu, J.-F.

    2003-01-01

    Full text: Objective: To observe the effects of abdominal lavage fluid from rats with radiation injury, burn injury and combined radiation-burn injury on growth of hematopoietic progenitor cells. Methods Rats were irradiated with a single dose of 12 Gy γ-ray of 60Co, combined with 30% of total body surface area (TBSA) generated under a 5 KW bromo-tungsten lamp for 25 s. Lavage fluid from the peritoneum was collected 3, 12, 24, 48 and 72 hours after injury. Then the lavage fluid was added to the culture media of erythrocyte progenitor cells (CFU-E, BFE-E) or of granulocyte-macrophage progenitor cells (CFU-GM) at 40 mg/ml final concentration. Results The formed clones of CFU-E, BFU-E and CFU-GM of the lavage fluid from rats with radiation injury or combined radiation-burn injury at 3h, 12h, 24h, 48h and 72h time points were significantly higher than those from normal. They reached their peaks at 24h after injury (215.7%, 202.3%, or 241.2% from burned rats and 188.1%, 202.3% or 204.6% from rats inflected with combined radiation-burn injury as compared with those from normal rats). However, few CFU-E, BFU-E or CFU-GM clones were found after addition of lavage fluid from irradiated rats. Conclusion Peritoneal lavage fluid from rats with burn injury or combined radiation-burn injury enhances the growth of erythrocytes and granulocyte progenitor cells. On the contrary, the lavage fluid from irradiated rats shows inhibitory effects

  1. Haematopoietic stem and progenitor cells from human pluripotent stem cells

    Science.gov (United States)

    Sugimura, Ryohichi; Jha, Deepak Kumar; Han, Areum; Soria-Valles, Clara; da Rocha, Edroaldo Lummertz; Lu, Yi-Fen; Goettel, Jeremy A.; Serrao, Erik; Rowe, R. Grant; Malleshaiah, Mohan; Wong, Irene; Sousa, Patricia; Zhu, Ted N.; Ditadi, Andrea; Keller, Gordon; Engelman, Alan N.; Snapper, Scott B.; Doulatov, Sergei; Daley, George Q.

    2018-01-01

    A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. PMID:28514439

  2. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    International Nuclear Information System (INIS)

    Wilson, J.M.; Danos, O.; Grossman, M.; Raulet, D.H.; Mulligan, R.C.

    1990-01-01

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans

  3. Potential Cellular Signatures of Viral Infections in Human Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    J. Mikovits

    2001-01-01

    Full Text Available Expression profiling of cellular genes was performed using a 10,000 cDNA human gene array in order to identify expression changes following chronic infection of human hematopoietic cells with Kapsosi’s Sarcoma -associated Virus (KSHV also known as Human Herpesvirus 8 (HHV8 and Human T cell leukemia virus-1 (HTLV-1. We performed cell-free {\\it in vitro} infection of primary bone marrow derived CD34+ cells using semi-purified HHV8 and a mature IL-2 dependent T cell line, KIT 225, using highly concentrated viral stocks prepared from an infectious molecular clone of HTLV-1. Thirty days post infection, mRNA was isolated from infected cultures and uninfected controls and submitted for microarray analysis. More than 400 genes were differentially expressed more than two-fold following HHV8 infection of primary bone marrow derived CD34+ cells. Of these 400, interferon regulatory factor 4 (IRF4, cyclin B2, TBP-associated factor, eukaryotic elongation factor and pim 2 were up-regulated more than 3.5 fold. In contrast, less than 100 genes were differentially expressed more than two-fold following chronic infection of a mature T cell line with HTLV-1. Of these, only cdc7 was up-regulated more than 3.5 fold. These data may provide insight into cellular signatures of infection useful for diagnosis of infection as well as potential targets for therapeutic intervention.

  4. Cord Blood-Derived Hematopoietic Stem/Progenitor Cells: Current Challenges in Engraftment, Infection, and Ex Vivo Expansion

    Directory of Open Access Journals (Sweden)

    Katsuhiro Kita

    2011-01-01

    Full Text Available Umbilical cord blood has served as an alternative to bone marrow for hematopoietic transplantation since the late 1980s. Numerous clinical studies have proven the efficacy of umbilical cord blood. Moreover, the possible immaturity of cells in umbilical cord blood gives more options to recipients with HLA mismatch and allows for the use of umbilical cord blood from unrelated donors. However, morbidity and mortality rates associated with hematopoietic malignancies still remain relatively high, even after cord blood transplantation. Infections and relapse are the major causes of death after cord blood transplantation in patients with hematopoietic diseases. Recently, new strategies have been introduced to improve these major problems. Establishing better protocols for simple isolation of primitive cells and ex vivo expansion will also be very important. In this short review, we discuss several recent promising findings related to the technical improvement of cord blood transplantation.

  5. The thrombopoietin receptor, c-Mpl, is a selective surface marker for human hematopoietic stem cells

    Directory of Open Access Journals (Sweden)

    Kerr William G

    2006-02-01

    Full Text Available Abstract Background Thrombopoietin (TPO, the primary cytokine regulating megakaryocyte proliferation and differentiation, exerts significant influence on other hematopoietic lineages as well, including erythroid, granulocytic and lymphoid lineages. We previously demonstrated that the receptor for TPO, c-mpl, is expressed by a subset of human adult bone marrow hematopoietic stem/progenitor cells (HSC/PC that are enriched for long-term multilineage repopulating ability in the SCID-hu Bone in vivo model of human hematopoiesis. Methods Here, we employ flow cytometry and an anti-c-mpl monoclonal antibody to comprehensively define the surface expression pattern of c-mpl in four differentiation stages of human CD34+ HSC/PC (I: CD34+38--, II: CD34+38dim, III: CD34+38+, IV: CD34dim38+ for the major sources of human HSC: fetal liver (FL, umbilical cord blood (UCB, adult bone marrow (ABM, and cytokine-mobilized peripheral blood stem cells (mPBSC. We use a surrogate in vivo model of human thymopoiesis, SCID-hu Thy/Liv, to compare the capacity of c-mpl+ vs. c-mpl-- CD34+38--/dim HSC/PC for thymocyte reconstitution. Results For all tissue sources, the percentage of c-mpl+ cells was significantly highest in stage I HSC/PC (FL 72 ± 10%, UCB 67 ± 19%, ABM 82 ± 16%, mPBSC 71 ± 15%, and decreased significantly through stages II, III, and IV ((FL 3 ± 3%, UCB 8 ± 13%, ABM 0.6 ± 0.6%, mPBSC 0.2 ± 0.1% [ANOVA: P I, decreasing through stage IV [ANOVA: P + cells [P = 0.89] or intensity of c-mpl expression [P = 0.21]. Primary Thy/Liv grafts injected with CD34+38--/dimc-mpl+ cells showed slightly higher levels of donor HLA+ thymocyte reconstitution vs. CD34+38--/dimc-mpl---injected grafts and non-injected controls (c-mpl+ vs. c-mpl--: CD2+ 6.8 ± 4.5% vs. 2.8 ± 3.3%, CD4+8-- 54 ± 35% vs. 31 ± 29%, CD4--8+ 29 ± 19% vs. 18 ± 14%. Conclusion These findings support the hypothesis that the TPO receptor, c-mpl, participates in the regulation of primitive human HSC

  6. Osteoclasts Are Required for Hematopoietic Stem and Progenitor Cell Mobilization but Not for Stress Erythropoiesis in Plasmodium chabaudi adami Murine Malaria

    Directory of Open Access Journals (Sweden)

    Hugo Roméro

    2016-01-01

    Full Text Available The anemia and inflammation concurrent with blood stage malaria trigger stress haematopoiesis and erythropoiesis. The activity of osteoclasts seems required for the mobilization of hematopoietic stem and progenitor cells (HSPC from the bone marrow to the periphery. Knowing that BALB/c mice with acute Plasmodium chabaudi adami malaria have profound alterations in bone remodelling cells, we evaluated the extent to which osteoclasts influence their hematopoietic response to infection. For this, mice were treated with osteoclast inhibiting hormone calcitonin prior to parasite inoculation, and infection as well as hematological parameters was studied. In agreement with osteoclast-dependent HSPC mobilization, administration of calcitonin led to milder splenomegaly, reduced numbers of HSPC in the spleen, and their retention in the bone marrow. Although C-terminal telopeptide (CTX levels, indicative of bone resorption, were lower in calcitonin-treated infected mice, they remained comparable in naive and control infected mice. Calcitonin-treated infected mice conveniently responded to anemia but generated less numbers of splenic macrophages and suffered from exacerbated infection; interestingly, calcitonin also decreased the number of macrophages generated in vitro. Globally, our results indicate that although osteoclast-dependent HSC mobilization from bone marrow to spleen is triggered in murine blood stage malaria, this activity is not essential for stress erythropoiesis.

  7. Cloning and Functional Analysis of cDNAs with Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

    Science.gov (United States)

    Zhang, Qing-Hua; Ye, Min; Wu, Xin-Yan; Ren, Shuang-Xi; Zhao, Meng; Zhao, Chun-Jun; Fu, Gang; Shen, Yu; Fan, Hui-Yong; Lu, Gang; Zhong, Ming; Xu, Xiang-Ru; Han, Ze-Guang; Zhang, Ji-Wang; Tao, Jiong; Huang, Qiu-Hua; Zhou, Jun; Hu, Geng-Xi; Gu, Jian; Chen, Sai-Juan; Chen, Zhu

    2000-01-01

    Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58–752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon–intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3′ UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation. [The sequence data described in this paper have been submitted to the GenBank data library under the accession nos. listed in Table 1, pp 1548–1552.] PMID:11042152

  8. Protein kinase B (PKB/c-akt) regulates homing of hematopoietic progenitors through modulation of their adhesive and migratory properties

    NARCIS (Netherlands)

    Buitenhuis, M.; van der Linden, E.; Ulfman, L.H.; Hofhuis, F.M.A.; Bierings, M.B.; Coffer, P.J

    2010-01-01

    Limited number of hematopoietic stem cells in umbilical cord blood (UCB) presents a problem when using UCB for stem cell transplantation. Improving their homing capacity could reduce the need for high initial cell numbers during transplantation procedures. Although it is evident that protein kinase

  9. Hematopoietic defects in response to reduced Arhgap21

    Directory of Open Access Journals (Sweden)

    Juliana Xavier-Ferrucio

    2018-01-01

    Full Text Available Arhgap21 is a member of the Rho GTPase activating protein (RhoGAP family, which function as negative regulators of Rho GTPases. Arhgap21 has been implicated in adhesion and migration of cancer cells. However, the role of Arhgap21 has never been investigated in hematopoietic cells. Herein, we evaluated functional aspects of hematopoietic stem and progenitor cells (HSPC using a haploinsufficient (Arhgap21+/− mouse. Our results show that Arhgap21+/− mice have an increased frequency of phenotypic HSC, impaired ability to form progenitor colonies in vitro and decreased hematopoietic engraftment in vivo, along with a decrease in LSK cell frequency during serial bone marrow transplantation. Arhgap21+/− hematopoietic progenitor cells have impaired adhesion and enhanced mobilization of immature LSK and myeloid progenitors. Arhgap21+/− mice also exhibit reduced erythroid commitment and differentiation, which was recapitulated in human primary cells, in which knockdown of ARHGAP21 in CMP and MEP resulted in decreased erythroid commitment. Finally, we observed enhanced RhoC activity in the bone marrow cells of Arhgap21+/− mice, indicating that Arhgap21 functions in hematopoiesis may be at least partially mediated by RhoC inactivation. Keywords: Arhgap21, Hematopoiesis, Erythroid cells, Hematopoietic stem and progenitor cells, Fate decision

  10. Human neonatal cardiovascular progenitors: unlocking the secret to regenerative ability.

    Directory of Open Access Journals (Sweden)

    Tania I Fuentes

    Full Text Available Although clinical benefit can be achieved after cardiac transplantation of adult c-kit+ or cardiosphere-derived cells for myocardial repair, these stem cells lack the regenerative capacity unique to neonatal cardiovascular stem cells. Unraveling the molecular basis for this age-related discrepancy in function could potentially transform cardiovascular stem cell transplantation. In this report, clonal populations of human neonatal and adult cardiovascular progenitor cells were isolated and characterized, revealing the existence of a novel subpopulation of endogenous cardiovascular stem cells that persist throughout life and co-express both c-kit and isl1. Epigenetic profiling identified 41 microRNAs whose expression was significantly altered with age in phenotypically-matched clones. These differences were correlated with reduced proliferation and a limited capacity to invade in response to growth factor stimulation, despite high levels of growth factor receptor on progenitors isolated from adults. Further understanding of these differences may provide novel therapeutic targets to enhance cardiovascular regenerative capacity.

  11. Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

    Energy Technology Data Exchange (ETDEWEB)

    Sugino, Noriko [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Yao, Hisayuki [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Iwasa, Masaki; Fujishiro, Aya [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Division of Gastroenterology and Hematology, Shiga University of Medical Science, Shiga 520-2192 (Japan); Fujii, Sumie [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Hirai, Hideyo [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Takaori-Kondo, Akifumi [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Ichinohe, Tatsuo [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Maekawa, Taira [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan)

    2016-01-22

    Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment

  12. Tapak liman (Elephantopus scaber L) extract-induced CD4+ and CD8+ differentiation from hematopoietic stem cells and progenitor cell proliferation in mice (Mus musculus L)

    Science.gov (United States)

    Djati, Muhammad Sasmito; Habibu, Hindun; Jatiatmaja, Nabilah A.; Rifa'i, Muhaimin

    2017-11-01

    Tapak Liman (Elephantopus scaber L) is a traditional medicinal plant containing several active compounds that potentially affecting hematopoietic stem cells, such as epifrieelinol, lupeol, stigmasterol, triacontane-1-ol, dotriacontane-1-ol, lupeol acetate, deoxyelephan-topin, isodeoxyelephantopin, polyphenol luteolin-7, as well as various flavonoids and glucosides. The aim of this study was to elucidate the effect of leaf extract of Tapak Liman on hematopoietic stem cells in mice BALB/c, by observation of the relative number of cells expressing CD4/CD8, CD4/CD62L, and TER119/B220 in the spleen, and TER119/B220, TER119/VLA-4 and TER119/CD34 in bone marrow, after being administered leaf extract for 2 weeks. This experiment used 12 female mice, which were divided into three treatment groups, P1= 0.5 g.g bw-1.day-1, P2= 1.0 g.g bw-1.day-1 and P3=2.0 g.g bw-1.day-1 Tapak Liman leaf extract as well as a control. The relative numbers of cells expressing surface molecules were analyzed by flowcytometry and quantitative data were tested using one-way ANOVA. The results showed that the leaf extract of Tapak Liman has no significant effect on erythrocyte proliferation; on the other hand, it had a significant effect on both proliferation and differentiation of B lymphocytes (B220+) in bone marrow (p=0.044) and increased the expression of CD4+, CD8+ molecule in B cells (p=0.026) and erythroid cells in spleen and bone marrow, based on the estimation of cells that expressed TER119+VLA-4+, identified as important in the development pathway of erythrocytes. An increased cell percentage of TER11+VLA-4+ occurred for treatment P2, 12% higher than the control. The increased expression of TER119+VLA-4+ was assumed to be due to the iron content in Tapak Liman, which functioned to stimulate the progenitor hematopoietic cells to proliferate and differentiate into a precursor of erythroid cells (TER119+VLA-4+). There was an increasing number of cells expressing the surface molecules TER119

  13. Effect of dioxin on normal and leukemic human hematopoietic cells

    Energy Technology Data Exchange (ETDEWEB)

    Lambertenghi-Deliliers, G.; Soligo, D. [Univ. degli Studi, Milan (Italy). Dipt. die Ematologia, Ospedale Maggiore Policlinico IRCCS; Fracchiolla, N.S. [Ospedale Maggiore Policlinico IRCCS, Milan (Italy). Dipt. di Ematologia; Servida, F. [Fondazione Matarelli, Milan (Italy); Bertazzi, P.A. [Istituti Clinici di Perfezionamento, Milan (Italy). Dipt. di Medicina del Lavoro

    2004-09-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) arises from chlorination of phenolic substrates or from partial combustion of organic materials in the presence of chlorine sources. TCDD has a large number of biological effects such as long-lasting skin disease, cardiovascular disease, diabete and cancer. TCDD is the prototypical agonist of the aryl hydrocarbon receptor (AhR), a member of the erb-A family that also includes the receptors for steroids, thyroid hormones, peroxisome proliferators and retinoids. When bound to dioxin, the AhR can bind to DNA and alter the expression of some genes including cytokines and growth factors. In this study, we analyzed the effect of escalating doses of TCDD on human CD34{sup +} progenitor cells from the leukapheresis of normal donors stimulated with G-CSF as well as the human myeloid leukemic cell lines HL60 (promyelocytic leukemia) and K562 (chronic myelogenous leukemia). The possible specific modulation of gene expression induced by the TCDD exposure was then tested by means of microarray analyses.

  14. A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

    Science.gov (United States)

    Beaudette-Zlatanova, Britte C; Knight, Katherine L; Zhang, Shubin; Stiff, Patrick J; Zúñiga-Pflücker, Juan Carlos; Le, Phong T

    2011-05-01

    A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  15. Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34 hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Ziegler, Benedikt L.; Sandor, Peter S.; Plappert, Ulla; Thoma, Stefan; Mueller, Robert; Bock, Thomas; Thomas, Christian A.; Nothdurft, Wilhelm; Fliedner, Theodor M.

    1998-01-01

    Purpose: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. Methods and Materials: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34 + cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34 + cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. Results: The radiobiological parameters (D 0 and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34 + cells compared to optimal two-factor combinations. The D 0 values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n = 1 for all tested GF(s). DNA repair capacity of CD34 + cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34 + cells was similar. Conclusion: Our data indicate that increased survival of irradiated CB CD34 + cells after short-term GF treatment is

  16. Covalent immobilization of stem cell factor and stromal derived factor 1α for in vitro culture of hematopoietic progenitor cells.

    Science.gov (United States)

    Cuchiara, Maude L; Horter, Kelsey L; Banda, Omar A; West, Jennifer L

    2013-12-01

    Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the system's capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  17. Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors

    Science.gov (United States)

    Kermani, Pouneh; Rafii, Dahlia; Jin, David K.; Whitlock, Paul; Schaffer, Wendy; Chiang, Anne; Vincent, Loic; Friedrich, Matthias; Shido, Koji; Hackett, Neil R.; Crystal, Ronald G.; Rafii, Shahin; Hempstead, Barbara L.

    2005-01-01

    The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels. PMID:15765148

  18. Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

    Science.gov (United States)

    Masiuk, Katelyn E; Brown, Devin; Laborada, Jennifer; Hollis, Roger P; Urbinati, Fabrizia; Kohn, Donald B

    2017-09-06

    Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 - cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 - cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 - cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 - cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  19. In Vivo Deletion of the Cebpa +37 kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis

    Science.gov (United States)

    Guo, Hong; Cooper, Stacy; Friedman, Alan D.

    2016-01-01

    The murine Cebpa gene contains a +37 kb, evolutionarily conserved 440 bp enhancer that directs high-level expression to myeloid progenitors in transgenic mice. The enhancer is bound and activated by Runx1, Scl, GATA2, C/EBPα, c-Myb, Pu.1, and additional Ets factors in myeloid cells. CRISPR/Cas9-mediated replacement of the wild-type enhancer with a variant mutant in its seven Ets sites leads to 20-fold reduction of Cebpa mRNA in the 32Dcl3 myeloid cell line. To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer. CMV-Cre mediated germline deletion resulted in diminution of the expected number of viable Enh(f/f);CMV-Cre offspring, with 28-fold reduction in marrow Cebpa mRNA but normal levels in liver, lung, adipose, intestine, muscle, and kidney. Cre-transduction of lineage-negative marrow cells in vitro reduced Cebpa mRNA 12-fold, with impairment of granulocytic maturation, morphologic blast accumulation, and IL-3 dependent myeloid colony replating for >12 generations. Exposure of Enh(f/f);Mx1-Cre mice to pIpC led to 14-fold reduction of Cebpa mRNA in GMP or CMP, 30-fold reduction in LSK, and deletion and confirmed marrow-intrinsic impairment of granulopoiesis and B cell generation with LSK and monocyte lineage expansion. These findings demonstrate a critical role for the +37 kb Cebpa enhancer for hematopoietic-specific Cebpa expression, with enhancer deletion leading to impaired myelopoiesis and potentially preleukemic progenitor expansion. PMID:26937964

  20. Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK) and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice.

    Science.gov (United States)

    Lancini, Cesare; Gargiulo, Gaetano; van den Berk, Paul C M; Citterio, Elisabetta

    2016-03-01

    The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2], [3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells" (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis.

  1. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  2. CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

    Science.gov (United States)

    Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

    2015-07-27

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. © 2015 Mende et al.

  3. Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells

    International Nuclear Information System (INIS)

    Dassé, E; Volpe, G; Walton, D S; Wilson, N; Del Pozzo, W; O'Neill, L P; Slany, R K; Frampton, J; Dumon, S

    2012-01-01

    The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells

  4. Rac1 GTPase Promotes Interaction of Hematopoietic Stem/Progenitor Cell with Niche and Participates in Leukemia Initiation and Maintenance in Mouse.

    Science.gov (United States)

    Chen, Shuying; Li, Huan; Li, Shouyun; Yu, Jing; Wang, Min; Xing, Haiyan; Tang, Kejing; Tian, Zheng; Rao, Qing; Wang, Jianxiang

    2016-07-01

    Interaction between hematopoietic stem/progenitor cells (HSPCs) with their niche is critical for HSPC function. The interaction also plays an important role in the multistep process of leukemogenesis. Rac1 GTPase has been found to be highly expressed and activated in leukemia patients. Here, by forced expression of constitutively active form of Rac1 (Rac1-V12) in HSPCs, we demonstrate that active Rac1 promotes interaction of HSPC with niche. We then established an active Rac1 associated acute myeloid leukemia (AML) model by expression of Rac1-V12 cooperated with AML1-ETO9a (AE9a) in mouse HSPCs. Compared with AE9a alone, Rac1-V12 cooperated with AE9a (AER) drives an AML with a short latency, demonstrating that activation of Rac1 GTPase in mice promotes AML development. The mechanism of this AML promotion is by a better homing and lodging of leukemia cells in niche, which further enhancing their colony formation, quiescence and preventing leukemia cells from apoptosis. Further study showed that an inhibitor targeting activated Rac1 can increase the efficacy of chemotherapeutic agents to leukemia cells. This study provides evidence that activation of Rac1 promotes leukemia development through enhancing leukemia cells' homing and retention in niche, and suggests that inhibition of Rac1 GTPase could be an effective way of eliminating AML cells. Stem Cells 2016;34:1730-1741. © 2016 AlphaMed Press.

  5. Iron overload may promote alteration of NK cells and hematopoietic stem/progenitor cells by JNK and P38 pathway in myelodysplastic syndromes.

    Science.gov (United States)

    Hua, Yanni; Wang, Chaomeng; Jiang, Huijuan; Wang, Yihao; Liu, Chunyan; Li, Lijuan; Liu, Hui; Shao, Zonghong; Fu, Rong

    2017-08-01

    The objective of the study was to examine levels of intracellular iron, reactive oxygen species (ROS) and the expression of JNK and p38MAPK in NK cells and hematopoietic stem/progenitor cells (HSPCs) in MDS patients, and explore potential mechanisms by which iron overload (IOL) promotes MDS progression. Thirty-four cases of MDS and six cases of AML transformed from MDS (MDS/AML) were included. HSPCs and NK cells were isolated by magnetic absorption cell sorting. We used flow cytometry to detect the levels of ROS and intracellular JNK and P38 in NK cells and HSPCs. Total RNA and protein were extracted from NK cells and CD34 + cells to examine the expression of JNK and p38MAPK using RT-PCR and Western blotting. Intracellular iron concentration was detected. Data were analyzed by SPSS 21 statistical software. Intracellular iron concentration and ROS were increased in both NK cells and HSPCs in MDS patients with iron overload (P overload had higher JNK expression and lower p38 expression in NK cells, and higher p38 expression in HSPCs compared with non-iron overload group. IOL may cause alterations in NK cells and HSPCs through the JNK and p38 pathways, and play a role in the transformation to AML from MDS.

  6. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Directory of Open Access Journals (Sweden)

    Hayato Fukusumi

    2016-01-01

    Full Text Available Human neural progenitor cells (hNPCs have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi. Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  7. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Garrison, Brian S; Rybak, Adrian P; Beerman, Isabel; Heesters, Balthasar; Mercier, Francois E; Scadden, David T; Bryder, David; Baron, Roland; Rossi, Derrick J

    2017-08-03

    The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521 / Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521 -deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

  8. Radiosensitivity of human haematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Kato, Kengo; Kashiwakura, Ikuo; Omori, Atsuko

    2013-01-01

    The haematopoietic system is regenerative tissue with a high proliferative potential; therefore, haematopoietic stem cells (HSCs) are sensitive to extracellular oxidative stress caused by radiation and chemotherapeutic agents. An understanding of this issue can help predict haematopoietic recovery from radiation exposure as well as the extent of radiation damage to the haematopoietic system. In the present study, the radiosensitivity of human lineage-committed myeloid haematopoietic stem/progenitor cells (HSPCs), including colony-forming unit–granulocyte macrophage, burst-forming unit–erythroid and colony-forming unit–granulocyte–erythroid–macrophage–megakaryocyte cells, which are contained in adult individual peripheral blood (PB) and fetus/neonate placental/umbilical cord blood (CB), were studied. The PB of 59 healthy individual blood donors and the CB of 42 neonates were investigated in the present study. HSPCs prepared from PB and CB were exposed to 0.5 or 2 Gy x-irradiation. The results showed that large individual differences exist in the surviving fraction of cells. In the case of adult PB, a statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of the blood donors; however, none of these correlations were observed after 2 Gy x-irradiation. In addition, seasonal and gender variation were observed in the surviving fraction of CB HSPCs. The present results suggest that there are large individual differences in the surviving fraction of HSPCs contained in both adult PB and fetus/neonate CB. In addition, some factors, including the gender, age and season of birth, affect the radiosensitivity of HSPCs, especially with a relatively low-dose exposure. (paper)

  9. Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

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    Young Yu

    Full Text Available The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

  10. Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential.

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    Francesca Oltolina

    Full Text Available A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM. We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.

  11. Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice.

    Science.gov (United States)

    Takahashi, Masayuki; Tsujimura, Noriyuki; Otsuka, Kensuke; Yoshino, Tomoko; Mori, Tetsushi; Matsunaga, Tadashi; Nakasono, Satoshi

    2012-04-01

    Recently, humanized animals whereby a part of the animal is biologically engineered using human genes or cells have been utilized to overcome interspecific differences. Herein, we analyzed the detail of the differentiation states of various human leukocyte subpopulations in humanized mouse and evaluated comprehensively the similarity of the leukocyte lineage between humanized mice and humans. Humanized mice were established by transplanting human CD34(+) cord blood cells into irradiated severely immunodeficient NOD/Shi-scid/IL2Rγ(null) (NOG) mice, and the phenotypes of human cells contained in bone marrow, thymus, spleen and peripheral blood from the mice were analyzed at monthly intervals until 4 months after cell transplantation. The analysis revealed that transplanted human hematopoietic stem cells via the caudal vein homed and engrafted themselves successfully at the mouse bone marrow. Subsequently, the differentiated leukocytes migrated to the various tissues. Almost all of the leukocytes within the thymus were human cells. Furthermore, analysis of the differentiation states of human leukocytes in various tissues and organs indicated that it is highly likely that the human-like leukocyte lineage can be developed in mice. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

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    Hayato Fukusumi; Tomoko Shofuda; Yohei Bamba; Atsuyo Yamamoto; Daisuke Kanematsu; Yukako Handa; Keisuke Okita; Masaya Nakamura; Shinya Yamanaka; Hideyuki Okano; Yonehiro Kanemura

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPS...

  13. Hematopoietic Substrate-1-Associated Protein X-1 Regulates the Proliferation and Apoptosis of Endothelial Progenitor Cells Through Akt Pathway Modulation.

    Science.gov (United States)

    Guo, Xin-Bin; Deng, Xin; Wei, Ying

    2018-03-01

    Endothelial precursor cells (EPCs) are involved in vasculogenesis of various physiological and pathological processes. The proliferation and survival mechanism of EPCs needs to be explored further for the purpose of developing an effective glioma treatment. Hematopoietic substrate-1-associated protein X-1 (HAX-1) has been reported as an anti-apoptotic protein that plays an important role in several malignant tumors. However, the effect and mechanism of HAX-1 on EPCs remains unknown. This study aims to investigate the effect of HAX-1 on the proliferation and apoptosis of EPCs and explore its mechanism. According to our results, HAX-1 was overexpressed in EPCs. The results of clone formation and 5-ethynyl-2'-deoxyuridine proliferation assay showed that HAX-1 promoted multiplication of EPCs. Flow cytometry showed HAX-1 knockout cell cycle arrest mainly in G0/G1 phase. Apoptosis analysis showed that HAX-1 could protect EPCs from apoptosis in oxidative stress. Western blot assay indicated that HAX-1 could inhibit the activation of caspase cascade and reduce the expression of p21, Bcl-2-associated X protein, and p53. HAX-1 also enhanced the degradation rate and ubiquitination of p53 through the promotion of phosphorylation of proteins MDM-2 and Akt1. Co-immunoprecipitation and immunofluorescent colocalization assays were performed to test the influence of HAX-1 on the interaction between Akt1 and heat shock protein 90 (Hsp90), which is crucial for the activity of Akt1. In conclusion, this novel study suggests that HAX-1 could facilitate the Akt1 pathway through Hsp90, which led to a decline in the levels of p53, and finally promoted the proliferation and inhibited the apoptosis of EPCs. Stem Cells 2018;36:406-419. © 2017 AlphaMed Press.

  14. Progenitor Hematopoietic Cells Implantation Improves Functional Capacity of End Stage Coronary Artery Disease Patients with Advanced Heart Failure

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    Yoga Yuniadi

    2016-01-01

    Full Text Available Background. Proangiogenic Hematopoietic Cells (PHC which comprise diverse mixture of cell types are able to secrete proangiogenic factors and interesting candidate for cell therapy. The aim of this study was to seek for benefit in implantation of PHC on functional improvement in end stage coronary artery disease patients with advanced heart failure. Methods. Patients with symptomatic heart failure despite guideline directed medical therapy and LVEF less than 35% were included. Peripheral blood mononuclear cells were isolated, cultivated for 5 days, and then harvested. Flow cytometry and cell surface markers were used to characterize PHC. The PHC were delivered retrogradely via sinus coronarius. Echocardiography, myocardial perfusion, and clinical and functional data were analyzed up to 1-year observation. Results. Of 30 patients (56.4±7.40 yo preimplant NT proBNP level is 5124.5±4682.50 pmol/L. Harvested cells characterized with CD133, CD34, CD45, and KDR showed 0.87±0.41, 0.63±0.66, 99.00±2.60, and 3.22±3.79%, respectively. LVEF was improved (22±5.68 versus 26.8±7.93, p<0.001 during short and long term observation. Myocardial perfusion significantly improved 6 months after treatment. NYHA Class and six-minute walk test are improved during short term and long term follow-up. Conclusion. Expanded peripheral blood PHC implantation using retrograde delivery approach improved LV systolic function, myocardial perfusion, and functional capacity.

  15. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments

    Energy Technology Data Exchange (ETDEWEB)

    LaBarge, Mark A; Nelson, Celeste M; Villadsen, Rene; Fridriksdottir, Agla; Ruth, Jason R; Stampfer, Martha R; Petersen, Ole W; Bissell, Mina J

    2008-09-19

    In adult tissues, multi-potent progenitor cells are some of the most primitive members of the developmental hierarchies that maintain homeostasis. That progenitors and their more mature progeny share identical genomes, suggests that fate decisions are directed by interactions with extrinsic soluble factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells. Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence, maintain the progenitor state, and guide progenitor differentiation towards myoepithelial and luminal lineages.

  16. Mobilization of hematopoietic progenitor cells with granulocyte colony stimulating factors for autologous transplant in hematologic malignancies: a single center experience

    Science.gov (United States)

    Gabús, Raul; Borelli, Gabriel; Ferrando, Martín; Bódega, Enrique; Citrín, Estela; Jiménez, Constanza Olivera; Álvarez, Ramón

    2011-01-01

    Background In 2006 the Hematology Service of Hospital Maciel published its experience with peripheral blood progenitor cell harvesting for autologous stem cell transplantation using Filgen JP (Clausen Filgrastim). After mobilization with a mean filgrastim dose of 78 mcg/Kg, 4.7 x 106 CD34+ cells/Kg were obtained by apheresis. Age above 50, multiple myeloma as underlying disease and a malignancy that was not in remission were identified as frequent characteristics among patients showing complex mobilization. Objective The aim of this study was to compare stem cell mobilization using different brands of filgrastim. Methods One hundred and fifty-seven mobilizations performed between 1997 and 2006 were analyzed. This retrospective analysis comparative two groups of patients: those mobilized with different brands of filgrastim (Group A) and those who received Filgen JP (Clausen Filgrastim) as mobilizing agent (Group B). A cluster analysis technique was used to identify four clusters of individuals with different behaviors differentiated by age, total dose of filgrastim required, number of apheresis and harvested CD34+ cells. Results The mean total dose of filgrastim administered was 105 mcg/Kg, the median number of apheresis was 2 procedures and the mean number of harvested stem cells was 4.98 x 106 CD34+ cells/Kg. No significant differences were observed between Groups A and B regarding the number of apheresis, harvested CD34+ cells and number of mobilization failures, however the total dose of filgrastim was significantly lower in Group B. Conclusions Among other factors, the origin of the cytokine used as mobilizing agent is an element to be considered when evaluating CD34+ cell mobilization results. PMID:23049356

  17. Hematopoietic Progenitor Cell Mobilization is More Robust in Healthy African American Compared to Caucasian Donors and is not Affected by the Presence of Sickle Cell Trait

    Science.gov (United States)

    Panch, Sandhya R.; Yau, Yu Ying; Fitzhugh, Courtney D.; Hsieh, Matthew M.; Tisdale, John F.; Leitman, Susan F.

    2016-01-01

    Background G-CSF-stimulated hematopoietic progenitor cells (HPCs) collected by apheresis have become the predominant graft source for HPC transplantation in adults. Among healthy allogeneic donors, demographic characteristics (age, sex, BMI) and baseline hematologic counts affect HPC mobilization, leading to variability in CD34+ apheresis yields. Racial differences in HPC mobilization are less well characterized. Methods We retrospectively analyzed data from 1,096 consecutive G-CSF-stimulated leukapheresis procedures in healthy allogeneic African American (AA) or Caucasian donors. Results In a multivariate analysis, after adjusting for age, sex, BMI, baseline platelet and MNC counts, and daily G-CSF dose, peak CD34+ cell mobilization was significantly higher among AAs (n=215) than Caucasians (n=881) (123 ± 87 vs 75 ± 47 cells/uL; p<0.0001). A ceiling effect was observed with increasing G-CSF dose (10 vs 16 mcg/kg/day) in AAs (123 ± 88 vs 123 ± 87) but not in Caucasians (74 ± 46 vs 93 ± 53, p<0.001). In AA donors, presence of sickle cell trait (SCT, n=41) did not affect CD34+ mobilization (peak CD34+ 123 ± 91 vs 107 ±72 cells/uL, HbAS vs HbAA, p=0.34). Adverse events were minimal and similar across race. Conclusions AAs demonstrated significantly better CD34 mobilization responses to G-CSF than Caucasians. This was independent of other demographic and hematologic parameters. Studying race-associated pharmacogenomics in relation to G-CSF may improve dosing strategies. Adverse event profile and CD34 mobilization were similar in AA donors with and without SCT. Our findings suggest that it would be safe to include healthy AA donors with SCT in unrelated donor registries. PMID:27167356

  18. Mobilization Characteristics and Strategies to Improve Hematopoietic Progenitor Cell Mobilization and Collection in Patients with Chronic Granulomatous Disease and Severe Combined Immunodeficiency

    Science.gov (United States)

    Panch, Sandhya R.; Yau, Yu Ying; Kang, Elizabeth M.; De Ravin, Suk See; Malech, Harry L.; Leitman, Susan F.

    2014-01-01

    Background G-CSF mobilized autologous hematopoietic progenitor cells (HPC) may be collected by apheresis of patients with chronic granulomatous disease (CGD) and severe combined immunodeficiency (SCID) for use in gene therapy trials. CD34+ cell mobilization has not been well characterized in such patients. Study Design and Methods We retrospectively evaluated CD34+ cell mobilization and collection in 73 consecutive CGD and SCID patients and in 99 age, weight and G-CSF dose-matched healthy allogeneic controls. Results In subjects aged ≤20 years, day 5 pre-apheresis circulating CD34+ counts were significantly lower in CGD and SCID than in controls; mean peak CD34+ cells 58, 64, and 87/uL, respectively, p=0.01. The SCIDs had lower CD34+ collection efficiency than CGDs and controls; mean efficiency 40%, 63% and 57%, respectively, p=0.003. In subjects >20 years, the CGDs had significantly lower CD34+ cell mobilization than controls; mean peak CD34+ cells 41 and 113/uL, respectively, p<0.0001. In a multivariate analysis, lower sedimentation rate (ESR) at mobilization was significantly correlated with better CD34+ cell mobilization, p=0.007. In SCIDs, CD34 collection efficiency was positively correlated with higher red cell indices (MCV: R2=0.77; MCH: R2=0.94; MCHC: R2=0.7, p<0.007) but not hemoglobin. Conclusions CGD and SCID populations are characterized by significantly less robust CD34+ HPC mobilization than healthy controls. The presence of active inflammation/infection as suggested by an elevated ESR may negatively impact mobilization. Among SCIDs, markedly reduced CD34 collection efficiencies were related to iron deficiency, wherein decreased red cell size and density may impair apheresis cell separation mechanics. PMID:25143186

  19. Increased Proportion of Hematopoietic Stem and Progenitor Cell Population in Cord Blood of Neonates Born to Mothers with Gestational Diabetes Mellitus.

    Science.gov (United States)

    Hadarits, Orsolya; Zóka, András; Barna, Gábor; Al-Aissa, Zahra; Rosta, Klára; Rigó, János; Kautzky-Willer, Alexandra; Somogyi, Anikó; Firneisz, Gábor

    2016-01-01

    We assessed the hematopoietic stem and progenitor cell (HSPC) population in the cord blood of neonates born to mothers with gestational diabetes mellitus (GDM) in a hypothesis generating pilot study, due to that, neonatal polycythemia may be the consequence of GDM pregnancy. Forty-five pregnant women with GDM (last trimester mean HbA1C = 33.9 mmol/mol) and 42 (nondiabetic) control pregnant women were enrolled after their routine 75 g oral glucose tolerance test (OGTT) between the 24th and 28th gestational week (with expected differences in their mean routine clinical characteristics: plasma glucose at OGTT: 0' = 5.07 vs. 4.62 mM, 120' = 8.9 vs. 5.76 mM, age = 35.07 vs. 31.66 years, prepregnancy body mass index = 27.9 vs. 23.9 kg/m(2), GDM vs. control, respectively) on a voluntary basis after signing the informed consent. EDTA-treated cord blood samples were analyzed by flow cytometry and the software Kaluza1.2 using CD45 and CD34-specific fluorescent antibodies to identify the HSPC population (CD34(+) cells within the CD45(dim) blast gate). The proportion of CD34(+)CD45(dim) HSPCs among the nucleated cells was significantly (P mothers with GDM (median 0.38%) compared to neonates born to nondiabetic mothers (median 0.32%) and according to treatment types (P cells in the cord blood may possibly be related to altered fetal stem cell mobilization in GDM pregnancy, yet these results should be interpreted only as preliminary due to the small sample sizes.

  20. VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

    Science.gov (United States)

    Oberlin, Estelle; Fleury, Maud; Clay, Denis; Petit-Cocault, Laurence; Candelier, Jean-Jacques; Mennesson, Benoît; Jaffredo, Thierry; Souyri, Michèle

    2010-11-25

    Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

  1. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid

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    Sanie-Jahromi Fatemeh

    2012-04-01

    Full Text Available Abstract Background Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers during treatment of human retinal pigment epithelium (RPE cells with amniotic fluid (AF, RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age. Results Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1 confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation. Conclusion Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

  2. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid.

    Science.gov (United States)

    Sanie-Jahromi, Fatemeh; Ahmadieh, Hamid; Soheili, Zahra-Soheila; Davari, Maliheh; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Deezagi, Abdolkhalegh; Pakravesh, Jalil; Bagheri, Abouzar

    2012-04-10

    Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers) during treatment of human retinal pigment epithelium (RPE) cells with amniotic fluid (AF), RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age. Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1) confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation. Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

  3. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

    Science.gov (United States)

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1 , an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1 . We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34 + selected hematopoietic stem and progenitor cells.

  4. Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential

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    Cindy J.M. Loomans

    2018-03-01

    Full Text Available Summary: Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi, express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC, and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+ cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential. : In the context of β cell replacement therapy for diabetes, de Koning and colleagues describe a 3D culture platform that supports ex vivo expansion of human pancreatic tissue as organoids. These organoids harbor a subpopulation of ALDHhi cells that display proliferative capacity and can differentiate to an endocrine fate. Keywords: pancreas, organoid, human, ALDH, endocrine differentiation, beta cells, insulin, progenitor, fetal, diabetes

  5. Hematopoietic Progenitor Cell Mobilization with Ifosfamide, Carboplatin, and Etoposide Chemotherapy versus Plerixafor-Based Strategies in Patients with Hodgkin and Non-Hodgkin Lymphoma.

    Science.gov (United States)

    Dhakal, Binod; Veltri, Lauren Westfall; Fenske, Timothy S; Eastwood, Daniel; Craig, Michael D; Cumpston, Aaron; Shillingburg, Alexandra; Esselman, Jean; Watkins, Kathy; Pasquini, Marcelo C; D'Souza, Anita; Hari, Parameswaran; Kanate, Abraham Sebastian; Hamadani, Mehdi

    2016-10-01

    Studies comparing the efficacy and safety of chemo-mobilization with ifosfamide, carboplatin, and etoposide (ICE) ± rituximab with plerixafor-based approaches in lymphoma patients have not been performed. We analyzed hematopoietic progenitor cell mobilization outcomes in lymphoma patients undergoing chemo-mobilization with ICE (n = 35) compared with either routine plerixafor (n = 30) or "just in time" (JIT) plerixafor-based mobilization (n = 33). Chemo-mobilization provided a significantly higher total CD34(+) cell yield (median collection, 5.35 × 10(6) cells/kg for ICE versus 3.15 × 10(6) cells/kg for routine plerixafor and 3.6 × 10(6) cells/kg for JIT plerixafor, P JIT plerixafor, P = .20). There was no significant difference in the 3 groups in terms of total number of apheresis sessions performed (median, 2 in each group; P = .78). There were no mobilization failures (inability to collect at least 2 × 10(6) cells/kg) in the chemo-mobilization group, whereas 5 patients (16.7%) in the routine plerixafor and 3 patients (9.1%) in JIT group had mobilization failure (P = .04). Mean time to neutrophil engraftment was faster in the chemo-mobilization group, 10.3 days (±1.2) compared with 12.1 days (±3.6) in the routine plerixafor group and 11.6 days (±3.0) in the JIT group (P JIT group (P JIT, P < .001). Our data suggests that chemo-mobilization with ICE provides a higher total CD34(+) cell yield, lower rates of mobilization failure, faster engraftment, and lower cost compared to plerixafor-based approaches with comparable toxicity profile between the groups, except for higher transfusion requirements with chemo-mobilization. Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  6. Mobilization and collection of CD34+ cells for autologous transplantation of peripheral blood hematopoietic progenitor cells in children: analysis of two different granulocyte-colony stimulating factor doses

    Directory of Open Access Journals (Sweden)

    Kátia Aparecida de Brito Eid

    2015-06-01

    Full Text Available Introduction: The use of peripheral hematopoietic progenitor cells (HPCs is the cell choice in autologous transplantation. The classic dose of granulocyte-colony stimulating factor (G- CSF for mobilization is a single daily dose of 10 µg/kg of patient body weight. There is a theory that higher doses of granulocyte-colony stimulating factor applied twice daily could increase the number of CD34+ cells collected in fewer leukapheresis procedures. Objective: The aim of this study was to compare a fractionated dose of 15 µg G-CSF/kg of body weight and the conventional dose of granulocyte-colony stimulating factor in respect to the number of leukapheresis procedures required to achieve a minimum collection of 3 × 106 CD34+ cells/kg body weight. Methods: Patients were divided into two groups: Group 10 - patients who received a single daily dose of 10 µg G-CSF/kg body weight and Group 15 - patients who received a fractioned dose of 15 µg G-CSF/kg body weight daily. The leukapheresis procedure was carried out in an automated cell separator. The autologous transplantation was carried out when a minimum number of 3 × 106 CD34+ cells/kg body weight was achieved. Results: Group 10 comprised 39 patients and Group 15 comprised 26 patients. A total of 146 apheresis procedures were performed: 110 (75.3% for Group 10 and 36 (24.7% for Group 15. For Group 10, a median of three (range: 1-7 leukapheresis procedures and a mean of 8.89 × 106 CD34+ cells/kg body weight (±9.59 were collected whereas for Group 15 the corresponding values were one (range: 1-3 and 5.29 × 106 cells/kg body weight (±4.95. A statistically significant difference was found in relation to the number of apheresis procedures (p-value <0.0001. Conclusions: To collect a minimum target of 3 × 106 CD34+ cells/kg body weight, the administration of a fractionated dose of 15 µg G-CSF/kg body weight significantly decreased the number of leukapheresis procedures performed.

  7. Hematopoietic Progenitor Cell Mobilization with “Just-in-Time” Plerixafor Approach is a Cost Effective Alternative to Routine Plerixafor Use

    Science.gov (United States)

    Veltri, Lauren; Cumpston, Aaron; Shillingburg, Alexandra; Wen, Sijin; Luo, Jin; Leadmon, Sonia; Watkins, Kathy; Craig, Michael; Hamadani, Mehdi; Kanate, Abraham S.

    2015-01-01

    Hematopoietic progenitor cell (HPC) mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor results in superior CD34+ cell yield, when compared to mobilization with G-CSF alone in patients with myeloma and lymphoma. However, plerixafor-based approaches are associated with high costs. To circumvent this, several institutions use a so-called “just-in-time” plerixafor (JIT-P) approach, where plerixafor is only administered to patients likely to fail mobilization with G-CSF alone. Whether such a JIT-P approach is cost effective has not been confirmed to date. We present here, results of 136 patients with myeloma or lymphoma who underwent mobilization with two different approaches of plerixafor utilization. Between Jan 2010-Oct 2012 (n=76) patients uniformly received mobilization with G-CSF and plerixafor (routine G+P cohort). To reduce mobilization costs, between Nov 2012-Jun 2014 (n=60) patients were mobilized with JIT-P where plerixafor was only administered to patients likely to fail mobilization with G-CSF alone. Patients in routine G+P group had a higher median peak peripheral blood CD34+ cell count (62 vs. 29 cells/μL, p<0.001) and a higher median day 1 CD34+ cell yield (2.9 × 106 CD34+ cells/kg vs. 2.1 × 106 CD34+ cells/kg, p=0.001). The median total CD34+ cell collection was also higher in routine G+P group (5.8 × 106 CD34+ cells/kg vs. 4.5 × 106 CD34+ cells/kg, p=0.007). In the JIT-P group 40% (n=24) completed adequate HPC collection without plerixafor. There was no difference in mobilization failure rates. The mean number of plerixafor doses utilized in JIT-P was lower (1.3 vs. 2.1, p=0.0002). The mean estimated cost in the routine G+P group was higher than that in the JIT-P group (USD 27,513 vs. USD 23,597, p=0.01). Our analysis demonstrates that mobilization with a JIT-P approach is a safe, effective and cost efficient strategy for HPC collection. PMID:26475754

  8. Daily propranolol prevents prolonged mobilization of hematopoietic progenitor cells in a rat model of lung contusion, hemorrhagic shock, and chronic stress.

    Science.gov (United States)

    Bible, Letitia E; Pasupuleti, Latha V; Gore, Amy V; Sifri, Ziad C; Kannan, Kolenkode B; Mohr, Alicia M

    2015-09-01

    Propranolol has been shown previously to decrease the mobilization of hematopoietic progenitor cells (HPCs) after acute injury in rodent models; however, this acute injury model does not reflect the prolonged period of critical illness after severe trauma. Using our novel lung contusion/hemorrhagic shock/chronic restraint stress model, we hypothesize that daily administration of propranolol will decrease prolonged mobilization of HPCs without worsening lung healing. Male Sprague-Dawley rats underwent 6 days of restraint stress after undergoing lung contusion or lung contusion/hemorrhagic shock. Restraint stress consisted of a daily 2-hour period of restraint interrupted every 30 minutes by alarms and repositioning. Each day after the period of restraint stress, the rats received intraperitoneal propranolol (10 mg/kg). On day 7, peripheral blood was analyzed for granulocyte-colony stimulating factor (G-CSF) and stromal cell-derived factor 1 via enzyme-linked immunosorbent assay and for mobilization of HPCs using c-kit and CD71 flow cytometry. The lungs were examined histologically to grade injury. Seven days after lung contusion and lung contusion/hemorrhagic shock, the addition of chronic restraint stress significantly increased the mobilization of HPC, which was associated with persistently increased levels of G-CSF and increased lung injury scores. The addition of propranolol to lung contusion/chronic restraint stress and lung contusion/hemorrhagic shock/chronic restraint stress models greatly decreased HPC mobilization and restored G-CSF levels to that of naïve animals without worsening lung injury scores. The daily administration of propranolol after both lung contusion and lung contusion/hemorrhagic shock subjected to chronic restraint stress decreased the prolonged mobilization of HPC from the bone marrow and decreased plasma G-CSF levels. Despite the decrease in mobilization of HPC, lung healing did not worsen. Alleviating chronic stress with propranolol

  9. The human and murine hematopoietic stem cell niches: are they comparable?

    Science.gov (United States)

    van Pel, Melissa; Fibbe, Willem E; Schepers, Koen

    2016-04-01

    Hematopoietic stem cells (HSCs) reside in specific niches that provide various instructive cues that regulate HSC self-renewal and their development into all mature cells of the peripheral blood. Progress in this research field has largely been guided by mouse studies. However, parallel studies with human subjects, tissues, and cells, in combination with xenotransplantation experiments in immunodeficient mice, have contributed to our increased understanding of the human HSC niche. Here, we summarize our current knowledge of the various specialized subsets of both stromal and hematopoietic cells that support HSCs through cell-cell interactions and secreted factors, and the many parallels between the murine and human HSC niches. Furthermore, we discuss recent technological advances that are likely to improve our understanding of the human HSC niche, a better understanding of which may allow further identification of unique molecular and cellular pathways in the HSC niche. This information may help to further improve the outcome of HSC transplantation and refine the treatment of hematopoietic diseases. © 2015 New York Academy of Sciences.

  10. Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

    Science.gov (United States)

    Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

    2009-07-01

    Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

  11. Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

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    Teruyo Oishi

    Full Text Available Heterotopic ossification (HO is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+ and PDGFRα(+ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+ cells and PDGFRα(+ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+ cells formed bone-like tissue and showed successful engraftment, while CD56(+ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+ cells. Our results suggest that PDGFRα(+ cells may be the major source of HO and that the newly identified mi

  12. Xenotransplantation of human neural progenitor cells to the subretinal space of nonimmunosuppressed pigs

    DEFF Research Database (Denmark)

    Warfvinge, Karin; Schwartz, Philip H; Kiilgaard, Jens Folke

    2011-01-01

    To investigate the feasibility of transplanting human neural progenitor cells (hNPCs) to the retina of nonimmunosuppressed pigs, cultured hNPCs were injected into the subretinal space of 5 adult pigs after laser burns were applied to promote donor cell integration. Postoperatively, the retinal ve...

  13. A Cell Model to Evaluate Chemical Effects on Adult Human Cardiac Progenitor Cell Differentiation and Function

    Science.gov (United States)

    Adult cardiac stem cells (CSC) and progenitor cells (CPC) represent a population of cells in the heart critical for its regeneration and function over a lifetime. The impact of chemicals on adult human CSC/CPC differentiation and function is unknown. Research was conducted to dev...

  14. Xenotransplantation of human neural progenitor cells to the subretinal space of nonimmunosuppressed pigs

    DEFF Research Database (Denmark)

    Warfvinge, Karin; Schwartz, Philip H; Kiilgaard, Jens Folke

    2011-01-01

    To investigate the feasibility of transplanting human neural progenitor cells (hNPCs) to the retina of nonimmunosuppressed pigs, cultured hNPCs were injected into the subretinal space of 5 adult pigs after laser burns were applied to promote donor cell integration. Postoperatively, the retinal ve...... that modulation of host immunity is likely necessary for prolonged xenograft survival in this model....

  15. Generation of hepatocyte- and endocrine pancreatic-like cells from human induced endodermal progenitor cells

    NARCIS (Netherlands)

    Sambathkumar, Rangarajan; Akkerman, Renate; Dastidar, Sumitava; Roelandt, Philip; Kumar, Manoj; Bajaj, Manmohan; Mestre Rosa, Ana Rita; Helsen, Nicky; Vanslembrouck, Veerle; Kalo, Eric; Khurana, Satish; Laureys, Jos; Gysemans, Conny; Faas, Marijke M; de Vos, Paul; Verfaillie, Catherine M

    2018-01-01

    Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14

  16. A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia

    DEFF Research Database (Denmark)

    Windrem, Martha S; Schanz, Steven J; Morrow, Carolyn

    2014-01-01

    Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia. The differentiat...

  17. Human Urine-Derived Renal Progenitors for Personalized Modeling of Genetic Kidney Disorders.

    Science.gov (United States)

    Lazzeri, Elena; Ronconi, Elisa; Angelotti, Maria Lucia; Peired, Anna; Mazzinghi, Benedetta; Becherucci, Francesca; Conti, Sara; Sansavini, Giulia; Sisti, Alessandro; Ravaglia, Fiammetta; Lombardi, Duccio; Provenzano, Aldesia; Manonelles, Anna; Cruzado, Josep M; Giglio, Sabrina; Roperto, Rosa Maria; Materassi, Marco; Lasagni, Laura; Romagnani, Paola

    2015-08-01

    The critical role of genetic and epigenetic factors in the pathogenesis of kidney disorders is gradually becoming clear, and the need for disease models that recapitulate human kidney disorders in a personalized manner is paramount. In this study, we describe a method to select and amplify renal progenitor cultures from the urine of patients with kidney disorders. Urine-derived human renal progenitors exhibited phenotype and functional properties identical to those purified from kidney tissue, including the capacity to differentiate into tubular cells and podocytes, as demonstrated by confocal microscopy, Western blot analysis of podocyte-specific proteins, and scanning electron microscopy. Lineage tracing studies performed with conditional transgenic mice, in which podocytes are irreversibly tagged upon tamoxifen treatment (NPHS2.iCreER;mT/mG), that were subjected to doxorubicin nephropathy demonstrated that renal progenitors are the only urinary cell population that can be amplified in long-term culture. To validate the use of these cells for personalized modeling of kidney disorders, renal progenitors were obtained from (1) the urine of children with nephrotic syndrome and carrying potentially pathogenic mutations in genes encoding for podocyte proteins and (2) the urine of children without genetic alterations, as validated by next-generation sequencing. Renal progenitors obtained from patients carrying pathogenic mutations generated podocytes that exhibited an abnormal cytoskeleton structure and functional abnormalities compared with those obtained from patients with proteinuria but without genetic mutations. The results of this study demonstrate that urine-derived patient-specific renal progenitor cultures may be an innovative research tool for modeling of genetic kidney disorders. Copyright © 2015 by the American Society of Nephrology.

  18. Ex vivo expansion of human umbilical cord blood hematopoietic stem and progenitor cells

    NARCIS (Netherlands)

    N. Kusadasi (Nuray)

    2002-01-01

    textabstractThe goal of this thesis research is to establish ex vivo expansion conditions for HSCs derived from UCB. To realize the expansion of HSCs, CD34+ or ACJ33+ UCB cells were cultured in the absence or presence of various cocktails of early acting cytokines including Flt3-L, Tpo, SCF or IL6

  19. Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures.

    Science.gov (United States)

    Cerdan, Chantal; Hong, Seok Ho; Bhatia, Mickie

    2007-10-01

    The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein-4 (BMP-4) was able to generate up to 90% of CD45(+) hematopoietic cells with colony-forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies.

  20. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

    Science.gov (United States)

    Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

    1997-03-01

    We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

  1. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

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    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  2. Establishment of a humanized APL model via the transplantation of PML-RARA-transduced human common myeloid progenitors into immunodeficient mice.

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    Hiromichi Matsushita

    Full Text Available Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC or leukemia-initiating cells (LIC appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL, a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34(+ hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34- fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP from CD34(+/CD38(+ cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34(- APL cells may share the ability to maintain the tumor.

  3. Using Proteomics to 1) Identify the Bone Marrow Homing Receptors Expressed on Human Hematopoietic Stem Cells and 2) Elucidate Critical Signaling Pathways Responsible for the Blockage of Hematopoietic Differentiation in Leukemia

    KAUST Repository

    Chin, Chee J.

    2011-05-22

    Successful hematopoiesis requires the trafficking of hematopoietic stem/progenitor cells (HSPCs) to their bone marrow (BM) niche, where they can differentiate to produce all blood lineages. Leukemia arises when there is a blockage of differentiation and uncontrolled proliferation in the hematopoietic cells during their development. To refine therapies for leukemia, this study sought to improve the homing of healthy donor HSPCs for better transplantation and to find new candidates for differentiating and blocking proliferation in leukemic cells. Characterizing the molecular effectors mediating cell migration forms the basis for improving clinical transplantation of HSPCs. E-selectin/ligand interactions play a critical role in the homing of HSPCs to the BM, however, the identity of E-selectin ligands remains elusive. We aimed to use mass spectrometry (MS) to fully analyze the E-selectin ligands expressed on HSPCs. Immunoprecipitation studies coupled with MS confirmed the expression of three known E-selectin ligands, the hematopoietic cell E-/L-selectin ligand (HCELL), P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, and revealed the presence of many interesting candidates on HSPCs-like cell line and on primary human BM CD34+ cells. The MS dataset represents a rich resource for further characterization of E-selectin ligands, which will lead to improvement of HSPCs transplantation. 4 Understanding the critical pathways underlying the initiation and maintenance of leukemia plays a key role in treating acute myeloid leukemia (AML). Ligation of the glycoprotein, CD44, using monoclonal antibodies or its natural ligand, hyaluronic acid, drives the differentiation of immature leukemic cells towards mature terminally differentiated cells, inhibits their proliferation and in some case induces their apoptosis. The aim of this study is to characterize the phosphoproteome of AML cells in response to CD44-induced differentiation. This will afford novel insights into the

  4. Lipofectamine and related cationic lipids strongly improve adenoviral infection efficiency of primitive human hematopoietic cells.

    Science.gov (United States)

    Byk, T; Haddada, H; Vainchenker, W; Louache, F

    1998-11-20

    Adenoviral vectors have the potential to infect a large number of cell types including quiescent cells. Their use in hematopoietic cells is limited by the episomal form of their DNA, leading to transgene loss in the progeny cells. However, the use of this vector may be interesting for short-term in vitro modifications of primitive human hematopoietic cells. Therefore, we have investigated the ability of adenovirus to transduce cord blood CD34+ cells. Several promoters were tested using the lacZ reporter gene. The PGK and CMV promoters induced transgene expression in 18-25% of the cells, whereas the HTLV-I and especially the RSV promoter were almost inactive. To improve infection efficiency, adenovirus was complexed with cationic lipids. Lipofectamine, Cellfectin, and RPR120535b, but not Lipofectin, Lipofectace, or DOTAP, markedly improved transgene expression in CD34+ cells (from 19 to 35%). Lipofectamine strongly enhanced infection efficiency of the poorly infectable primitive CD34+CD38low cells (from 11 to 28%) whereas the more mature CD34+CD38+ cells were only slightly affected (from 24 to 31%). Lipofectamine tripled the infection of CFU-GMs and LTC-ICs derived from the CD34+CD38low cell fraction (from 4 to 12% and from 5 to 16%, respectively) and doubled that of BFU-Es (from 13 to 26%). We conclude that cationic lipids can markedly increase the efficiency of adenovirus-mediated gene transfer into primitive hematopoietic cells.

  5. Obtención y procesamiento de células progenitoras hematopoyéticas periféricas Obtention and processing of peripheral hematopoietic progenitor cells

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    Jesús Diego de la Campa

    2003-12-01

    Full Text Available Se realizó un estudio descriptivo de la totalidad de las colectas de células progenitoras hematopoyéticas realizadas en el Banco de Sangre del Hospital "Hermanos Ameijeiras" desde abril del 2001 hasta abril del 2002, mediante leucoféresis. Se utilizó un equipo de flujo continuo (FRESENIUS ASTEC 204 y se empleó el programa mononucleares con el set P1Y. Se determinó peso, talla, hematócrito, hemoglobina, conteo de leucocitos y plaquetas de los donantes previo a la colecta. En el producto de la leucoféresis se analizó el conteo de leucocitos con diferencial, conteo de células CD34+/ CD45+ (mediante técnica de inmunofluorescencia en citómetro de flujo y determinación de viabilidad celular con yoduro de propidio en este mismo equipo (Facscan, Becton Dickinson. Recibieron trasplante autólogo 5 pacientes y 2 trasplantes alogénicos. Los primeros con diagnósticos de: linfoma no hodgkiniano (3, enfermedad de Hodgkin (1, artritis reumatoidea (1; y los segundos afectados de leucemia mieloide crónica (1 y linfoma no hodgkiniano (1 ; todos ellos atendidos en el Servicio de Hematología de nuestro centro. La media del conteo de células CD34+ por cada leucoféresis para trasplante autólogo fue 2,4 x 106 Kg y en el alogénico 2,96 x 106 Kg . Los resultados evidencian que mediante el ajuste del volumen de los ciclos, la velocidad centrífuga, el volumen de rebosado y el volumen celular, se obtuvo un conteo de células precursoras hematopoyéticas en la colecta similar a lo reportado por otros autores, lo que garantiza que el producto transfundido cuente con la celularidad necesaria para repoblar la médula ósea, luego que el paciente sea tratado con altas dosis de terapia inmunosupresora y/o radiacionesA descriptive study of all the collections of hematopoietic progenitor cells obtained by leukophoresis at the Blood Bank of "Hermanos Ameijeiras" Hospital from April, 2001, to April, 2002, was conducted. A continual flow equipment (FRESENIUS

  6. Late-occurring chromosome aberrations and global DNA methylation in hematopoietic stem/progenitor cells of CBA/CaJ mice exposed to silicon ({sup 28}Si) ions

    Energy Technology Data Exchange (ETDEWEB)

    Rithidech, Kanokporn Noy, E-mail: kanokporn.rithidech@stonybrookmedicine.edu [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Honikel, Louise M. [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Reungpathanaphong, Paiboon [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Department of Applied Radiation and Isotopes, Faculty of Sciences, Kasetsart University, Chatuchuck, Bangkok 10900 (Thailand); Tungjai, Montree [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Department of Radiologic Technology, Faculty of Associated Medical Sciences, Center of Excellence for Molecular Imaging, Chiang Mai University, Chiang Mai 50200 (Thailand); Jangiam, Witawat [Pathology Department, Stony Brook University, Stony Brook, NY 11794-8691 (United States); Department of Chemical Engineering, Faculty of Engineering, Burapha University, Chonburi 20131 (Thailand); Whorton, Elbert B. [StatCom, PO Box 3041, Galveston, TX 77551 (United States)

    2015-11-15

    Highlights: • Late-occurring chromosome aberrations were found in HSPCs of exposed CBA/CaJ mice. • A dose-dependent reduction in the level of global 5hmC was detected in HSPCs. • There is a link between reduced global 5hmC levels and genomic instability in vivo. • The level of global 5hmC is a better marker of radiation exposure than that of 5mC. - Abstract: Although myeloid leukemia (ML) is one of the major health concerns from exposure to space radiation, the risk prediction for developing ML is unsatisfactory. To increase the reliability of predicting ML risk, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is important. We focused on the in vivo induction of late-occurring damage in HSPCs of mice exposed to {sup 28}Si ions since such damage is associated with radiation-induced genomic instability (a key event of carcinogenesis). We gave adult male CBA/CaJ mice, known to be sensitive to radiation-induced ML, a whole-body exposure (2 fractionated exposures, 15 days apart, that totaled each selected dose, delivered at the dose-rate of 1 cGy/min) to various doses of 300 MeV/n {sup 28}Si ions, i.e. 0 (sham controls), 0.1, 0.25, or 0.5 Gy. At 6 months post-irradiation, we collected bone marrow cells from each mouse (five mice per treatment-group) for obtaining the myeloid-lineage of HSPC-derived clones for analyses. We measured the frequencies of late-occurring chromosome aberrations (CAs), using the genome-wide multicolor fluorescence in situ hybridization method. The measurement of CAs was coupled with the characterization of the global DNA methylation patterns, i.e. 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). A dose-dependent increase in the frequencies of CAs was detected (Analysis of Variance or ANOVA, p < 0.01), indicating the induction of genomic instability after exposure of mice to 300 MeV/n {sup 28}Si ions. Slight increases in the levels of 5m

  7. Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium (48Ti Ions

    Directory of Open Access Journals (Sweden)

    Kanokporn Noy Rithidech

    2015-07-01

    Full Text Available Myeloid leukemia (ML is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e., hematopoietic stem/progenitor cells (HSPCs, is critically important. We used the label-free quantitative mass spectrometry (LFQMS proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week and a late time-point (six months after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium (48Ti ions, which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk post-irradiation reflects acute effects of exposure to 48Ti ions, while those detected in samples collected at six months (mos post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability. Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and

  8. Immortalization of human myogenic progenitor cell clone retaining multipotentiality

    International Nuclear Information System (INIS)

    Hashimoto, Naohiro; Kiyono, Tohru; Wada, Michiko R.; Shimizu, Shirabe; Yasumoto, Shigeru; Inagawa, Masayo

    2006-01-01

    Human myogenic cells have limited ability to proliferate in culture. Although forced expression of telomerase can immortalize some cell types, telomerase alone delays senescence of human primary cultured myogenic cells, but fails to immortalize them. In contrast, constitutive expression of both telomerase and the E7 gene from human papillomavirus type 16 immortalizes primary human myogenic cells. We have established an immortalized primary human myogenic cell line preserving multipotentiality by ectopic expression of telomerase and E7. The immortalized human myogenic cells exhibit the phenotypic characteristics of their primary parent, including an ability to undergo myogenic, osteogenic, and adipogenic terminal differentiation under appropriate culture conditions. The immortalized cells will be useful for both basic and applied studies aimed at human muscle disorders. Furthermore, immortalization by transduction of telomerase and E7 represents a useful method by which to expand human myogenic cells in vitro without compromising their ability to differentiate

  9. In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

    DEFF Research Database (Denmark)

    Barraud, Perrine; Stott, Simon; Møllgård, Kjeld

    2007-01-01

    The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression....... Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain....... decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we...

  10. The development of human mast cells. An historical reappraisal

    International Nuclear Information System (INIS)

    Ribatti, Domenico

    2016-01-01

    The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34"+/CD117"+/CD13"+multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, different MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.

  11. IL-7 Enhances Thymic Human T Cell Development in "Human Immune System" Rag2-/-IL-2R{gamma}c-/- Mice without Affecting Peripheral T Cell Homeostasis

    NARCIS (Netherlands)

    van Lent, Anja U.; Dontje, Wendy; Nagasawa, Maho; Siamari, Rachida; Bakker, Arjen Q.; Pouw, Stephan M.; Maijoor, Kelly A.; Weijer, Kees; Cornelissen, Jan J.; Blom, Bianca; Di Santo, James P.; Spits, Hergen; Legrand, Nicolas

    2009-01-01

    IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported. By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7 is approximately 100-fold less potent than human IL-7

  12. CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

    Science.gov (United States)

    Mende, Nicole; Kuchen, Erika E.; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D.; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico

    2015-01-01

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. PMID:26150472

  13. Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Khazaei, Mohamad; Ahuja, Christopher S; Fehlings, Michael G

    2017-08-14

    This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  14. Interleukin-3 Does Not Affect the Differentiation of Mast Cells Derived from Human Bone Marrow Progenitors

    Science.gov (United States)

    Shimizu, Yuji; Matsumoto, Kenji; Okayama, Yoshimichi; Kentaro, Sakai; Maeno, Toshitaka; Suga, Tatsuo; Miura, Toru; Takai, Shinji; Kurabayashi, Masahiko; Saito, Hirohisa

    2008-01-01

    Although IL-3 is commonly used for culture of human progenitor-derived mast cells together with Stem cell factor (SCF) and IL-6, the effect of IL-3 on human mast cell differentiation has not been well elucidated. Human bone marrow CD34+ progenitors were cultured for up to 12 weeks in the presence of rhSCF and rhIL-6 either with rhIL-3 (IL-3 (+)) or without rhIL-3 (IL-3 (−)) for the initial 1-week of culture. Total cell number increased at 2 weeks in IL-3 (+), as compared to IL-3 (−), but changes in the appearance of mast cells were delayed. When IL-3 was present for the initial 1-week culture, granules looked more mature with IL-3 than without IL-3. However, tryptase and chymase contents, and surface antigen expression (CD18, CD51, CD54, and CD117) were not altered by IL-3. Surface expression and mRNA level of FcεRIα and histamine release by crosslinking of FcεRIα did not differ from one preparation to the next. GeneChip analysis revealed that no significant differences were observed between IL-3 (+) and IL-3 (−) cells either when inactivated or activated by aggregation of FcεRIα. These findings indicate that initial incubation of human bone marrow CD34+ progenitors with IL-3 does not affect the differentiation of mast cells. PMID:18214796

  15. JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Sihui Li

    2018-04-01

    Full Text Available Summary: The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B−/− mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis. : Li et al. identify the arginine demethylase (RDM activity of JMJD1B, a known lysine demethylase (KDM. They reveal that JMJD1B actively mediates demethylation of histone markers H4R3me2s and H3K9me2 in hematopoietic stem/progenitor cells (HSPCs. Keywords: JMJD1B, KDM3B, PRMT5, arginine demethylase, histone, epigenetic programming, gene expression, hematopoiesis

  16. Further phenotypic characterization of the primitive lineage− CD34+CD38−CD90+CD45RA− hematopoietic stem cell/progenitor cell sub-population isolated from cord blood, mobilized peripheral blood and patients with chronic myelogenous leukemia

    International Nuclear Information System (INIS)

    Wisniewski, D; Affer, M; Willshire, J; Clarkson, B

    2011-01-01

    The most primitive hematopoietic stem cell (HSC)/progenitor cell (PC) population reported to date is characterized as being Lin−CD34+CD38−CD90+CD45R. We have a long-standing interest in comparing the characteristics of hematopoietic progenitor cell populations enriched from normal subjects and patients with chronic myelogenous leukemia (CML). In order to investigate further purification of HSCs and for potential targetable differences between the very primitive normal and CML stem/PCs, we have phenotypically compared the normal and CML Lin−CD34+CD38−CD90+CD45RA− HSC/PC populations. The additional antigens analyzed were HLA-DR, the receptor tyrosine kinases c-kit and Tie2, the interleukin-3 cytokine receptor, CD33 and the activation antigen CD69, the latter of which was recently reported to be selectively elevated in cell lines expressing the Bcr-Abl tyrosine kinase. Notably, we found a strikingly low percentage of cells from the HSC/PC sub-population isolated from CML patients that were found to express the c-kit receptor (<1%) compared with the percentages of HSC/PCs expressing the c-kitR isolated from umbilical cord blood (50%) and mobilized peripheral blood (10%). Surprisingly, Tie2 receptor expression within the HSC/PC subset was extremely low from both normal and CML samples. Using in vivo transplantation studies, we provide evidence that HLA-DR, c-kitR, Tie2 and IL-3R may not be suitable markers for further partitioning of HSCs from the Lin−CD34+CD38−CD90+CD45RA− sub-population

  17. Roles of CDX2 and EOMES in human induced trophoblast progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503 (United States); Wang, Kai [Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503 (United States); Gong, Yun Guo; Khoo, Sok Kean [Genomic Microarray Core Facility, Van Andel Research Institute, Grand Rapids, MI 49503 (United States); Leach, Richard, E-mail: Richard.Leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group, Grand Rapids, MI 49503 (United States)

    2013-02-08

    Highlights: ► CDX2 and EOMES play critical roles in human induced trophoblast progenitors (iTP). ► iTP cells directly transformed from fibroblasts. ► Differentiation of iTP cells into extravillous trophoblasts and syncytiotrophoblasts. -- Abstract: Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, can be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2 months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and

  18. Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

    Science.gov (United States)

    Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

    2016-01-01

    In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

  19. Effects of topography on the functional development of human neural progenitor cells.

    Science.gov (United States)

    Wu, Ze-Zhi; Kisaalita, William S; Wang, Lina; Zachman, Angela L; Zhao, Yiping; Hasneen, Kowser; Machacek, Dave; Stice, Steven L

    2010-07-01

    We have fabricated a topographical substrate with a packed polystyrene bead array for the development of cell-based assay systems targeting voltage-gated calcium channels (VGCCs). Human neural progenitor cells (H945RB.3) cultured on both flat and topographical substrates were analyzed in terms of morphological spreading, neuronal commitment, resting membrane potential (V(m)) establishment and VGCC function development. We found, by SEM imaging, that arrayed substrates, formed with both sub-micrometer (of 0.51 microm in mean diameter) and micrometer (of 1.98 microm in mean diameter) beads, were capable of promoting the spreading of the progenitor cells as compared with the flat polystyrene surfaces. With the micrometer beads, it was found that arrayed substrates facilitated the neural progenitor cells' maintenance of less negative V(m) values upon differentiation with bFGF starvation, which favored predominant neuronal commitment. Almost all the progenitor cells were responsive to 50 mM K(+) depolarization with an increase in [Ca(2+)](i) either before or upon differentiation, suggesting the expression of functional VGCCs. Compared to the flat polystyrene surfaces, microbead arrayed substrates facilitated the development of higher VGCC responsiveness by the progenitor cells upon differentiation. The enhancement of both VGCC responsiveness and cell spreading by arrays of micrometer beads was most significant on day 14 into differentiation, which was the latest time point of measurement in this study. This study thus rationalized the possibility for future substrate topography engineering to manipulate ion channel function and to meet the challenge of low VGCC responsiveness found in early drug discovery.

  20. Increasing Hematopoietic Stem Cell Yield to Develop Mice with Human Immune Systems

    Directory of Open Access Journals (Sweden)

    Juan-Carlos Biancotti

    2013-01-01

    Full Text Available Hematopoietic stem cells (HSCs are unique in their capacity to give rise to all mature cells of the immune system. For years, HSC transplantation has been used for treatment of genetic and neoplastic diseases of the hematopoietic and immune systems. The sourcing of HSCs from human umbilical cord blood has salient advantages over isolation from mobilized peripheral blood. However, poor sample yield has prompted development of methodologies to expand HSCs ex vivo. Cytokines, trophic factors, and small molecules have been variously used to promote survival and proliferation of HSCs in culture, whilst strategies to lower the concentration of inhibitors in the culture media have recently been applied to promote HSC expansion. In this paper, we outline strategies to expand HSCs in vitro, and to improve engraftment and reconstitution of human immune systems in immunocompromised mice. To the extent that these “humanized” mice are representative of the endogenous human immune system, they will be invaluable tools for both basic science and translational medicine.

  1. Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.

    Science.gov (United States)

    Shelton, Michael; Kocharyan, Avetik; Liu, Jun; Skerjanc, Ilona S; Stanford, William L

    2016-05-15

    Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Differentiation of insulin-producing cells from human neural progenitor cells.

    Directory of Open Access Journals (Sweden)

    Yuichi Hori

    2005-04-01

    Full Text Available BACKGROUND: Success in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin. METHODS AND FINDINGS: Here we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs. During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors. CONCLUSION: Production of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.

  3. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Carolyn A. de Graaf

    2016-09-01

    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  4. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  5. Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells.

    Science.gov (United States)

    Raghavan, Shreya; Miyasaka, Eiichi A; Gilmont, Robert R; Somara, Sita; Teitelbaum, Daniel H; Bitar, Khalil N

    2014-04-01

    The internal anal sphincter (IAS) is a major contributing factor to pressure within the anal canal and is required for maintenance of rectoanal continence. IAS damage or weakening results in fecal incontinence. We have demonstrated that bioengineered, intrinsically innervated, human IAS tissue replacements possess key aspects of IAS physiology, such as the generation of spontaneous basal tone and contraction/relaxation in response to neurotransmitters. The objective of this study is to demonstrate the feasibility of implantation of bioengineered IAS constructs in the perianal region of athymic rats. Human IAS tissue constructs were bioengineered from isolated human IAS circular smooth muscle cells and human enteric neuronal progenitor cells. After maturation of the bioengineered constructs in culture, they were implanted operatively into the perianal region of athymic rats. Platelet-derived growth factor was delivered to the implanted constructs through a microosmotic pump. Implanted constructs were retrieved from the animals 4 weeks postimplantation. Animals tolerated the implantation well, and there were no early postoperative complications. Normal stooling was observed during the implantation period. At harvest, implanted constructs were adherent to the perirectal rat tissue and appeared healthy and pink. Immunohistochemical analysis revealed neovascularization. Implanted smooth muscle cells maintained contractile phenotype. Bioengineered constructs responded in vitro in a tissue chamber to neuronally evoked relaxation in response to electrical field stimulation and vasoactive intestinal peptide, indicating the preservation of neuronal networks. Our results indicate that bioengineered innervated IAS constructs can be used to augment IAS function in an animal model. This is a regenerative medicine based therapy for fecal incontinence that would directly address the dysfunction of the IAS muscle. Copyright © 2014 Mosby, Inc. All rights reserved.

  6. A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia.

    Science.gov (United States)

    Windrem, Martha S; Schanz, Steven J; Morrow, Carolyn; Munir, Jared; Chandler-Militello, Devin; Wang, Su; Goldman, Steven A

    2014-11-26

    Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia. The differentiation of the donor cells is influenced by the host environment, such that more donor cells differentiated as oligodendrocytes in the hypomyelinated shiverer brain than in myelin wild-types, in which hGPCs were more likely to remain as progenitors. Yet in each recipient, both the number and relative proportion of mouse GPCs fell as a function of time, concomitant with the mitotic expansion and spread of donor hGPCs. By a year after neonatal xenograft, the forebrain GPC populations of implanted mice were largely, and often entirely, of human origin. Thus, neonatally implanted hGPCs outcompeted and ultimately replaced the host population of mouse GPCs, ultimately generating mice with a humanized glial progenitor population. These human glial chimeric mice should permit us to define the specific contributions of glia to a broad variety of neurological disorders, using human cells in vivo. Copyright © 2014 the authors 0270-6474/14/3416153-09$15.00/0.

  7. Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

    Science.gov (United States)

    Saenko, Vladimir; Suzuki, Masatoshi; Matsuse, Michiko; Ohtsuru, Akira; Kumagai, Atsushi; Uga, Tatsuya; Yano, Hiroshi; Nagayama, Yuji; Yamashita, Shunichi

    2011-01-01

    While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases. PMID:21556376

  8. Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

    Directory of Open Access Journals (Sweden)

    Ryo Kurita

    Full Text Available Transfusion of red blood cells (RBCs is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

  9. Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells

    Science.gov (United States)

    Katsura, Mari; Cyou-Nakamine, Hiromasa; Zen, Qin; Zen, Yang; Nansai, Hiroko; Amagasa, Shota; Kanki, Yasuharu; Inoue, Tsuyoshi; Kaneki, Kiyomi; Taguchi, Akashi; Kobayashi, Mika; Kaji, Toshiyuki; Kodama, Tatsuhiko; Miyagawa, Kiyoshi; Wada, Youichiro; Akimitsu, Nobuyoshi; Sone, Hideko

    2016-01-01

    The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

  10. Human Migratory Meniscus Progenitor Cells Are Controlled via the TGF-β Pathway

    Science.gov (United States)

    Muhammad, Hayat; Schminke, Boris; Bode, Christa; Roth, Moritz; Albert, Julius; von der Heyde, Silvia; Rosen, Vicki; Miosge, Nicolai

    2014-01-01

    Summary Degeneration of the knee joint during osteoarthritis often begins with meniscal lesions. Meniscectomy, previously performed extensively after meniscal injury, is now obsolete because of the inevitable osteoarthritis that occurs following this procedure. Clinically, meniscus self-renewal is well documented as long as the outer, vascularized meniscal ring remains intact. In contrast, regeneration of the inner, avascular meniscus does not occur. Here, we show that cartilage tissue harvested from the avascular inner human meniscus during the late stages of osteoarthritis harbors a unique progenitor cell population. These meniscus progenitor cells (MPCs) are clonogenic and multipotent and exhibit migratory activity. We also determined that MPCs are likely to be controlled by canonical transforming growth factor β (TGF-β) signaling that leads to an increase in SOX9 and a decrease in RUNX2, thereby enhancing the chondrogenic potential of MPC. Therefore, our work is relevant for the development of novel cell biological, regenerative therapies for meniscus repair. PMID:25418724

  11. Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

    Science.gov (United States)

    Davari, Maliheh; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

    2013-01-01

    Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

  12. Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.

    Science.gov (United States)

    Ortinau, Stefanie; Schmich, Jürgen; Block, Stephan; Liedmann, Andrea; Jonas, Ludwig; Weiss, Dieter G; Helm, Christiane A; Rolfs, Arndt; Frech, Moritz J

    2010-11-11

    3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3

  13. The complexity of the calretinin-expressing progenitors in the human cerebral cortex

    Directory of Open Access Journals (Sweden)

    Nevena V Radonjic

    2014-08-01

    Full Text Available The complex structure and function of the cerebral cortex critically depend on the balance of excitation and inhibition provided by the pyramidal projection neurons and GABAergic interneurons, respectively. The calretinin-expressing (CalR+ cell is a subtype of GABAergic cortical interneurons that is more prevalent in humans than in rodents. In rodents, CalR+ interneurons originate in the caudal ganglionic eminence (CGE from Gsx2+ progenitors, but in humans it has been suggested that a subpopulation of CalR+ cells can also be generated in the cortical ventricular/subventricular zone (VZ/SVZ. The progenitors for cortically generated CalR+ subpopulation in primates are not yet characterized. Hence, the aim of this study was to identify patterns of expression of the transcription factors (TFs that commit cortical stem cells to the CalR fate, with a focus on Gsx2. First, we studied the expression of Gsx2 and its downstream effectors, Ascl1 and Sp8 in the cortical regions of the fetal human forebrain at midgestation. Next, we established that a subpopulation of cells expressing these TFs are proliferating in the cortical SVZ, and can be co-labeled with CalR. The presence and proliferation of Gsx2+ cells, not only in the ventral telencephalon (GE as previously reported, but also in the cerebral cortex suggests cortical origin of a subpopulation of CalR+ neurons in humans. In vitro treatment of human cortical progenitors with Sonic hedgehog (Shh, an important morphogen in the specification of interneurons, decreased levels of Ascl1 and Sp8 proteins, but did not affect Gsx2 levels. Taken together, our ex-vivo and in vitro results on human fetal brain suggest complex endogenous and exogenous regulation of TFs implied in the specification of different subtypes of CalR+ cortical interneurons.

  14. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  15. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells

    International Nuclear Information System (INIS)

    Lim, B.; Apperley, J.F.; Orkin, S.H.; Williams, D.A.

    1989-01-01

    Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. The authors have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. They report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy

  16. CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin.

    Science.gov (United States)

    Gattei, V; Degan, M; Gloghini, A; De Iuliis, A; Improta, S; Rossi, F M; Aldinucci, D; Perin, V; Serraino, D; Babare, R; Zagonel, V; Gruss, H J; Carbone, A; Pinto, A

    1997-03-15

    CD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals leading to either cell death or proliferation through its specific counterstructure CD30. Although several lines of evidence indicate that CD30L plays a key role as a paracrine- or autocrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known regarding its distribution and biologic significance in other human hematopoietic malignancies. By analyzing tumor cells from 181 patients with RNA studies and immunostaining by the anti-CD30L monoclonal antibody M80, we were able to show that human hematopoietic malignancies of different lineage and maturation stage display a frequent and broad expression of the ligand. CD30L mRNA and surface protein were detected in 60% of acute myeloid leukemias (AMLs), 54% of B-lineage acute lymphoblastic leukemias (ALLs), and in a consistent fraction (68%) of B-cell lymphoproliferative disorders. In this latter group, hairy cell leukemia and high-grade B-cell non-Hodgkin's lymphoma (B-NHL) expressed a higher surface density of CD30L as compared with B-cell chronic lymphocytic leukemia and low-grade B-NHL. Purified plasmacells from a fraction of multiple myeloma patients also displayed CD30L mRNA and protein. A more restricted expression of CD30L was found in T-cell tumors that was mainly confined to neoplasms with an activated peripheral T-cell phenotype, such as T-cell prolymphocytic leukemia, peripheral T-NHL, and adult T-cell leukemia/lymphoma. In contrast, none of the T-lineage ALLs analyzed expressed the ligand. In AML, a high cellular density of CD30L was detected in French-American-British M3, M4, and M5 phenotypes, which are directly associated with the presence on tumor cells of certain surface structures, including the p55 interleukin-2 receptor alpha-chain, the alpha(M) (CD11b) chain of beta2 integrins, and the intercellular adhesion molecule-1 (CD54). Analysis of normal hematopoietic cells

  17. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells

    International Nuclear Information System (INIS)

    Dontu, Gabriela; Jackson, Kyle W; McNicholas, Erin; Kawamura, Mari J; Abdallah, Wissam M; Wicha, Max S

    2004-01-01

    suggest that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells, affecting self-renewal and lineage-specific differentiation. Based on these findings we propose that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells

  18. Activation of adenosine A3 receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Vacek, Antonín; Pospíšil, Milan; Holá, Jiřina; Štreitová, Denisa; Znojil, V.

    2009-01-01

    Roč. 58, č. 2 (2009), s. 247-252 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA305/06/0015; GA ČR(CZ) GA305/08/0158 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : hematopoiesis * adenosine A3 receptor agonist * hematopoietic growth factors Subject RIV: BO - Biophysics Impact factor: 1.430, year: 2009

  19. CRISPR/Cas9 system and its applications in human hematopoietic cells.

    Science.gov (United States)

    Hu, Xiaotang

    2016-11-01

    Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors

    International Nuclear Information System (INIS)

    Zanocco-Marani, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio

    2006-01-01

    The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage

  1. Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

    Science.gov (United States)

    Tian, Xinghui; Kaufman, Dan S

    2008-07-01

    Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

  2. Efficient Generation of NKX6-1+ Pancreatic Progenitors from Multiple Human Pluripotent Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    M. Cristina Nostro

    2015-04-01

    Full Text Available Human pluripotent stem cells (hPSCs represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF and nicotinamide signaling induces the generation of NKX6-1+ progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1+ population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10, and inhibitors of bone morphogenetic protein (BMP and hedgehog signaling pathways. When transplanted into NOD scid gamma (NSG recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, including monohormonal insulin+ cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.

  3. Progressive Recruitment of Mesenchymal Progenitors Reveals a Time-Dependent Process of Cell Fate Acquisition in Mouse and Human Nephrogenesis.

    Science.gov (United States)

    Lindström, Nils O; De Sena Brandine, Guilherme; Tran, Tracy; Ransick, Andrew; Suh, Gio; Guo, Jinjin; Kim, Albert D; Parvez, Riana K; Ruffins, Seth W; Rutledge, Elisabeth A; Thornton, Matthew E; Grubbs, Brendan; McMahon, Jill A; Smith, Andrew D; McMahon, Andrew P

    2018-06-04

    Mammalian nephrons arise from a limited nephron progenitor pool through a reiterative inductive process extending over days (mouse) or weeks (human) of kidney development. Here, we present evidence that human nephron patterning reflects a time-dependent process of recruitment of mesenchymal progenitors into an epithelial nephron precursor. Progressive recruitment predicted from high-resolution image analysis and three-dimensional reconstruction of human nephrogenesis was confirmed through direct visualization and cell fate analysis of mouse kidney organ cultures. Single-cell RNA sequencing of the human nephrogenic niche provided molecular insights into these early patterning processes and predicted developmental trajectories adopted by nephron progenitor cells in forming segment-specific domains of the human nephron. The temporal-recruitment model for nephron polarity and patterning suggested by direct analysis of human kidney development provides a framework for integrating signaling pathways driving mammalian nephrogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Parvovirus B19 integration into human CD36+ erythroid progenitor cells.

    Science.gov (United States)

    Janovitz, Tyler; Wong, Susan; Young, Neal S; Oliveira, Thiago; Falck-Pedersen, Erik

    2017-11-01

    The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Hematopoietic stem cell expansion : challenges and opportunities

    NARCIS (Netherlands)

    Walasek, Marta A.; van Os, Ronald; de Haan, Gerald; Kanz, L; Fibbe, WE; Lengerke, C; Dick, JE

    2012-01-01

    Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell

  6. Growth regulation on human acute myeloid leukemia effects of five recombinant hematopoietic factors in a serum-free culture system

    NARCIS (Netherlands)

    Delwel, E.; Salem, M.; Pellens, C.; Dorssers, L.; Wagemaker, G.; Clark, S.; Loewenberg, B

    1988-01-01

    The response of human acute myeloid leukemia (AML) cells to the distinct hematopoietic growth factors (HGFs), ie, recombinant interleukin-3 (IL-3), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), and erythropoietin (Epo) was investigated under well-defined

  7. A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.

    Science.gov (United States)

    Magistri, Marco; Khoury, Nathalie; Mazza, Emilia Maria Cristina; Velmeshev, Dmitry; Lee, Jae K; Bicciato, Silvio; Tsoulfas, Pantelis; Faghihi, Mohammad Ali

    2016-11-01

    Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  8. The fps/fes proto-oncogene regulates hematopoietic lineage output.

    Science.gov (United States)

    Sangrar, Waheed; Gao, Yan; Zirngibl, Ralph A; Scott, Michelle L; Greer, Peter A

    2003-12-01

    The fps/fes proto-oncogene is abundantly expressed in myeloid cells, and the Fps/Fes cytoplasmic protein-tyrosine kinase is implicated in signaling downstream from hematopoietic cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). Studies using leukemic cell lines have previously suggested that Fps/Fes contributes to granulomonocytic differentiation, and that it might play a more selective role in promoting survival and differentiation along the monocytic pathway. In this study we have used a genetic approach to explore the role of Fps/Fes in hematopoiesis. We used transgenic mice that tissue-specifically express a mutant human fps/fes transgene (fps(MF)) that was engineered to encode Fps/Fes kinase that is activated through N-terminal myristoylation (MFps). Hematopoietic function was assessed using lineage analysis, hematopoietic progenitor cell colony-forming assays, and biochemical approaches. fps(MF) transgenic mice displayed a skewed hematopoietic output reflected by increased numbers of circulating granulocytic and monocytic cells and a corresponding decrease in lymphoid cells. Bone marrow colony assays of progenitor cells revealed a significant increase in the number of both granulomonocytic and multi-lineage progenitors. A molecular analysis of signaling in mature monocytic cells showed that MFps promoted GM-CSF-induced STAT3, STAT5, and ERK1/2 activation. These observations support a role for Fps/Fes in signaling pathways that contribute to lineage determination at the level of multi-lineage hematopoietic progenitors as well as the more committed granulomonocytic progenitors.

  9. Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.

    Directory of Open Access Journals (Sweden)

    Rebecca Williams

    Full Text Available BACKGROUND: Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC, are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage. METHODS AND FINDINGS: Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect. CONCLUSIONS: In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell

  10. Brief Report: Robo1 Regulates the Migration of Human Subventricular Zone Neural Progenitor Cells During Development.

    Science.gov (United States)

    Guerrero-Cazares, Hugo; Lavell, Emily; Chen, Linda; Schiapparelli, Paula; Lara-Velazquez, Montserrat; Capilla-Gonzalez, Vivian; Clements, Anna Christina; Drummond, Gabrielle; Noiman, Liron; Thaler, Katrina; Burke, Anne; Quiñones-Hinojosa, Alfredo

    2017-07-01

    Human neural progenitor cell (NPC) migration within the subventricular zone (SVZ) of the lateral ganglionic eminence is an active process throughout early brain development. The migration of human NPCs from the SVZ to the olfactory bulb during fetal stages resembles what occurs in adult rodents. As the human brain develops during infancy, this migratory stream is drastically reduced in cell number and becomes barely evident in adults. The mechanisms regulating human NPC migration are unknown. The Slit-Robo signaling pathway has been defined as a chemorepulsive cue involved in axon guidance and neuroblast migration in rodents. Slit and Robo proteins expressed in the rodent brain help guide neuroblast migration from the SVZ through the rostral migratory stream to the olfactory bulb. Here, we present the first study on the role that Slit and Robo proteins play in human-derived fetal neural progenitor cell migration (hfNPC). We describe that Robo1 and Robo2 isoforms are expressed in the human fetal SVZ. Furthermore, we demonstrate that Slit2 is able to induce a chemorepellent effect on the migration of hfNPCs derived from the human fetal SVZ. In addition, when Robo1 expression is inhibited, hfNPCs are unable to migrate to the olfactory bulb of mice when injected in the anterior SVZ. Our findings indicate that the migration of human NPCs from the SVZ is partially regulated by the Slit-Robo axis. This pathway could be regulated to direct the migration of NPCs in human endogenous neural cell therapy. Stem Cells 2017;35:1860-1865. © 2017 AlphaMed Press.

  11. Dissociation of Survival, Proliferation, and State Control in Human Hematopoietic Stem Cells.

    Science.gov (United States)

    Knapp, David J H F; Hammond, Colin A; Miller, Paul H; Rabu, Gabrielle M; Beer, Philip A; Ricicova, Marketa; Lecault, Véronique; Da Costa, Daniel; VanInsberghe, Michael; Cheung, Alice M; Pellacani, Davide; Piret, James; Hansen, Carl; Eaves, Connie J

    2017-01-10

    The role of growth factors (GFs) in controlling the biology of human hematopoietic stem cells (HSCs) remains limited by a lack of information concerning the individual and combined effects of GFs directly on the survival, Mitogenesis, and regenerative activity of highly purified human HSCs. We show that the initial input HSC activity of such a purified starting population of human cord blood cells can be fully maintained over a 21-day period in serum-free medium containing five GFs alone. HSC survival was partially supported by any one of these GFs, but none were essential, and different combinations of GFs variably stimulated HSC proliferation. However, serial transplantability was not detectably compromised by many conditions that reduced human HSC proliferation and/or survival. These results demonstrate the dissociated control of these three human HSC bio-responses, and set the stage for future improvements in strategies to modify and expand human HSCs ex vivo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Human mammary progenitor cell fate decisions are productsof interactions with combinatorial microenvironments

    DEFF Research Database (Denmark)

    LaBarge, Mark A.; Nelson, Celeste M.; Villadsen, René

    2009-01-01

    factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify...... combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells.Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well...

  13. Effects of radiation and porphyrin on mitosis and chromosomes in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Tan, J.C.; Huang, C.C.; Fiel, R.J.

    1976-01-01

    The effect on mitosis of a human hematopoietic cell line RPMI-1788 treated with a metal chelate (Zn ++ ) of meso-tetra (p-carboxyphenyl) porphine (Zn-TCPP) alone at various concentrations or in combination with gamma-irradiation at various doses were studied. The results showed that both Zn-TCPP and radiation were effective in interfering with normal mitosis and that the effect of radiation was relatively more effective. Data also suggest interacting effects between Zn-TCPP and gamma-irradiation. At low doses of radiation, Zn-TCPP potentiated the effect of radiation. The reverse seemed to be true at a high dose of radiation. The effects of two porphyrins (Zn-TCPP and hematoporphyrin) and radiation on chromosomes were also studied. Chromosomal aberrations characteristic of radiation were observed. The porphyrins were found not to be effective chromosome-breaking agents under the experimental conditions tested

  14. Prolonged Shedding of Human Coronavirus in Hematopoietic Cell Transplant Recipients: Risk Factors and Viral Genome Evolution.

    Science.gov (United States)

    Ogimi, Chikara; Greninger, Alexander L; Waghmare, Alpana A; Kuypers, Jane M; Shean, Ryan C; Xie, Hu; Leisenring, Wendy M; Stevens-Ayers, Terry L; Jerome, Keith R; Englund, Janet A; Boeckh, Michael

    2017-07-15

    Recent data suggest that human coronavirus (HCoV) pneumonia is associated with significant mortality in hematopoietic cell transplant (HCT) recipients. Investigation of risk factors for prolonged shedding and intrahost genome evolution may provide critical information for development of novel therapeutics. We retrospectively reviewed HCT recipients with HCoV detected in nasal samples by polymerase chain reaction (PCR). HCoV strains were identified using strain-specific PCR. Shedding duration was defined as time between first positive and first negative sample. Logistic regression analyses were performed to evaluate factors for prolonged shedding (≥21 days). Metagenomic next-generation sequencing (mNGS) was conducted when ≥4 samples with cycle threshold values of Genome changes were consistent with the expected molecular clock of HCoV. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  15. Granulocyte-colony stimulating factor (G-CSF)-primed, delayed marrow harvests as a source of hematopoietic stem and progenitor cells for allogeneic transplantation.

    Science.gov (United States)

    Phillips, G L; Davey, D D; Hale, G A; Marshall, K W; Munn, R K; Nath, R; Reece, D E; Van Zant, G

    1999-10-01

    We evaluated the ability of G-CSF to increase the number of hematopoietic stem cells obtained by "delayed" BM harvest for allogeneic transplantation. Five normal donors received G-CSF @ 10 mcg/kg/day x 5 followed by repeat PB and BM assays at day 6 and 16, and BM harvest at day 16. Stem cells were not increased in the BM at day 16. Five patients underwent BMT and engrafted at +10 to +19 days. While the tested strategy offers no intrinsic advantages, its potential cannot be evaluated fully without alternative timing and/or additional, "early acting" growth factors.

  16. Autonomous Agents on Expedition: Humans and Progenitor Ants and Planetary Exploration

    Science.gov (United States)

    Rilee, M. L.; Clark, P. E.; Curtis, S. A.; Truszkowski, W. F.

    2002-01-01

    The Autonomous Nano-Technology Swarm (ANTS) is an advanced mission architecture based on a social insect analog of many specialized spacecraft working together to achieve mission goals. The principal mission concept driving the ANTS architecture is a Main Belt Asteroid Survey in the 2020s that will involve a thousand or more nano-technology enabled, artificially intelligent, autonomous pico-spacecraft (architecture. High level, mission-oriented behaviors are to be managed by a control / communications layer of the swarm, whereas common low level functions required of all spacecraft, e.g. attitude control and guidance and navigation, are handled autonomically on each spacecraft. At the higher levels of mission planning and social interaction deliberative techniques are to be used. For the asteroid survey, ANTS acts as a large community of cooperative agents while for precursor missions there arises the intriguing possibility of Progenitor ANTS and humans acting together as agents. For optimal efficiency and responsiveness for individual spacecraft at the lowest levels of control we have been studying control methods based on nonlinear dynamical systems. We describe the critically important autonomous control architecture of the ANTS mission concept and a sequence of partial implementations that feature increasingly autonomous behaviors. The scientific and engineering roles that these Progenitor ANTS could play in human missions or remote missions with near real time human interactions, particularly to the Moon and Mars, will be discussed.

  17. A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

    Science.gov (United States)

    Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

    2016-01-01

    The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

  18. Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

    Science.gov (United States)

    Rostovskaya, Maria; Bredenkamp, Nicholas; Smith, Austin

    2015-10-19

    Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification. © 2015 The Authors.

  19. What is the potential of oligodendrocyte progenitor cells to successfully treat human spinal cord injury?

    Directory of Open Access Journals (Sweden)

    Yeung Trevor M

    2011-09-01

    Full Text Available Abstract Background Spinal cord injury is a serious and debilitating condition, affecting millions of people worldwide. Long seen as a permanent injury, recent advances in stem cell research have brought closer the possibility of repairing the spinal cord. One such approach involves injecting oligodendrocyte progenitor cells, derived from human embryonic stem cells, into the injured spinal cord in the hope that they will initiate repair. A phase I clinical trial of this therapy was started in mid 2010 and is currently underway. Discussion The theory underlying this approach is that these myelinating progenitors will phenotypically replace myelin lost during injury whilst helping to promote a repair environment in the lesion. However, the importance of demyelination in the pathogenesis of human spinal cord injury is a contentious issue and a body of literature suggests that it is only a minor factor in the overall injury process. Summary This review examines the validity of the theory underpinning the on-going clinical trial as well as analysing published data from animal models and finally discussing issues surrounding safety and purity in order to assess the potential of this approach to successfully treat acute human spinal cord injury.

  20. Involvement of urokinase receptor in the cross-talk between human hematopoietic stem cells and bone marrow microenvironment

    DEFF Research Database (Denmark)

    Selleri, Carmine; Montuori, Nunzia; Salvati, Annamaria

    2016-01-01

    Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and can be induced to mobilize into the circulation for transplantation. Homing and lodgement into BM of transplanted HSCs are the first critical steps in their engraftment and involve multiple interactions between HSCs and the BM...... Culture (LTC)-Initiating Cells (ICs) and in the release of clonogenic progenitors from LTCs of CD34+ HSCs. Further, suPAR increases adhesion and survival of CD34+ KG1 AML cells, whereas uPAR84-95 increases their proliferation.Thus, circulating DIIDIII-suPAR, strongly increased in HSC mobilization...... microenvironment.uPAR is a three domain receptor (DIDIIDIII) which binds urokinase, vitronectin, integrins. uPAR can be cleaved and shed from the cell surface generating full-length and cleaved soluble forms (suPAR and DIIDIII-suPAR). DIIDIII-suPAR can bind fMLF receptors through the SRSRY sequence (residues 88...

  1. Controle de esterilidade de produtos de células progenitoras hematopoéticas do sangue periférico Sterility control of hematopoietic progenitor cells from peripheral blood products

    Directory of Open Access Journals (Sweden)

    Igor D. Almeida

    2010-02-01

    Full Text Available A taxa de contaminação microbiana dos produtos de células progenitoras hematopoéticas do sangue periférico é baixa. Neste estudo pesquisou-se a prevalência de hemoculturas positivas em células progenitoras hematopoéticas do sangue periférico (CPHSP no Serviço de Hemoterapia do Hospital de Clínicas de Porto Alegre. Do total de 618 coletas realizadas no período de 2000 a 2007, 26 (4,2% apresentaram contaminação por bactérias. O Staphylococcus coagulase-negativo foi predominantemente isolado nas hemoculturas. A antibioticoterapia pré e pós-infusão foi estabelecida de acordo com o microorganismo e seu antibiograma, sendo que, em cinco das doze infusões contaminadas realizadas, não foram administrados antimicrobianos profilaticamente. Episódios febris foram observados em sete pacientes (58%, enquanto cinco (42% não apresentaram febre. Das doze infusões contaminadas realizadas, seis (50% apresentaram hemocultura pós-descongelamento positivas, enquanto as restantes (50% foram negativas. Isto se deve às propriedades bactericidas do DMSO, de células fagocitose-ativas e de temperaturas muito baixas atingidas na criopreservação. Autores têm relatado sucesso neste procedimento após a infusão desses produtos contaminados com o mínimo de consequências clínicas.The rate of microbial contamination of hematopoietic progenitor cell products from peripheral blood is low. In this study, we investigated the prevalence of positive blood cultures of hematopoietic progenitor cells from peripheral blood in a hemotherapy service. Of a total of 618 samples taken during the period from 2000 to 2007, 26 (4.2% were contaminated by bacteria. Staphylococcus coagulase-negative was the predominant bacterium isolated in blood cultures. Pre- and post-infusion antibiotic therapy was established depending on the microorganism and antibiogram, whereas in five out of twelve contaminated infusions, no antibiotics were administered prophylactically

  2. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Romeo Cecchelli

    Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

  3. Protection of visual functions by human neural progenitors in a rat model of retinal disease.

    Directory of Open Access Journals (Sweden)

    David M Gamm

    2007-03-01

    Full Text Available A promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat.Animals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90-100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed.Wild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in

  4. Spontaneous calcium transients in human neural progenitor cells mediated by transient receptor potential channels.

    Science.gov (United States)

    Morgan, Peter J; Hübner, Rayk; Rolfs, Arndt; Frech, Moritz J

    2013-09-15

    Calcium signals affect many developmental processes, including proliferation, migration, survival, and apoptosis, processes that are of particular importance in stem cells intended for cell replacement therapies. The mechanisms underlying Ca(2+) signals, therefore, have a role in determining how stem cells respond to their environment, and how these responses might be controlled in vitro. In this study, we examined the spontaneous Ca(2+) activity in human neural progenitor cells during proliferation and differentiation. Pharmacological characterization indicates that in proliferating cells, most activity is the result of transient receptor potential (TRP) channels that are sensitive to Gd(3+) and La(3+), with the more subtype selective antagonist Ruthenium red also reducing activity, suggesting the involvement of transient receptor potential vanilloid (TRPV) channels. In differentiating cells, Gd(3+) and La(3+)-sensitive TRP channels also appear to underlie the spontaneous activity; however, no sub-type-specific antagonists had any effect. Protein levels of TRPV2 and TRPV3 decreased in differentiated cells, which is demonstrated by western blot. Thus, it appears that TRP channels represent the main route of Ca(2+) entry in human neural progenitor cells (hNPCs), but the responsible channel types are subject to substitution under differentiating conditions. The level of spontaneous activity could be increased and decreased by lowering and raising the extracellular K(+) concentration. Proliferating cells in low K(+) slowed the cell cycle, with a disproportionate increased percentage of cells in G1 phase and a reduction in S phase. Taken together, these results suggest a link between external K(+) concentration, spontaneous Ca(2+) transients, and cell cycle distribution, which is able to influence the fate of stem and progenitor cells.

  5. Human endothelial progenitor cells rescue cortical neurons from oxygen-glucose deprivation induced death.

    Science.gov (United States)

    Bacigaluppi, Susanna; Donzelli, Elisabetta; De Cristofaro, Valentina; Bragazzi, Nicola Luigi; D'Amico, Giovanna; Scuteri, Arianna; Tredici, Giovanni

    2016-09-19

    Cerebral ischemia is characterized by both acute and delayed neuronal injuries. Neuro-protection is a major issue that should be properly addressed from a pharmacological point of view, and cell-based treatment approaches are of interest due to their potential pleiotropic effects. Endothelial progenitor cells have the advantage of being mobilized from the bone marrow into the circulation, but have been less studied than other stem cells, such as mesenchymal stem cells. Therefore, the comparison between human endothelial progenitor cells (hEPC) and human mesenchymal progenitor cells (hMSC) in terms of efficacy in rescuing neurons from cell death after transitory ischemia is the aim of the current study, in the effort to address further directions. In vitro model of oxygen-glucose deprivation (OGD) on a primary culture of rodent cortical neurons was set up with different durations of exposure: 1, 2 and 3hrs with assessment of neuron survival. The 2hrs OGD was chosen for the subsequent experiments. After 2hrs OGD neurons were either placed in indirect co-culture with hMSC or hEPC or cultured in hMSC or hEPC conditioned medium and cell viability was evaluated by MTT assay. At day 2 after 2hrs OGD exposure, mean neuronal survival was 47.9±24.2%. In contrast, after treatment with hEPC and hMSC indirect co-culture was 74.1±27.3%; and 69.4±18.8%, respectively. In contrast, treatment with conditioned medium did not provide any advantage in terms of survival to OGD neurons The study shows the efficacy of hEPC in indirect co-culture to rescue neurons from cell death after OGD, comparable to that of hMSC. hEPC deserve further studies given their potential interest for ischemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Desai, Amar; Qing, Yulan; Gerson, Stanton L

    2014-02-01

    Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long-term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5' HR end resection. We use cultured Exo1(mut) fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1(mut) mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1(mut) mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1(mut) HSCs were pushed into cell cycle in vivo with 5-fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1-mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. © AlphaMed Press.

  7. Recruitment of host's progenitor cells to sites of human amniotic fluid stem cells implantation.

    Science.gov (United States)

    Mirabella, Teodelinda; Poggi, Alessandro; Scaranari, Monica; Mogni, Massimo; Lituania, Mario; Baldo, Chiara; Cancedda, Ranieri; Gentili, Chiara

    2011-06-01

    The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host's progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys.

    Science.gov (United States)

    Sagrinati, Costanza; Netti, Giuseppe Stefano; Mazzinghi, Benedetta; Lazzeri, Elena; Liotta, Francesco; Frosali, Francesca; Ronconi, Elisa; Meini, Claudia; Gacci, Mauro; Squecco, Roberta; Carini, Marco; Gesualdo, Loreto; Francini, Fabio; Maggi, Enrico; Annunziato, Francesco; Lasagni, Laura; Serio, Mario; Romagnani, Sergio; Romagnani, Paola

    2006-09-01

    Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowman's capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell-specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24+CD133+ PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24+CD133+ PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24+CD133+ PEC but not of CD24-CD133- renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24+CD133+ PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases.

  9. Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.

    Science.gov (United States)

    Hamed, Saher; Ben-Nun, Ohad; Egozi, Dana; Keren, Aviad; Malyarova, Nastya; Kruchevsky, Danny; Gilhar, Amos; Ullmann, Yehuda

    2012-10-01

    Bone marrow-derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that was isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell-treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell-treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.

  10. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thomas C Schulz

    Full Text Available Development of a human embryonic stem cell (hESC-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

  11. Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

    Directory of Open Access Journals (Sweden)

    Xianming Wang

    2018-03-01

    Full Text Available Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4. Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult β-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC line that efficiently differentiates into human pancreatic progenitors (PPs. Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation

  12. Hematopoietic Stem Cell Therapy as a Counter-Measure for Human Exploration of Deep Space

    Science.gov (United States)

    Ohi, S.; Roach, A.-N.; Ramsahai, S.; Kim, B. C.; Fitzgerald, W.; Riley, D. A.; Gonda, S. R.

    2004-01-01

    Human exploration of deep space depends, in part, on our ability to counter severe/invasive disorders that astronauts experience in space environments. The known symptoms include hematological/cardiac abnormalities,bone and muscle losses, immunodeficiency, neurological disorders, and cancer. Exploiting the extraordinary plasticity of hematopoietic stem cells (HSCs), which differentiate not only to all types of blood cells, but also to various tissues, we have advanced a hypothesis that ome of the space-caused disorders maybe amenable to hematopoietis stem cell therapy(HSCT) so as to maintain promote human exploration of deep space. Using mouse models of human anemia beta-thaiassemia) as well as spaceflight (hindlimb unloading system), we have obtained feasibility results of HSCT for space anemia, muscle loss, and immunodeficiency. For example, in the case of HSCT for muscle loss, the beta-galactosidese marked HSCs were detected in the hindlimbs of unloaded mouse following transplantation by -X-gal wholemaunt staining procedure. Histochemicaland physical analyses indicated structural contribution of HSCs to the muscle. HSCT for immunodeficiency was investigated ising beta-galactosidese gene-tagged Escherichia coli as the infectious agent. Results of the X-gal staining procedure indicated the rapeutic role of the HSCT. To facilitate the HSCT in space, growth of HSCs were optimized in the NASA Rotating Wall Vessel (RWV) culture systems, including Hydrodynamic Focusing Bioreactor (HFB).

  13. Transplantation of Human Chorion-Derived Cholinergic Progenitor Cells: a Novel Treatment for Neurological Disorders.

    Science.gov (United States)

    Mohammadi, Alireza; Maleki-Jamshid, Ali; Sanooghi, Davood; Milan, Peiman Brouki; Rahmani, Arash; Sefat, Farshid; Shahpasand, Koorosh; Soleimani, Mansoureh; Bakhtiari, Mehrdad; Belali, Rafie; Faghihi, Faezeh; Joghataei, Mohammad Taghi; Perry, George; Mozafari, Masoud

    2018-03-16

    A neurological disorder is any disorder or abnormality in the nervous system. Among different neurological disorders, Alzheimer's disease (AD) is recognized as the sixth leading cause of death globally. Considerable research has been conducted to find pioneer treatments for this devastating disorder among which cell therapy has attracted remarkable attentions over the last decade. Up to now, targeted differentiation into specific desirable cell types has remained a major obstacle to clinical application of cell therapy. Also, potential risks including uncontrolled growth of stem cells could be disastrous. In our novel protocol, we used basal forebrain cholinergic progenitor cells (BFCN) derived from human chorion-derived mesenchymal stem cells (hC-MSCs) which made it possible to obtain high-quality population of cholinergic neurons and in vivo in much shorter time period than previous established methods. Remarkably, the transplanted progenitors fully differentiated to cholinergic neurons which in turn integrated in higher cortical networks of host brains, resulting in significant improvement in cognitive assessments. This method may have profound implications in cell therapies for any other neurodegenerative disorders. Graphical Abstract ᅟ.

  14. Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Jamie Trott

    2017-06-01

    Full Text Available Pluripotent stem cells have been proposed as an unlimited source of pancreatic β cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional β cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ β-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.

  15. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  16. Prolonged Expansion Induces Spontaneous Neural Progenitor Differentiation from Human Gingiva-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Rajan, Thangavelu Soundara; Scionti, Domenico; Diomede, Francesca; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-12-01

    Neural crest-derived mesenchymal stem cells (MSCs) obtained from dental tissues received considerable interest in regenerative medicine, particularly in nerve regeneration owing to their embryonic origin and ease of harvest. Proliferation efficacy and differentiation capacity into diverse cell lineages propose dental MSCs as an in vitro tool for disease modeling. In this study, we investigated the spontaneous differentiation efficiency of dental MSCs obtained from human gingiva tissue (hGMSCs) into neural progenitor cells after extended passaging. At passage 41, the morphology of hGMSCs changed from typical fibroblast-like shape into sphere-shaped cells with extending processes. Next-generation transcriptomics sequencing showed increased expression of neural progenitor markers such as NES, MEIS2, and MEST. In addition, de novo expression of neural precursor genes, such as NRN1, PHOX2B, VANGL2, and NTRK3, was noticed in passage 41. Immunocytochemistry results showed suppression of neurogenesis repressors TP53 and p21, whereas Western blot results revealed the expression of neurotrophic factors BDNF and NT3 at passage 41. Our results showed the spontaneous efficacy of hGMSCs to differentiate into neural precursor cells over prolonged passages and that these cells may assist in producing novel in vitro disease models that are associated with neural development.

  17. Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia.

    Directory of Open Access Journals (Sweden)

    Naeem Khan

    Full Text Available In acute myeloid leukemia (AML quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC with treatment-resistance. LSC in CD34+ and more mature CD34- AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS. In control BM (n = 24, ROS levels were highest in granulocyte-macrophage progenitors (GMP and CD34- myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12. Erythroid CD34- precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93 and MDS-with-excess-blasts (MDS-RAEB (n = 14, immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34- SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs but also in CD34- AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

  18. Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture.

    Science.gov (United States)

    Glen, Katie E; Workman, Victoria L; Ahmed, Forhad; Ratcliffe, Elizabeth; Stacey, Adrian J; Thomas, Robert J

    2013-09-01

    Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Human progenitor cells rapidly mobilized by AMD3100 repopulate NOD/SCID mice with increased frequency in comparison to cells from the same donor mobilized by granulocyte colony stimulating factor

    DEFF Research Database (Denmark)

    Hess, David A; Bonde, Jesper; Craft, Timothy P

    2007-01-01

    ) or purified CD34(+) cells was compared at limiting dilution into NOD/SCID mice. Human AMD3100-mobilized MNC possessed enhanced repopulating frequency in comparison to G-CSF-mobilized MNC from paired donors, and purified CD34(+) progenitors were at least as efficient as the G-CSF mobilized cells....... The frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 8.7 x 10(6) AMD3100-mobilized MNC compared to 1 SRC in 29.0 x 10(6) G-CSF-mobilized MNC, and 1 SRC in 1.2 x 10(5) AMD3100-mobilized CD34(+) cells compared to 1 SRC in 1.8 x 10(5) G-CSF-mobilized CD34(+) cells. Hematopoietic differentiation...

  20. Noncoding RNA in the Transcriptional Landscape of Human Neural Progenitor Cell Differentiation

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    Patrick eHecht

    2015-10-01

    Full Text Available Increasing evidence suggests that noncoding RNAs play key roles in cellular processes, particularly in the brain. The present study used RNA sequencing to identify the transcriptional landscape of two human neural progenitor cell lines, SK-N-SH and ReNcell CX, as they differentiate into human cortical projection neurons. Protein coding genes were found to account for 54.8% and 57.0% of expressed genes, respectively, and alignment of RNA sequencing reads revealed that only 25.5-28.1% mapped to exonic regions of the genome. Differential expression analysis in the two cell lines identified altered gene expression in both protein coding and noncoding RNAs as they undergo neural differentiation with 222 differentially expressed genes observed in SK-N-SH cells and 19 differentially expressed genes in ReNcell CX. Interestingly, genes showing differential expression in SK-N-SH cells are enriched in genes implicated in autism spectrum disorder, but not in gene sets related to cancer or Alzheimer’s disease. Weighted gene co-expression network analysis (WGCNA was used to detect modules of co-expressed protein coding and noncoding RNAs in SK-N-SH cells and found four modules to be associated with neural differentiation. These modules contain varying levels of noncoding RNAs ranging from 10.7% to 49.7% with gene ontology suggesting roles in numerous cellular processes important for differentiation. These results indicate that noncoding RNAs are highly expressed in human neural progenitor cells and likely hold key regulatory roles in gene networks underlying neural differentiation and neurodevelopmental disorders.

  1. AF10 plays a key role in the survival of uncommitted hematopoietic cells.

    Directory of Open Access Journals (Sweden)

    Raquel Chamorro-Garcia

    Full Text Available Hematopoiesis is a complex process regulated by both cell intrinsic and cell extrinsic factors. Alterations in the expression of critical genes during hematopoiesis can modify the balance between stem cell differentiation and proliferation, and may ultimately give rise to leukemia and other diseases. AF10 is a transcription factor that has been implicated in the development of leukemia following chromosomal rearrangements between the AF10 gene and one of at least two other genes, MLL and CALM. The link between AF10 and leukemia, together with the known interactions between AF10 and hematopoietic regulators, suggests that AF10 may be important in hematopoiesis and in leukemic transformation. Here we show that AF10 is important for proper hematopoietic differentiation. The induction of hematopoietic differentiation in both human hematopoietic cell lines and murine total bone marrow cells triggers a decrease of AF10 mRNA and protein levels, particularly in stem cells and multipotent progenitors. Gain- and loss-of-function studies demonstrate that over- or under-expression of AF10 leads to apoptotic cell death in stem cells and multipotent progenitors. We conclude that AF10 plays a key role in the maintenance of multipotent hematopoietic cells.

  2. High-level Gpr56 expression is dispensable for the maintenance and function of hematopoietic stem and progenitor cells in mice

    Directory of Open Access Journals (Sweden)

    Tata Nageswara Rao

    2015-05-01

    Full Text Available Blood formation by hematopoietic stem cells (HSCs is regulated by a still incompletely defined network of general and HSC-specific regulators. In this study, we analyzed the role of G-protein coupled receptor 56 (Gpr56 as a candidate HSC regulator based on its differential expression in quiescent relative to proliferating HSCs and its common targeting by core HSC regulators. Detailed expression analysis revealed that Gpr56 is abundantly expressed by HSPCs during definitive hematopoiesis in the embryo and in the adult bone marrow, but its levels are reduced substantially as HSPCs differentiate. However, despite enriched expression in HSPCs, Gpr56-deficiency did not impair HSPC maintenance or function during steady-state or myeloablative stress-induced hematopoiesis. Gpr56-deficient HSCs also responded normally to physiological and pharmacological mobilization signals, despite the reported role of this GPCR as a regulator of cell adhesion and migration in neuronal cells. Moreover, Gpr56-deficient bone marrow engrafted with equivalent efficiency as wild-type HSCs in primary recipients; however, their reconstituting ability was reduced when subjected to serial transplantation. These data indicate that although GPR56 is abundantly and selectively expressed by primitive HSPCs, its high level expression is largely dispensable for steady-state and regenerative hematopoiesis.

  3. SIMPL enhancement of tumor necrosis factor-α dependent p65-MED1 complex formation is required for mammalian hematopoietic stem and progenitor cell function.

    Directory of Open Access Journals (Sweden)

    Weina Zhao

    Full Text Available Significant insight into the signaling pathways leading to activation of the Rel transcription factor family, collectively termed NF-κB, has been gained. Less well understood is how subsets of NF-κB-dependent genes are regulated in a signal specific manner. The SIMPL protein (signaling molecule that interacts with mouse pelle-like kinase is required for full Tumor Necrosis Factor-α (TNFα induced NF-κB activity. We show that SIMPL is required for steady-state hematopoiesis and the expression of a subset of TNFα induced genes whose products regulate hematopoietic cell activity. To gain insight into the mechanism through which SIMPL modulates gene expression we focused on the Tnf gene, an immune response regulator required for steady-state hematopoiesis. In response to TNFα SIMPL localizes to the Tnf gene promoter where it modulates the initiation of Tnf gene transcription. SIMPL binding partners identified by mass spectrometry include proteins involved in transcription and the interaction between SIMPL and MED1 was characterized in more detail. In response to TNFα, SIMPL is found in p65-MED1 complexes where SIMPL enhances p65/MED1/SIMPL complex formation. Together our results indicate that SIMPL functions as a TNFα-dependent p65 co-activator by facilitating the recruitment of MED1 to p65 containing transcriptional complexes to control the expression of a subset of TNFα-induced genes.

  4. Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.

    Directory of Open Access Journals (Sweden)

    Enas R Yassin

    2009-08-01

    Full Text Available NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2. Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding.

  5. Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons

    Science.gov (United States)

    García-Castro, Irma Lydia; García-López, Guadalupe; Ávila-González, Daniela; Flores-Herrera, Héctor; Molina-Hernández, Anayansi; Portillo, Wendy; Ramón-Gallegos, Eva; Díaz, Néstor Fabián

    2015-01-01

    Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC. PMID:26720151

  6. Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia

    DEFF Research Database (Denmark)

    Garbe, James C; Pepin, Francois; Pelissier, Fanny A

    2012-01-01

    of the cellular and molecular mechanisms that underlies these observations is lacking. In this study, we generated a large collection of normal human mammary epithelial cell strains from women ages 16 to 91 years, derived from primary tissues, to investigate the molecular changes that occur in aging breast cells....... We found that in finite lifespan cultured and uncultured epithelial cells, aging is associated with a reduction of myoepithelial cells and an increase in luminal cells that express keratin 14 and integrin-a6, a phenotype that is usually expressed exclusively in myoepithelial cells in women younger...... than 30 years. Changes to the luminal lineage resulted from age-dependent expansion of defective multipotent progenitors that gave rise to incompletely differentiated luminal or myoepithelial cells. The aging process therefore results in both a shift in the balance of luminal/myoepithelial lineages...

  7. Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells

    DEFF Research Database (Denmark)

    Vincent, P.; Benedikz, Eirikur; Uhlén, Per

    2017-01-01

    Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast...... to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem...... cells (CD133+/CD24lo), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology...

  8. [Teratologic cranio-encephalic effects of chronic thinner inhalation in progenitors, in rats and humans].

    Science.gov (United States)

    Barroso-Moguel, R; Villeda-Hernández, J; Méndez-Armenta, M

    1991-01-01

    Inhalation of thinner by youngsters and adolescents is an increasing drug abuse problem in Mexico. It presents serious repercussions upon socio-economic, cultural, legal and health (neurologic and psychiatric) problems. We report a comparative study in humans and rats which demonstrate the embryotoxic and craneo encephalic teratologic effects in the children and brood of progenitors who have chronically inhaled thinner (in the case of pregnant women, before, at the beginning and throughout pregnancy). Inhaled thinner passes directly to the blood stream and crosses the placentary barrier freely reaching the embryo. It may cause craneal bone and partial or total encephalon agenesia, added to macro and microscopic lesions secondary to direct aggression to the neuroepithelial germ cells. Abortions and premature labor with weight and size underdeveloped products and placentary hemorrhages occur. Usually these die, but if they survive they show trascendental mental retardation, as well as neurologic and psychiatric sequels.

  9. Histone deacetylase inhibition regulates inflammation and enhances Tregs after allogeneic hematopoietic cell transplantation in humans

    NARCIS (Netherlands)

    Choi, S.W.; Gatza, E.; Hou, G.; Sun, Y; Whitfield, J.; Song, Y.; Oravecz-Wilson, K.; Tawara, I.; Dinarello, C.A.; Reddy, P.

    2015-01-01

    We examined immunological responses in patients receiving histone deacetylase (HDAC) inhibition (vorinostat) for graft-versus-host disease prophylaxis after allogeneic hematopoietic cell transplant. Vorinostat treatment increased histone acetylation in peripheral blood mononuclear cells (PBMCs) from

  10. Protective Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through Attenuating Oxidative Stress and the Wnt/β-Catenin Signaling Pathway in a Mouse Model of d-Galactose-induced Aging.

    Science.gov (United States)

    Li, Jing; Cai, Dachuan; Yao, Xin; Zhang, Yanyan; Chen, Linbo; Jing, Pengwei; Wang, Lu; Wang, Yaping

    2016-06-09

    Stem cell senescence is an important and current hypothesis accounting for organismal aging, especially the hematopoietic stem cell (HSC). Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng, which is a traditional Chinese medicine. This study explored the protective effect of ginsenoside Rg1 on Sca-1⁺ hematopoietic stem/progenitor cells (HSC/HPCs) in a mouse model of d-galactose-induced aging. The mimetic aging mouse model was induced by continuous injection of d-gal for 42 days, and the C57BL/6 mice were respectively treated with ginsenoside Rg1, Vitamin E or normal saline after 7 days of d-gal injection. Compared with those in the d-gal administration alone group, ginsenoside Rg1 protected Sca-1⁺ HSC/HPCs by decreasing SA-β-Gal and enhancing the colony forming unit-mixture (CFU-Mix), and adjusting oxidative stress indices like reactive oxygen species (ROS), total anti-oxidant (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA). In addition, ginsenoside Rg1 decreased β-catenin and c-Myc mRNA expression and enhanced the phosphorylation of GSK-3β. Moreover, ginsenoside Rg1 down-regulated advanced glycation end products (AGEs), 4-hydroxynonenal (4-HNE), phospho-histone H2A.X (r-H2A.X), 8-OHdG, p16(Ink4a), Rb, p21(Cip1/Waf1) and p53 in senescent Sca-1⁺ HSC/HPCs. Our findings indicated that ginsenoside Rg1 can improve the resistance of Sca-1⁺ HSC/HPCs in a mouse model of d-galactose-induced aging through the suppression of oxidative stress and excessive activation of the Wnt/β-catenin signaling pathway, and reduction of DNA damage response, p16(Ink4a)-Rb and p53-p21(Cip1/Waf1) signaling.

  11. Expression and function of β-adrenergic receptors in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Maeki, T.; Andersson, L.C.; Kontula, K.K.

    1992-01-01

    We investigated the expression and functional characteristics of β-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [ 125 I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of β-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimala or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of β-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32 P-labelled β 2 -adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their β-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage. (au)

  12. Clonal evolution of pre-leukemic hematopoietic stem cells precedes human acute myeloid leukemia.

    Science.gov (United States)

    Majeti, Ravindra

    2014-01-01

    Massively parallel DNA sequencing has uncovered recurrent mutations in many human cancers. In acute myeloid leukemia (AML), cancer genome/exome resequencing has identified numerous recurrently mutated genes with an average of 5 mutations in each case of de novo AML. In order for these multiple mutations to accumulate in a single lineage of cells, they are serially acquired in clones of self-renewing hematopoietic stem cells (HSC), termed pre-leukemic HSC. Isolation and characterization of pre-leukemic HSC have shown that their mutations are enriched in genes involved in regulating DNA methylation, chromatin modifications, and the cohesin complex. On the other hand, genes involved in regulating activated signaling are generally absent. Pre-leukemic HSC have been found to persist in clinical remission and may ultimately give rise to relapsed disease through the acquisition of novel mutations. Thus, pre-leukemic HSC may constitute a key cellular reservoir that must be eradicated for long-term cures. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Milestones of Hematopoietic Stem Cell Transplantation – From First Human Studies to Current Developments

    Science.gov (United States)

    Juric, Mateja Kralj; Ghimire, Sakhila; Ogonek, Justyna; Weissinger, Eva M.; Holler, Ernst; van Rood, Jon J.; Oudshoorn, Machteld; Dickinson, Anne; Greinix, Hildegard T.

    2016-01-01

    Since the early beginnings, in the 1950s, hematopoietic stem cell transplantation (HSCT) has become an established curative treatment for an increasing number of patients with life-threatening hematological, oncological, hereditary, and immunological diseases. This has become possible due to worldwide efforts of preclinical and clinical research focusing on issues of transplant immunology, reduction of transplant-associated morbidity, and mortality and efficient malignant disease eradication. The latter has been accomplished by potent graft-versus-leukemia (GvL) effector cells contained in the stem cell graft. Exciting insights into the genetics of the human leukocyte antigen (HLA) system allowed improved donor selection, including HLA-identical related and unrelated donors. Besides bone marrow, other stem cell sources like granulocyte-colony stimulating-mobilized peripheral blood stem cells and cord blood stem cells have been established in clinical routine. Use of reduced-intensity or non-myeloablative conditioning regimens has been associated with a marked reduction of non-hematological toxicities and eventually, non-relapse mortality allowing older patients and individuals with comorbidities to undergo allogeneic HSCT and to benefit from GvL or antitumor effects. Whereas in the early years, malignant disease eradication by high-dose chemotherapy or radiotherapy was the ultimate goal; nowadays, allogeneic HSCT has been recognized as cellular immunotherapy relying prominently on immune mechanisms and to a lesser extent on non-specific direct cellular toxicity. This chapter will summarize the key milestones of HSCT and introduce current developments. PMID:27881982

  14. Hematopoietic progenitor cell mobilization with "just-in-time" plerixafor approach is a cost-effective alternative to routine plerixafor use.

    Science.gov (United States)

    Veltri, Lauren; Cumpston, Aaron; Shillingburg, Alexandra; Wen, Sijin; Luo, Jin; Leadmon, Sonia; Watkins, Kathy; Craig, Michael; Hamadani, Mehdi; Kanate, Abraham S

    2015-12-01

    Hematopoietic cell mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor results in superior CD34+ cell yield compared with G-CSF alone in patients with myeloma and lymphoma. However, plerixafor-based approaches may be associated with high costs. Several institutions use a "just-in-time" plerixafor approach, in which plerixafor is only administered to patients likely to fail mobilization with G-CSF alone. Whether such an approach is cost-effective is unknown. We evaluated 136 patients with myeloma or lymphoma who underwent mobilization with 2 approaches of plerixafor utilization. Between January 2010 and October 2012, 76 patients uniformly received mobilization with G-CSF and plerixafor. Between November 2012 and June 2014, 60 patients were mobilized with plerixafor administered only to those patients likely to fail mobilization with G-CSF alone. The routine plerixafor group had a higher median peak peripheral blood CD34+ cell count (62 versus 29 cells/μL, P just-in-time" group, 40% (n = 24) completed adequate collection without plerixafor. There was no difference in mobilization failure rates. The mean plerixafor doses used was lower with "just-in-time" approach (1.3 versus 2.1, P = 0.0002). The mean estimated cost in the routine plerixafor group was higher (USD 27,513 versus USD 23,597, P = 0.01). Our analysis demonstrates that mobilization with a just-in-time plerixafor approach is a safe, effective, and cost-efficient strategy for HPC collection. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. High purity of human oligodendrocyte progenitor cells obtained from neural stem cells: suitable for clinical application.

    Science.gov (United States)

    Wang, Caiying; Luan, Zuo; Yang, Yinxiang; Wang, Zhaoyan; Wang, Qian; Lu, Yabin; Du, Qingan

    2015-01-30

    Recent studies have suggested that the transplantation of oligodendrocyte progenitor cells (OPCs) may be a promising potential therapeutic strategy for a broad range of diseases affecting myelin, such as multiple sclerosis, periventricular leukomalacia, and spinal cord injury. Clinical interest arose from the potential of human stem cells to be directed to OPCs for the clinical application of treating these diseases since large quantities of high quality OPCs are needed. However, to date, there have been precious few studies about OPC induction from human neural stem cells (NSCs). Here we successfully directed human fetal NSCs into highly pure OPCs using a cocktail of basic fibroblast growth factor, platelet-derived growth factor, and neurotrophic factor-3. These cells had typical morphology of OPCs, and 80-90% of them expressed specific OPC markers such as A2B5, O4, Sox10 and PDGF-αR. When exposed to differentiation medium, 90% of the cells differentiated into oligodendrocytes. The OPCs could be amplified in our culture medium and passaged at least 10 times. Compared to a recent published method, this protocol had much higher stability and repeatability, and OPCs could be obtained from NSCs from passage 5 to 38. It also obtained more highly pure OPCs (80-90%) via simpler and more convenient manipulation. This study provided an easy and efficient method to obtain large quantities of high-quality human OPCs to meet clinical demand. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. A curated transcriptome dataset collection to investigate the functional programming of human hematopoietic cells in early life.

    Science.gov (United States)

    Rahman, Mahbuba; Boughorbel, Sabri; Presnell, Scott; Quinn, Charlie; Cugno, Chiara; Chaussabel, Damien; Marr, Nico

    2016-01-01

    Compendia of large-scale datasets made available in public repositories provide an opportunity to identify and fill gaps in biomedical knowledge. But first, these data need to be made readily accessible to research investigators for interpretation. Here we make available a collection of transcriptome datasets to investigate the functional programming of human hematopoietic cells in early life. Thirty two datasets were retrieved from the NCBI Gene Expression Omnibus (GEO) and loaded in a custom web application called the Gene Expression Browser (GXB), which was designed for interactive query and visualization of integrated large-scale data. Quality control checks were performed. Multiple sample groupings and gene rank lists were created allowing users to reveal age-related differences in transcriptome profiles, changes in the gene expression of neonatal hematopoietic cells to a variety of immune stimulators and modulators, as well as during cell differentiation. Available demographic, clinical, and cell phenotypic information can be overlaid with the gene expression data and used to sort samples. Web links to customized graphical views can be generated and subsequently inserted in manuscripts to report novel findings. GXB also enables browsing of a single gene across projects, thereby providing new perspectives on age- and developmental stage-specific expression of a given gene across the human hematopoietic system. This dataset collection is available at: http://developmentalimmunology.gxbsidra.org/dm3/geneBrowser/list.

  17. HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells

    Directory of Open Access Journals (Sweden)

    Chang Li

    2018-06-01

    Full Text Available We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR. The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ null (NSG mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35++ vector that expressed two anti-CRISPR (Acr peptides (AcrII4 and AcrII2 capable of binding to the CRISPR/Cas9 complex (HDAd-Acr. CD34+ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34+ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.

  18. Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality.

    Science.gov (United States)

    Dijkstra-Tiekstra, Margriet J; Setroikromo, Airies C; Kraan, Marcha; Gkoumassi, Effimia; de Wildt-Eggen, Janny

    2014-12-01

    Adding dimethyl sulfoxide (DMSO) to hematopoietic progenitor cells (HPCs) causes an exothermic reaction, potentially affecting their viability. The freezing method might also influence this. The aim was to investigate the effect of 1) precooling of DMSO and plasma (D/P) and white blood cell (WBC)-enriched product, 2) DMSO concentration of D/P, 3) freezing program, and 4) storage method on WBC quality. WBC-enriched product without CD34+ cells was used instead of HPCs. This was divided into six or eight portions. D/P (20 or 50%; precooled or room temperature [RT]) was added to the WBC-enriched product (precooled or RT), resulting in 10% DMSO, while monitoring temperature. The product was frozen using controlled-rate freezing ("fast-rate" or "slow-rate") and placed in vapor-phase or liquid nitrogen. After thawing, WBC recovery and viability were determined. Temperature increased most for precooled D/P to precooled WBC-enriched product, without influence of 20 or 50% D/P, but remained for all variations below 30°C. WBC recovery for both freezing programs was more than 95%. Recovery of WBC viability was higher for slow-rate freezing compared to fast-rate freezing (74% vs. 61%; p liquid nitrogen was marginal. Based on these results, precooling is not necessary. Fifty percent D/P is preferred over 20% D/P. Slow-rate freezing is preferred over fast-rate freezing. For safety reasons storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. Additional testing using real HPCs might be necessary. © 2014 AABB.

  19. Evolution of the clonogenic potential of human epidermal stem/progenitor cells with age

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    Zobiri O

    2012-02-01

    Full Text Available Olivia Zobiri, Nathalie Deshayes, Michelle Rathman-JosserandDepartment of Biological Research, L'Oréal Advanced Research, Clichy Cedex, FranceAbstract: A number of clinical observations have indicated that the regenerative potential and overall function of the epidermis is modified with age. The epidermis becomes thinner, repairs itself less efficiently after wounding, and presents modified barrier function recovery. In addition, the dermal papillae flatten out with increasing age, suggesting a modification in the interaction between epidermal and dermal compartments. As the epidermal regenerative capacity is dependent upon stem and progenitor cell function, it is naturally of interest to identify and understand age-related changes in these particular keratinocyte populations. Previous studies have indicated that the number of stem cells does not decrease with age in mouse models but little solid evidence is currently available concerning human skin. The objective of this study was to evaluate the clonogenic potential of keratinocyte populations isolated from the epidermis of over 50 human donors ranging from 18 to 71 years old. The data indicate that the number of epidermal cells presenting high regenerative potential does not dramatically decline with age in human skin. The authors believe that changes in the microenvironment controlling epidermal basal cell activity are more likely to explain the differences in epidermal function observed with increasing age.Keywords: skin, epidermal stem cells, aging, colony-forming efficiency test

  20. Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.

    Science.gov (United States)

    Korytnikov, Roman; Nostro, Maria Cristina

    2016-05-15

    Generation of pancreatic β-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate β-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors

    DEFF Research Database (Denmark)

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have...... analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors....

  2. Proof of region-specific multipotent progenitors in human breast epithelia

    DEFF Research Database (Denmark)

    Fridriksdottir, Agla J; Villadsen, René; Morsing, Mikkel

    2017-01-01

    in luminal progenitors to interrogate the differentiation repertoire of candidate stem cells in TDLUs. We show that stem-like activity in serial passage culture and in vivo breast morphogenesis relies on the preservation of a myoepithelial phenotype. By enrichment for region-specific progenitors, we identify...

  3. Gene expression profiling identifies HOXB4 as a direct downstream target of GATA-2 in human CD34+ hematopoietic cells.

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    Tohru Fujiwara

    Full Text Available Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10 and idiopathic thrombocytopenic purpura (n = 13, and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01. Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.

  4. Mechanism of recombinant human bone morphogenetic protein-2 in repairing hematopoietic injury in mice exposed to γ-rays

    International Nuclear Information System (INIS)

    Liu Shuibing; Hu Peizhen; Hou Ying; Li Xubo; Tian Qiong; Shi Mei

    2009-01-01

    Objective: To investigate the mechanism of recombinant human bone morphogenetic protein-2 (rhBMP-2) in repairing hematopoietic injury in mice irradiated with γ-ray. To prepare SRY gene probe and study the effect of rhBMP-2 in repairing hematopoietic injury in mice by in situ hybridization. Methods: Twenty-two BALB/c female mice were randomly divided into the irradiated group and BMP treated group, respectively. Bone marrow cells of normal male mice were transplanted into 22 female mice post-irradiation to 8.5 Gy of 60 Co γ rays. The left femurs of the survived female mice were re-irradiated with 9 Gy 14 days later. Mice in BMP treated group were given rhBMP-2 20 mg/kg while those in control group were treated with 0.9% saline by intraperitoneal injection every day for 6 days. These mice were killed 14 days later and paraffin sections of femurs were made. The SRY gene was detected with in situ hybridization. Results: There were more positive blots in the left femurs of the mice in irradiated group than those in BMP treated group (T=155.0, P 0.05). The number of positive blots in the left femurs of the mice in BMPtreated group was significantly less than those in the right femurs of the mice in two groups (T=155.0, 55.0, P<0.05). Conclusions: No donor cell of male mice was detected in the left femurs of BMP treated group, suggesting that rhBMP-2 promoted the restoration of residuary bone marrow cells. Thus, rhBMP-2 promotes the proliferation or differentiation of residuary mesenchymal stem cells, improves hematopoietic microenvironment and accelerates the hematopoietic restoration. (authors)

  5. Focal Transplantation of Human iPSC-Derived Glial-Rich Neural Progenitors Improves Lifespan of ALS Mice

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    Takayuki Kondo

    2014-08-01

    Full Text Available Transplantation of glial-rich neural progenitors has been demonstrated to attenuate motor neuron degeneration and disease progression in rodent models of mutant superoxide dismutase 1 (SOD1-mediated amyotrophic lateral sclerosis (ALS. However, translation of these results into a clinical setting requires a renewable human cell source. Here, we derived glial-rich neural progenitors from human iPSCs and transplanted them into the lumbar spinal cord of ALS mouse models. The transplanted cells differentiated into astrocytes, and the treated mouse group showed prolonged lifespan. Our data suggest a potential therapeutic mechanism via activation of AKT signal. The results demonstrated the efficacy of cell therapy for ALS by the use of human iPSCs as cell source.

  6. Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells

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    Renata Santos

    2017-06-01

    Full Text Available Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.

  7. Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Santos, Renata; Vadodaria, Krishna C; Jaeger, Baptiste N; Mei, Arianna; Lefcochilos-Fogelquist, Sabrina; Mendes, Ana P D; Erikson, Galina; Shokhirev, Maxim; Randolph-Moore, Lynne; Fredlender, Callie; Dave, Sonia; Oefner, Ruth; Fitzpatrick, Conor; Pena, Monique; Barron, Jerika J; Ku, Manching; Denli, Ahmet M; Kerman, Bilal E; Charnay, Patrick; Kelsoe, John R; Marchetto, Maria C; Gage, Fred H

    2017-06-06

    Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Human Traumatic Brain Injury Results in Oligodendrocyte Death and Increases the Number of Oligodendrocyte Progenitor Cells.

    Science.gov (United States)

    Flygt, Johanna; Gumucio, Astrid; Ingelsson, Martin; Skoglund, Karin; Holm, Jonatan; Alafuzoff, Irina; Marklund, Niklas

    2016-06-01

    Oligodendrocyte (OL) death may contribute to white matter pathology, a common cause of network dysfunction and persistent cognitive problems in patients with traumatic brain injury (TBI). Oligodendrocyte progenitor cells (OPCs) persist throughout the adult CNS and may replace dead OLs. OL death and OPCs were analyzed by immunohistochemistry of human brain tissue samples, surgically removed due to life-threatening contusions and/or focal brain swelling at 60.6 ± 75 hours (range 4-192 hours) postinjury in 10 severe TBI patients (age 51.7 ± 18.5 years). Control brain tissue was obtained postmortem from 5 age-matched patients without CNS disorders. TUNEL and CC1 co-labeling was used to analyze apoptotic OLs, which were increased in injured brain tissue (p The OPC markers Olig2, A2B5, NG2, and PDGFR-α were used. In contrast to the number of single-labeled Olig2, A2B5, NG2, and PDGFR-α-positive cells, numbers of Olig2 and A2B5 co-labeled cells were increased in TBI samples (p human TBI results in OL death and increases in OPCs postinjury, which may influence white matter function following TBI. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

  9. ERK-dependent and -independent pathways trigger human neural progenitor cell migration

    International Nuclear Information System (INIS)

    Moors, Michaela; Cline, Jason E.; Abel, Josef; Fritsche, Ellen

    2007-01-01

    Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways

  10. Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.

    Science.gov (United States)

    Matsuoka, Yoshikazu; Nakatsuka, Ryusuke; Sumide, Keisuke; Kawamura, Hiroshi; Takahashi, Masaya; Fujioka, Tatsuya; Uemura, Yasushi; Asano, Hiroaki; Sasaki, Yutaka; Inoue, Masami; Ogawa, Hiroyasu; Takahashi, Takayuki; Hino, Masayuki; Sonoda, Yoshiaki

    2015-05-01

    Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. © 2014 AlphaMed Press.

  11. The Polycomb Group Protein L3MBTL1 Represses a SMAD5-Mediated Hematopoietic Transcriptional Program in Human Pluripotent Stem Cells

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    Fabiana Perna

    2015-04-01

    Full Text Available Epigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia.

  12. Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions

    Directory of Open Access Journals (Sweden)

    Agnete Kirkeby

    2012-06-01

    Full Text Available To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.

  13. Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment.

    Science.gov (United States)

    Perlin, Julie R; Sporrij, Audrey; Zon, Leonard I

    2017-08-01

    Cells of the hematopoietic system undergo rapid turnover. Each day, humans require the production of about one hundred billion new blood cells for proper function. Hematopoietic stem cells (HSCs) are rare cells that reside in specialized niches and are required throughout life to produce specific progenitor cells that will replenish all blood lineages. There is, however, an incomplete understanding of the molecular and physical properties that regulate HSC migration, homing, engraftment, and maintenance in the niche. Endothelial cells (ECs) are intimately associated with HSCs throughout the life of the stem cell, from the specialized endothelial cells that give rise to HSCs, to the perivascular niche endothelial cells that regulate HSC homeostasis. Recent studies have dissected the unique molecular and physical properties of the endothelial cells in the HSC vascular niche and their role in HSC biology, which may be manipulated to enhance hematopoietic stem cell transplantation therapies.

  14. Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

    Science.gov (United States)

    Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

    2017-05-01

    Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

  15. Viral infection of human progenitor and liver-derived cells encapsulated in three-dimensional PEG-based hydrogel

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Nam-Joon; Elazar, Menashe; Xiong, Anming; Glenn, Jeffrey S [Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CCSR Building Room 3115A, 269 Campus Drive, Stanford, CA 94305 (United States); Lee, Wonjae [Mechanical Engineering, Stanford University, Stanford, CA 94305 (United States); Chiao, Eric; Baker, Julie [Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 (United States); Frank, Curtis W, E-mail: jeffrey.glenn@stanford.ed, E-mail: curt.frank@stanford.ed [Department of Chemical Engineering, Stanford University, Stanford, CA 94305 (United States)

    2009-02-15

    We have studied the encapsulation of human progenitor cells into 3D PEG hydrogels. Replication-incompetent lentivirus promoter reporter vectors were found to efficiently detect the in vivo expression of human hepatic genes in hydrogel-encapsulated liver progenitor cells. Similarly, hydrogel-encapsulated cells could be efficiently infected with hepatitis C virus, and progeny infectious virus could be recovered from the media supernatants of the hydrogels. Provocatively, the diameters of these virus particles range from {approx}50 to 100 nm, while the calculated mesh size of the 8 k hydrogel is 44.6 +- 1.7 A. To reconcile how viral particles can penetrate the hydrogels to infect the encapsulated cells, we propose that microfractures/defects of the hydrogel result in a functional pore size of up to 20 fold greater than predicted by theoretical mesh calculations. These results suggest a new model of hydrogel structure, and have exciting implications for tissue engineering and hepatitis virus studies. (communication)

  16. Viral infection of human progenitor and liver-derived cells encapsulated in three-dimensional PEG-based hydrogel

    International Nuclear Information System (INIS)

    Cho, Nam-Joon; Elazar, Menashe; Xiong, Anming; Glenn, Jeffrey S; Lee, Wonjae; Chiao, Eric; Baker, Julie; Frank, Curtis W

    2009-01-01

    We have studied the encapsulation of human progenitor cells into 3D PEG hydrogels. Replication-incompetent lentivirus promoter reporter vectors were found to efficiently detect the in vivo expression of human hepatic genes in hydrogel-encapsulated liver progenitor cells. Similarly, hydrogel-encapsulated cells could be efficiently infected with hepatitis C virus, and progeny infectious virus could be recovered from the media supernatants of the hydrogels. Provocatively, the diameters of these virus particles range from ∼50 to 100 nm, while the calculated mesh size of the 8 k hydrogel is 44.6 ± 1.7 A. To reconcile how viral particles can penetrate the hydrogels to infect the encapsulated cells, we propose that microfractures/defects of the hydrogel result in a functional pore size of up to 20 fold greater than predicted by theoretical mesh calculations. These results suggest a new model of hydrogel structure, and have exciting implications for tissue engineering and hepatitis virus studies. (communication)

  17. Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Okolicsanyi, Rachel K; Oikari, Lotta E; Yu, Chieh; Griffiths, Lyn R; Haupt, Larisa M

    2018-01-01

    Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation. Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression. Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs. Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis.

  18. Lesion-induced increase in survival and migration of human neural progenitor cells releasing GDNF

    Science.gov (United States)

    Behrstock, Soshana; Ebert, Allison D.; Klein, Sandra; Schmitt, Melanie; Moore, Jeannette M.; Svendsen, Clive N.

    2009-01-01

    The use of human neural progenitor cells (hNPC) has been proposed to provide neuronal replacement or astrocytes delivering growth factors for brain disorders such as Parkinson’s and Huntington’s disease. Success in such studies likely requires migration from the site of transplantation and integration into host tissue in the face of ongoing damage. In the current study, hNPC modified to release glial cell line derived neurotrophic factor (hNPCGDNF) were transplanted into either intact or lesioned animals. GDNF release itself had no effect on the survival, migration or differentiation of the cells. The most robust migration and survival was found using a direct lesion of striatum (Huntington’s model) with indirect lesions of the dopamine system (Parkinson’s model) or intact animals showing successively less migration and survival. No lesion affected differentiation patterns. We conclude that the type of brain injury dictates migration and integration of hNPC which has important consequences when considering transplantation of these cells as a therapy for neurodegenerative diseases. PMID:19044202

  19. Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

    Science.gov (United States)

    Yan, Yuanwei; Song, Liqing; Tsai, Ang-Chen; Ma, Teng; Li, Yan

    2016-01-01

    Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

  20. A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Laura Giusti

    2017-01-01

    Full Text Available Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs, bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG, on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation.

  1. Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

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    Vincent, Per Henrik; Benedikz, Eirikur; Uhlén, Per; Hovatta, Outi; Sundström, Erik

    2017-06-15

    Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133 + /CD24 lo ), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

  2. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

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    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  3. Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation

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    Meyer Anne K

    2009-06-01

    Full Text Available Abstract Despite a comprehensive mapping of the Parkinson's disease (PD-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2 in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

  4. Butein Inhibits Angiogenesis of Human Endothelial Progenitor Cells via the Translation Dependent Signaling Pathway

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    Ching-Hu Chung

    2013-01-01

    Full Text Available Compelling evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs can contribute to postnatal neovascularization and tumor angiogenesis. EPCs have been shown to play a “catalytic” role in metastatic progression by mediating the angiogenic switch. Understanding the pharmacological functions and molecular targets of natural products is critical for drug development. Butein, a natural chalcone derivative, has been reported to exert potent anticancer activity. However, the antiangiogenic activity of butein has not been addressed. In this study, we found that butein inhibited serum- and vascular endothelial growth factor- (VEGF- induced cell proliferation, migration, and tube formation of human EPCs in a concentration dependent manner without cytotoxic effect. Furthermore, butein markedly abrogated VEGF-induced vessels sprouting from aortic rings and suppressed microvessel formation in the Matrigel implant assay in vivo. In addition, butein concentration-dependently repressed the phosphorylation of Akt, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in EPCs. Taken together, our results demonstrate for the first time that butein exhibits the antiangiogenic effect both in vitro and in vivo by targeting the translational machinery. Butein is a promising angiogenesis inhibitor with the potential for treatment of cancer and other angiogenesis-related diseases.

  5. The effects of (-)-epigallocatechin-3-gallate on the proliferation and differentiation of human megakaryocytic progenitor cells

    International Nuclear Information System (INIS)

    Monzen, Satoru; Takahashi, Kenji; Abe, Yoshinao; Kashiwakura, Ikuo; Mori, Takao; Inanami, Osamu; Kuwabara, Mikinori

    2006-01-01

    Epigallocatechin-3-gallate (EGCg) has been widely recognized as a powerful antioxidant and free radical scavenger. The effects of EGCg on the proliferation and differentiation of X-irradiated megakaryocytic progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) using CD34 + cells prepared from human placental and umbilical cord blood have been shown. In the absence of exogenous thrombopoietin (TPO), no colonies are observed in cultures containing or lacking EGCg (1 nM-100 μM). In the presence of TPO, in contrast, EGCg significantly promotes CFU-Meg-derived colony formations within the 10-100 nM range. A 1.5-fold increase in the total number of CFU-Meg has been counted compared with the control. These favorable effects of EGCg are also observed in the culture of CD34 + cells before and after X irradiation with 2 Gy. Moreover, in order to investigate the function of EGCg promoting megakaryocyto-poiesis and thrombopoiesis in ex vivo cultures, both non-irradiated and X-irradiated CD34 + cells are grown in liquid cultures supplemented with TPO. In both cultures, EGCg increases the total number of cells and megakaryocytes. It has been suggested that the favorable effects of EGCg reduce the risk factor from radiation damage in megakaryocytopoiesis. (author)

  6. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

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    Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

    2006-07-01

    Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

  7. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

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    Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

    2013-07-19

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

  8. Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

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    Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

    2009-11-01

    Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

  9. Incidence of human herpes virus-6 and human cytomegalovirus infections in donated bone marrow and umbilical cord blood hematopoietic stem cells

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    Behzad-Behbahani A

    2008-01-01

    Full Text Available This study examined the incidence of human herpes virus-6 (HHV-6 and human cytomegalovirus (HCMV infections that are potentially transmitted to haematopoietic stem cells (HSC transplant recipients via bone marrow (BM or umbilical cord blood (UCB. Bone marrow progenitor cells were collected from 30 allogenic BM donors. UCB HSC were collected from 34 subjects. The extracted DNA was then processed using nested polymerase chain reaction (nPCR technique. HCMV and HHV-6 serological status were determined by enzyme immunoassay (EIA. Nested PCR identified HCMV in 22 (73% of 30 samples of BM progenitor cells but in only eight (23.5% of 34 samples of UBC HSC ( P = 0.001. HHV-6 DNA was detected in 11 (36.6% of 30 BM progenitor cells and in only one (2.9% of 34 UBC cells ( P = 0.002. Both HHV-6 and HCMV infections were determined in nine (26.5% of 34 bone marrow samples. The results indicate that, the risk of HCMV and HHV-6 via BM progenitor cells is higher than transmission by UCB cells ( P= 0.04.

  10. Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.

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    Benedetti, Sara; Uno, Narumi; Hoshiya, Hidetoshi; Ragazzi, Martina; Ferrari, Giulia; Kazuki, Yasuhiro; Moyle, Louise Anne; Tonlorenzi, Rossana; Lombardo, Angelo; Chaouch, Soraya; Mouly, Vincent; Moore, Marc; Popplewell, Linda; Kazuki, Kanako; Katoh, Motonobu; Naldini, Luigi; Dickson, George; Messina, Graziella; Oshimura, Mitsuo; Cossu, Giulio; Tedesco, Francesco Saverio

    2018-02-01

    Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  11. Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination.

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    Alireza Pouya

    Full Text Available BACKGROUND: This study aims to differentiate human induced pluripotent stem cells (hiPSCs into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats. METHODOLOGY/PRINCIPAL FINDINGS: We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials. CONCLUSIONS/SIGNIFICANCE: These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.

  12. Alternative splicing events identified in human embryonic stem cells and neural progenitors.

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    Gene W Yeo

    2007-10-01

    Full Text Available Human embryonic stem cells (hESCs and neural progenitor (NP cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders. While much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hESC to NP, very little work has been performed to understand post-transcriptional changes during neuronal differentiation. Alternative RNA splicing (AS, a major form of post-transcriptional gene regulation, is important in mammalian development and neuronal function. Human ESC, hESC-derived NP, and human central nervous system stem cells were compared using Affymetrix exon arrays. We introduced an outlier detection approach, REAP (Regression-based Exon Array Protocol, to identify 1,737 internal exons that are predicted to undergo AS in NP compared to hESC. Experimental validation of REAP-predicted AS events indicated a threshold-dependent sensitivity ranging from 56% to 69%, at a specificity of 77% to 96%. REAP predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved AS events. Our results also reveal that focusing on differentially expressed genes between hESC and NP will overlook 14% of potential AS genes. In addition, we found that REAP predictions are enri