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Sample records for human erythroid progenitor

  1. Gene expression profiling of human erythroid progenitors by micro-serial analysis of gene expression.

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    Fujishima, Naohito; Hirokawa, Makoto; Aiba, Namiko; Ichikawa, Yoshikazu; Fujishima, Masumi; Komatsuda, Atsushi; Suzuki, Yoshiko; Kawabata, Yoshinari; Miura, Ikuo; Sawada, Ken-ichi

    2004-10-01

    We compared the expression profiles of highly purified human CD34+ cells and erythroid progenitor cells by micro-serial analysis of gene expression (microSAGE). Human CD34+ cells were purified from granulocyte colony-stimulating factor-mobilized blood stem cells, and erythroid progenitors were obtained by cultivating these cells in the presence of stem cell factor, interleukin 3, and erythropoietin. Our 10,202 SAGE tags allowed us to identify 1354 different transcripts appearing more than once. Erythroid progenitor cells showed increased expression of LRBA, EEF1A1, HSPCA, PILRB, RANBP1, NACA, and SMURF. Overexpression of HSPCA was confirmed by real-time polymerase chain reaction analysis. MicroSAGE revealed an unexpected preferential expression of several genes in erythroid progenitor cells in addition to the known functional genes, including hemoglobins. Our results provide reference data for future studies of gene expression in various hematopoietic disorders, including myelodysplastic syndrome and leukemia.

  2. Determination of the hemoglobin F program in human progenitor-derived erythroid cells.

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    Friedman, A.D.; Linch, D. C.; Miller, B.; Lipton, J M; Javid, J; Nathan, D G

    1985-01-01

    The absolute adult and fetal hemoglobin (HbF) contents of the erythroid cells derived from the differentiation of normal human and simian erythroid progenitors and of the peripheral blood erythroid burst-forming units (BFU-E) of patients with nondeletion hemoglobinopathies have been measured with a sensitive radioligand immunoassay. The HbF content varied between 0.13 and 2.96 pg/cell, representing between 0.7% and 19.6% of the total hemoglobin with a mean value of 7.0%. The absolute content ...

  3. Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.

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    Li, Jie; Hale, John; Bhagia, Pooja; Xue, Fumin; Chen, Lixiang; Jaffray, Julie; Yan, Hongxia; Lane, Joseph; Gallagher, Patrick G; Mohandas, Narla; Liu, Jing; An, Xiuli

    2014-12-04

    Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) and CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity of ∼90%. The BFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) cells required stem cell factor and erythropoietin, while the CFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) cells required only erythropoietin. Bioinformatic analysis of the RNA-sequencing data revealed unique transcriptomes at each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow, cord blood, and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematologic disease. Our data provides an important resource for future studies of human erythropoiesis.

  4. Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TU 67).

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    Herrmann, F; Griffin, J D; Sabbath, K D; Oster, W; Wernet, P; Mertelsmann, R

    1988-04-01

    Anti-TU 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TU 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TU 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TU 67 antibody.

  5. Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

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    Wang, X S; Yoder, M C; Zhou, S Z; Srivastava, A

    1995-01-01

    The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies. Images Fig. 2 Fig. 3 Fig. 4 PMID:8618912

  6. No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

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    Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

    2017-07-20

    Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. A method for enriching myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from human cord blood by accessory cell depletion.

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    Dowton, L A; Ma, D D

    1992-10-01

    Human cord blood provides a convenient alternative to bone marrow as a rich source of hemopoietic progenitor cells. This study reports a simple means for enriching a cord blood progenitor cell population by accessory cell depletion. Two methods of monocyte depletion were tested. A Cytodex 3 microcarrier system using collagen coated dextran beads was compared to the more commonly used method of plastic plate adhesion. The method of plastic plate adhesion gave a significantly higher cell recovery. T cell depletion using a recently characterized rat monoclonal antibody which fixes human complement was also investigated. A combined method of monocyte depletion by plate adhesion and T cell depletion resulted in the removal of > 96% of monocytes and > 98% of T cells. This led to a significant enrichment of myeloid (CFU-GM) and erythroid (BFU-E) colony growth. Such enriched progenitor cell populations provide a useful starting population for any study on hemopoiesis.

  8. THE EFFECTS OF IL-1 AND IL-4 ON THE EPO-INDEPENDENT ERYTHROID PROGENITOR IN POLYCYTHEMIA-VERA

    NARCIS (Netherlands)

    DEWOLF, JTM; HENDRIKS, DW; ESSELINK, MT; HALIE, MR; VELLENGA, E

    1994-01-01

    Human recombinant interleukin-1 (IL-1) was studied for its effects on the erythroid progenitors from normal subjects and from patients with polycythaemia vera (PV). No supportive effect of IL-1 was noticed on the normal, erythropoietin (Epo) dependent, erythroid burst-forming unit (BFU-E) using peri

  9. Loss of Forkhead box M1 promotes erythropoiesis through increased proliferation of erythroid progenitors.

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    Youn, Minyoung; Wang, Nan; LaVasseur, Corinne; Bibikova, Elena; Kam, Sharon; Glader, Bertil; Sakamoto, Kathleen M; Narla, Anupama

    2017-05-01

    Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34(+) cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis. Copyright© Ferrata Storti Foundation.

  10. PPAR-α and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal.

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    Lee, Hsiang-Ying; Gao, Xiaofei; Barrasa, M Inmaculada; Li, Hu; Elmes, Russell R; Peters, Luanne L; Lodish, Harvey F

    2015-06-25

    Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists, additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid

  11. PPARα and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal

    Science.gov (United States)

    Lee, Hsiang-Ying; Gao, Xiaofei; Barrasa, M. Inmaculada; Li, Hu; Elmes, Russell R.; Peters, Luanne L.; Lodish, Harvey F.

    2015-01-01

    Summary Many acute and chronic anemias, including hemolysis, sepsis, and genetic bone marrow failure diseases such as Diamond-Blackfan Anemia (DBA), are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production 1,2,3–5,6,7,8,9. Treatment of these anemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently we showed that glucocorticoids specifically stimulate self-renewal of the early erythroid progenitor, the burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells 10,11. Here we demonstrate that activation of the peroxisome proliferator-activated receptor alpha (PPARα) by PPARα agonists, GW7647 and fenofibrate, synergizes with glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures both of mouse fetal liver BFU-Es and of mobilized human adult CD34+ peripheral blood progenitors, the latter employing a new and effective culture system that generates normal enucleated reticulocytes. While PPARα−/− mice show no hematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPARα agonists facilitate recovery of wild-type mice, but not PPARα−/− mice, from PHZ-induced acute hemolytic anemia. We also showed that PPARα alleviates anemia in a mouse model of chronic anemia. Finally, both in control and corticosteroid-treated BFU-E cells PPARα co-occupies many chromatin sites with GR; when activated by PPARα agonists, additional PPARα is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPARα agonists in stimulating self

  12. Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells

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    Koury, M.J.; Bondurant, M.C. (Vanderbilt Univ. Medical Center, Nashville, TN (USA) Veterans Administration Medical Center, Nashville, TN (USA))

    1990-04-20

    The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro with ({sup 3}H) thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis. Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus, apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA breakdown and preventing apoptosis in erythroid progenitor cells.

  13. Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice.

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    Lin, Kou-Ray; Yang-Yen, Hsin-Fang; Lien, Huang-Wei; Liao, Wei-Hao; Huang, Chang-Jen; Lin, Liang-In; Li, Chung-Leung; Yen, Jeffrey Jong-Young

    2016-08-23

    Tribbles homolog 2 (Trib2) is a member of Tribbles protein pseudokinases and involves in apoptosis, autoimmunity, cancer, leukemia and erythropoiesis, however, the physiological function of Trib2 in hematopoietic system remains to be elucidated. Here, we report that Trib2 knockout (KO) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis. Endogenous Trib2 mRNA is expressed in early hematopoietic progenitors, erythroid precursors, and lymphoid lineages, but not in mature RBCs, myeloid progenitors and granulocytes. Consistently, flow cytometric analysis and in vitro colony forming assay revealed that deletion of Trib2 mainly affected erythroid lineage development, and had no effect on either granulocyte or megakaryocyte lineages in bone marrow. Furthermore, a genetic approach using double knockout of Trib2 and C/ebpα genes in mice suggested that Trib2 promotes erythropoiesis independent of C/ebpα proteins in vivo. Finally, ectopic expression of human Trib2 in zebrafish embryos resulted in increased expression of erythropoiesis-related genes and of hemoglobin. Taking all data together, our results suggest that Trib2 positively promotes early erythrocyte differentiation and is essential for tolerance to hemolysis.

  14. Cooperative Effect of Erythropoietin and TGF-β Inhibition on Erythroid Development in Human Pluripotent Stem Cells.

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    Xie, Yinliang; Bai, Hao; Liu, Yanfeng; Hoyle, Dixie L; Cheng, Tao; Wang, Zack Z

    2015-12-01

    Patient-specific human induced-pluripotent stem cells (hiPSCs) represent important cell sources to treat patients with acquired blood disorders. To realize the therapeutic potential of hiPSCs, it is crucial to understand signals that direct hiPSC differentiation to a hematopoietic lineage fate. Our previous study demonstrated that CD34(+)CD31(+) cells derived from human pluripotent stem cells (hPSCs) contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic cells and endothelial cells. Here, we established a serum-free and feeder-free system to induce the differentiation of hPSC-derived CD34(+)CD31(+) progenitor cells to erythroid cells. We show that extracellular matrix (ECM) proteins promote the differentiation of CD34(+)CD31(+) progenitor cells into CD235a(+) erythroid cells through CD41(+)CD235a(+) megakaryocyte-erythroid progenitors (MEP). Erythropoietin (EPO) is a predominant factor for CD34(+)CD31(+) progenitor differentiation to erythroid cells, whereas transforming growth factor beta (TGF-β) inhibits the development of CD34(+)CD31(+) progenitor cells. Apoptosis of progenitor cells is induced by TGF-β in early erythroid differentiation. Suppression of TGF-β signaling by SB431542 at early stage of CD34(+)CD31(+) progenitor differentiation induces the erythroid cell generation. Together, these findings suggest that TGF-β suppression and EPO stimulation promote erythropoiesis of CD34(+)CD31(+) progenitor cells derived from hPSCs.

  15. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation.

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    Anuradha Tarafdar

    Full Text Available The generation of hematopoietic stem cells (HSCs during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

  16. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation.

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    Tarafdar, Anuradha; Dobbin, Edwina; Corrigan, Pamela; Freeburn, Robin; Wheadon, Helen

    2013-01-01

    The generation of hematopoietic stem cells (HSCs) during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

  17. Transcriptome dynamics during human erythroid differentiation and development.

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    Yang, Yadong; Wang, Hai; Chang, Kai-Hsin; Qu, Hongzhu; Zhang, Zhaojun; Xiong, Qian; Qi, Heyuan; Cui, Peng; Lin, Qiang; Ruan, Xiuyan; Yang, Yaran; Li, Yajuan; Shu, Chang; Li, Quanzhen; Wakeland, Edward K; Yan, Jiangwei; Hu, Songnian; Fang, Xiangdong

    2013-01-01

    To explore the mechanisms controlling erythroid differentiation and development, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human embryonic stem cells (HESCs) into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. HESCs and erythroid cells at three developmental stages: ESER (embryonic), FLER (fetal), and PBER (adult) were analyzed. Our findings revealed that the number of expressed genes decreased during differentiation, whereas the total expression intensity increased. At each of the three transitions (HESCs-ESERs, ESERs-FLERs, and FLERs-PBERs), many differentially expressed genes were observed, which were involved in maintaining pluripotency, early erythroid specification, rapid cell growth, and cell-cell adhesion and interaction. We also discovered dynamic networks and their central nodes in each transition. Our study provides a fundamental basis for further investigation of erythroid differentiation and development, and has implications in using ESERs for transfusion product in clinical settings.

  18. The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro

    NARCIS (Netherlands)

    W. Zauner; G. Mellitzer; P. Steinlein (Peter); G. Fritsch; K. Huber; H. Beug (Hartmut); B. Löwenberg (Bob); M.M. von Lindern (Marieke)

    1999-01-01

    textabstractAlthough erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormone

  19. ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors.

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    Zhang, Lingbo; Prak, Lina; Rayon-Estrada, Violeta; Thiru, Prathapan; Flygare, Johan; Lim, Bing; Lodish, Harvey F

    2013-07-04

    Stem cells and progenitors in many lineages undergo self-renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red blood cell formation by promoting self-renewal of early burst-forming unit-erythroid (BFU-E) progenitors. Here we show that the RNA-binding protein ZFP36L2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU-Es and is required for BFU-E self-renewal. ZFP36L2 is normally downregulated during erythroid differentiation from the BFU-E stage, but its expression is maintained by all tested GR agonists that stimulate BFU-E self-renewal, and the GR binds to several potential enhancer regions of ZFP36L2. Knockdown of ZFP36L2 in cultured BFU-E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU-E self-renewal, and knockdown of ZFP36L2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. ZFP36L2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. ZFP36L2 therefore functions as part of a molecular switch promoting BFU-E self-renewal and a subsequent increase in the total numbers of colony-forming unit-erythroid (CFU-E) progenitors and erythroid cells that are generated.

  20. Identification of biologically relevant enhancers in human erythroid cells.

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    Su, Mack Y; Steiner, Laurie A; Bogardus, Hannah; Mishra, Tejaswini; Schulz, Vincent P; Hardison, Ross C; Gallagher, Patrick G

    2013-03-22

    Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.

  1. TGF-β inhibitors stimulate red blood cell production by enhancing self-renewal of BFU-E erythroid progenitors.

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    Gao, Xiaofei; Lee, Hsiang-Ying; da Rocha, Edroaldo Lummertz; Zhang, Cheng; Lu, Yi-Fen; Li, Dandan; Feng, Yuxiong; Ezike, Jideofor; Elmes, Russell R; Barrasa, M Inmaculada; Cahan, Patrick; Li, Hu; Daley, George Q; Lodish, Harvey F

    2016-12-08

    Burst-forming unit erythroid progenitors (BFU-Es) are so named based on their ability to generate in methylcellulose culture large colonies of erythroid cells that consist of "bursts" of smaller erythroid colonies derived from the later colony-forming unit erythroid progenitor erythropoietin (Epo)-dependent progenitors. "Early" BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do "late" BFU-Es forming small colonies, but the mechanism underlying this heterogeneity remains unknown. We show that the type III transforming growth factor β (TGF-β) receptor (TβRIII) is a marker that distinguishes early and late BFU-Es. Transient elevation of TβRIII expression promotes TGF-β signaling during the early BFU-E to late BFU-E transition. Blocking TGF-β signaling using a receptor kinase inhibitor increases early BFU-E cell self-renewal and total erythroblast production, suggesting the usefulness of this type of drug in treating Epo-unresponsive anemias.

  2. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

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    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  3. Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells.

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    Gloria Bua

    Full Text Available The pathogenic Parvovirus B19 (B19V is characterized by a strict adaptation to erythroid progenitor cells (EPCs, a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi and lower levels at day 18 (+1.5 Log from 2 to 48 hpi. B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18. Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6-15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage.

  4. 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

  5. Increase of microRNA-210, decrease of raptor gene expression and alteration of mammalian target of rapamycin regulated proteins following mithramycin treatment of human erythroid cells.

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    Nicoletta Bianchi

    Full Text Available Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3'-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells.

  6. Endogenous K-ras signaling in erythroid differentiation.

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    Zhang, Jing; Lodish, Harvey F

    2007-08-15

    K-ras is one of the most frequently mutated genes in virtually all types of human cancers. Using mouse fetal liver erythroid progenitors as a model system, we studied the role of endogenous K-ras signaling in erythroid differentiation. When oncogenic K-ras is expressed from its endogenous promoter, it hyperactivates cytokine-dependent signaling pathways and results in a partial block in erythroid differentiation. In erythroid progenitors deficient in K-ras, cytokine-dependent Akt activation is greatly reduced, leading to delays in erythroid differentiation. Thus, both loss- and gain-of-Kras functions affect erythroid differentiation through modulation of cytokine signaling. These results support the notion that in human cancer patients oncogenic Ras signaling might be controlled by antagonizing essential cytokines.

  7. Eos negatively regulates human γ-globin gene transcription during erythroid differentiation.

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    Hai-Chuan Yu

    Full Text Available BACKGROUND: Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4, a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs. DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3 of the β-globin locus control region (LCR, the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.

  8. JAK2 V617F stimulates proliferation of erythropoietin-dependent erythroid progenitors and delays their differentiation by activating Stat1 and other nonerythroid signaling pathways.

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    Shi, Jiahai; Yuan, Bingbing; Hu, Wenqian; Lodish, Harvey

    2016-11-01

    JAK2 V617F is a mutant-activated JAK2 kinase found in most polycythemia vera (PV) patients; it skews normal proliferation and differentiation of hematopoietic stem and progenitor cells and simulates aberrant expansion of erythroid progenitors. JAK2 V617F is known to activate some signaling pathways not normally activated in mature erythroblasts, but there has been no systematic study of signal transduction pathways or gene expression in erythroid cells expressing JAK2 V617F undergoing erythropoietin (Epo)-dependent terminal differentiation. Here we report that expression of JAK2 V617F in murine fetal liver Epo-dependent progenitors allows them to divide approximately six rather than the normal approximately four times in the presence of Epo, delaying their exit from the cell cycle. Over time, the number of red cells formed from each Epo-dependent progenitor increases fourfold, and these cells eventually differentiate into normal enucleated reticulocytes. We report that purified fetal liver Epo-dependent progenitors express many cytokine receptors additional to the EpoR. Expression of JAK2 V617F triggers activation of Stat5, the only STAT normally activated by Epo, as well as activation of Stat1 and Stat3. Expression of JAK2 V617F also leads to transient induction of many genes not normally activated in terminally differentiating erythroid cells and that are characteristic of other hematopoietic lineages. Inhibition of Stat1 activation blocks JAK2 V617F hyperproliferation of erythroid progenitors, and we conclude that Stat1-mediated activation of nonerythroid signaling pathways delays terminal erythroid differentiation and permits extended cell divisions.

  9. CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells.

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    Laurie A Steiner

    Full Text Available CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC and primary human erythroid cells from single donors.Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner.These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.

  10. Human uroporphyrinogen-III synthase: genomic organization, alternative promoters, and erythroid-specific expression.

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    Aizencang, G; Solis, C; Bishop, D F; Warner, C; Desnick, R J

    2000-12-01

    Uroporphyrinogen-III (URO) synthase is the heme biosynthetic enzyme defective in congenital erythropoietic porphyria. The approximately 34-kb human URO-synthase gene (UROS) was isolated, and its organization and tissue-specific expression were determined. The gene had two promoters that generated housekeeping and erythroid-specific transcripts with unique 5'-untranslated sequences (exons 1 and 2A) followed by nine common coding exons (2B to 10). Expression arrays revealed that the housekeeping transcript was present in all tissues, while the erythroid transcript was only in erythropoietic tissues. The housekeeping promoter lacked TATA and SP1 sites, consistent with the observed low level expression in most cells, whereas the erythroid promoter contained GATA1 and NF-E2 sites for erythroid specificity. Luciferase reporter assays demonstrated that the housekeeping promoter was active in both erythroid K562 and HeLa cells, while the erythroid promoter was active only in erythroid cells and its activity was increased during hemin-induced erythroid differentiation. Thus, human URO-synthase expression is regulated during erythropoiesis by an erythroid-specific alternative promoter.

  11. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

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    Ghaffari, Saghi; Kitidis, Claire; Zhao, Wei; Marinkovic, Dragan; Fleming, Mark D; Luo, Biao; Marszalek, Joseph; Lodish, Harvey F

    2006-03-01

    AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

  12. Qualitative and quantitative comparison of the proteome of erythroid cells differentiated from human iPSCs and adult erythroid cells by multiplex TMT labelling and nanoLC-MS/MS.

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    Trakarnsanga, Kongtana; Wilson, Marieangela C; Griffiths, Rebecca E; Toye, Ashley M; Carpenter, Lee; Heesom, Kate J; Parsons, Steve F; Anstee, David J; Frayne, Jan

    2014-01-01

    Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified 30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10-15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.

  13. Qualitative and quantitative comparison of the proteome of erythroid cells differentiated from human iPSCs and adult erythroid cells by multiplex TMT labelling and nanoLC-MS/MS.

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    Kongtana Trakarnsanga

    Full Text Available Induced pluripotent stem cells (iPSC are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19 and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2 with that of adult and cord blood progenitors. Of the 1989 proteins quantified 30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10-15% of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.

  14. Effect of Tumor Necrosis Factor-Alpha on Erythropoietinand Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in β Thalassemia/Hemoglobin E Patients

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    Dalina I Tanyong

    2015-12-01

    Full Text Available Objective: Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels of several inflammatory cytokines have been reported in β thalassemia and might contribute to ineffective erythropoiesis. However, the mechanism by which tumor necrosis factor-alpha (TNF-α is involved in ineffective erythropoiesis in thalassemic patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO and erythropoietin receptor (EPOR expression involved in proliferation of β-thalassemia/hemoglobin (Hb E erythroid progenitor cells compared with cells from healthy subjects. Materials and Methods: CD34-positive cells were isolated from heparinized blood by using the EasySep® CD34 selection kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR protein were measured by flow cytometry. Results: Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants and β-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for β-thalassemia/ Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic erythroid progenitor cells. Conclusion: TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation. It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in β-thalassemia/Hb E patients.

  15. Application of high-performance liquid chromatographic methodology to the analysis of hemoglobins synthesized in erythroid progenitor cells.

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    Bhaumik, K; Huisman, T H

    1989-11-10

    High-performance liquid chromatography (HPLC) has been successfully used in the quantitation of the relatively minute amounts of hemoglobin types recovered from in vitro cultures of hemoglobin-synthesizing erythroid progenitor (BFU-E) cells. This reversed-phase HPLC method uses the Vydac C4 column and water-acetonitrile-trifluoroacetic acid as mobile phases; it has been applied to the study of fetal hemoglobin synthesis patterns in ten homozygous sickle cell anemia patients and a similar number of their heterozygous relatives along with a few normal control subjects. A significant increase in the total gamma chain level was observed in the BFU-E lysate samples corresponding to the whole blood lysates of all the patients and their heterozygous relatives, except in one patient with the beta S haplotype Mor. On the other hand, the relative level of the G gamma chains appeared to be decreased in the BFU-E lysate samples of all except the individuals carrying the Mor haplotype, where it is reversed. The method has considerable advantages over other chromatographic and electrophoretic procedures; it is extremely sensitive and allows quantitation of all different globin chains in one single chromatogram.

  16. Differentiation Potential of O Bombay Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells into Fetal Erythroid-Like Cells

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    Fatemeh Ganji,

    2015-01-01

    Full Text Available Objective: There is constant difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of "universal" red blood cells. Advances in somatic cell reprogramming of human-induced pluripotent stem cells (hiPSCs have provided a valuable alternative source to differentiate into any desired cell type as a therapeutic promise to cure many human disease. Materials and Methods: In this experimental study, we examined the erythroid differentiation potential of normal Bombay hiPSCs (B-hiPSCs and compared results to human embryonic stem cell (hESC lines. Because of lacking ABO blood group expression in B-hiPSCs, it has been highlighted as a valuable source to produce any cell type in vitro. Results: Similar to hESC lines, hemangioblasts derived from B-hiPSCs expressed approximately 9% KDR+CD31+ and approximately 5% CD31+CD34+. In semisolid media, iPSC and hESC-derived hemangioblast formed mixed type of hematopoietic colony. In mixed colonies, erythroid progenitors were capable to express CD71+GPA+HbF+ and accompanied by endothelial cells differentiation. Conclusion: Finally, iPS and ES cells have been directly induced to erythropoiesis without hemangioblast formation that produced CD71+HbF+erythroid cells. Although we observed some variations in the efficiency of hematopoietic differentiation between iPSC and ES cells, the pattern of differentiation was similar among all three tested lines.

  17. Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production.

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    Wang, Weijia; Akbarian, Vahe; Audet, Julie

    2013-02-02

    Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.

  18. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

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    Aimola, Idowu A.; Inuwa, Hajiya M.; Nok, Andrew J.; Mamman, Aisha I.; Bieker, James J.

    2017-01-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24 h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer PMID:26879870

  19. High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules.

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    Olivier, Emmanuel N; Marenah, Lamin; McCahill, Angela; Condie, Alison; Cowan, Scott; Mountford, Joanne C

    2016-10-01

    : This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000-200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%-95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs. This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red

  20. In vitro myelotoxicity of propanil and 3,4-dichloroaniline on murine and human CFU-E/BFU-E progenitors.

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    Malerba, I; Castoldi, A F; Parent-Massin, D; Gribaldo, L

    2002-10-01

    Because of the wide use of pesticides for domestic and industrial purposes, the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice, but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study, an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E, CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors, has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M, and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand, human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and >500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors, as was found for the murine cells, emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose.

  1. Real time imaging of human progenitor neurogenesis.

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    Thomas M Keenan

    Full Text Available Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer's disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2(+ neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their

  2. Reprogramming of human peripheral blood monocytes to erythroid lineage by blocking of the PU-1 gene expression.

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    Nouri, Masoumeh; Deezagi, Abdolkhalegh; Ebrahimi, Marzieh

    2016-03-01

    In hematopoietic system development, PU.1 and GATA-1 as lineage-specific transcription factors (TF) are expressed in common myeloid progenitors. The cross antagonism between them ascertains gene expression programs of monocytic and erythroid cells, respectively. This concept in transdifferentiation approaches has not been well considered yet, especially in intralineage conversion systems. To demonstrate whether PU.1 suppression induces monocyte lineage conversion into red blood cells, a combination of three PU.1-specific siRNAs was implemented to knock down PU.1 gene expression and generate the balance in favor of GATA-1 expression to induce erythroid differentiation. For this purpose, monocytes were isolated from human peripheral blood and transfected by PU.1 siRNAs. In transfected monocytes, the rate of PU.1 expression in mRNA level was significantly decreased until 0.38 ± 0.118 when compared to untreated monocytes at 72 h (p value ≤0.05) which resulted in significant overexpression of GATA1 of 16.1 ± 0.343-fold compared to the untreated group (p value ≤0.01). Subsequently, overexpression of hemoglobin (α 13.26 ± 1.34-fold; p value≤0.0001) and β-globin (37.55 ± 16.56-fold; p value≤0.0001) was observed when compared to control groups. The results of western immunoblotting confirm those findings too. While, reduced expression of monocyte, CD14 gene, was observed in qRT-PCR and flow cytometry results. Our results suggest that manipulating the ratio of the two TFs in bifurcation differentiation pathways via applying siRNA technology can possibly change the cells' fate as a safe way for therapeutics application.

  3. Analysis of the erythroid differentiation effect of flavonoid apigenin on K562 human chronic leukemia cells.

    Science.gov (United States)

    Isoda, Hiroko; Motojima, Hideko; Onaga, Shoko; Samet, Imen; Villareal, Myra O; Han, Junkyu

    2014-09-05

    The erythroid differentiation-inducing effect of apigenin and its derivatives on human chronic myeloid leukemia K562 has been reported but the functional group in its structure responsible for the effect has not yet been elucidated. Here, we determined the moiety responsible for the erythroid differentiation induction effect of apigenin by using different flavonoids to represent the functional groups in its structure. In addition, we compared apigenin and apigetrin, a flavonoid similar in structure to apigenin except for the glycoside in its structure. Morphological changes as well as expressions of specific markers in K562 cells treated with apigenin were compared with those treated with apigetrin, flavone, 7-hydroxyflavone, chrysin, luteolin, or naringenin. The anti-proliferative and erythroid differentiation-inducing effect of apigenin and the five flavonoids were then investigated and their effects on the α, β, and γ globin genes expressions were compared using real-time PCR. Results of the comparison between apigenin and apigetrin revealed that the glycoside part of apigetrin does not have a role in the induction of cell differentiation. Based on glycophorin A expression, the potency of the other flavonoids for induction of differentiation, was: apigenin>chrysin>flavone/7-hydroxyflavone>luteolin/naringenin. Results of the analysis of the relationship between the structure and function of the flavonoids suggest that the apigenin-induced K562 cell differentiation was due to the 2-3 double bond and hydroxyl groups in its structure. This is the first study that identified the specific functional group in apigenin that impact the erythroid differentiation effect in K562 cells.

  4. Targeting human oligodendrocyte progenitors for myelin repair.

    Science.gov (United States)

    Dietz, Karen C; Polanco, Jessie J; Pol, Suyog U; Sim, Fraser J

    2016-09-01

    Oligodendrocyte development has been studied for several decades, and has served as a model system for both neurodevelopmental and stem/progenitor cell biology. Until recently, the vast majority of studies have been conducted in lower species, especially those focused on rodent development and remyelination. In humans, the process of myelination requires the generation of vastly more myelinating glia, occurring over a period of years rather than weeks. Furthermore, as evidenced by the presence of chronic demyelination in a variety of human neurologic diseases, it appears likely that the mechanisms that regulate development and become dysfunctional in disease may be, in key ways, divergent across species. Improvements in isolation techniques, applied to primary human neural and oligodendrocyte progenitors from both fetal and adult brain, as well as advancements in the derivation of defined progenitors from human pluripotent stem cells, have begun to reveal the extent of both species-conserved signaling pathways and potential key differences at cellular and molecular levels. In this article, we will review the commonalities and differences in myelin development between rodents and man, describing the approaches used to study human oligodendrocyte differentiation and myelination, as well as heterogeneity within targetable progenitor pools, and discuss the advances made in determining which conserved pathways may be both modeled in rodents and translate into viable therapeutic strategies to promote myelin repair.

  5. Eto2/MTG16 and MTGR1 are heteromeric corepressors of the TAL1/SCL transcription factor in murine erythroid progenitors

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Ying; Xu, Zhixiong; Xie, Jingping [Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Ham, Amy-Joan L. [Department of Biochemistry, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Koury, Mark J. [Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Tennessee Valley VA Healthcare System, Nashville, TN 37212 (United States); Hiebert, Scott W. [Department of Biochemistry, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Brandt, Stephen J., E-mail: stephen.brandt@vanderbilt.edu [Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Tennessee Valley VA Healthcare System, Nashville, TN 37212 (United States)

    2009-12-11

    The TAL1 (or SCL) gene, originally discovered through its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. To identify its interaction partners, we expressed a tandem epitope-tagged protein in murine erythroleukemia (MEL) cells and characterized affinity-purified Tal1-containing complexes by liquid chromatography-tandem mass spectrometry analysis. In addition to known interacting proteins, two proteins related to the Eight-Twenty-One (ETO) corepressor, Eto2/Mtg16 and Mtgr1, were identified from the peptide fragments analyzed. Tal1 interaction with Eto2 and Mtgr1 was verified by coimmunoprecipitation analysis in Tal1, Eto2-, and Mtgr1-transfected COS-7 cells, MEL cells expressing V5 epitope-tagged Tal1 protein, and non-transfected MEL cells. Mapping analysis with Gal4 fusion proteins demonstrated a requirement for the bHLH domain of Tal1 and TAF110 domain of Eto2 for their interaction, and transient transfection and glutathione S-transferase pull-down analysis showed that Mtgr1 and Eto2 enhanced the other's association with Tal1. Enforced expression of Eto2 in differentiating MEL cells inhibited the promoter of the Protein 4.2 (P4.2) gene, a direct target of TAL1 in erythroid progenitors, and transduction of Eto2 and Mtgr1 augmented Tal1-mediated gene repression. Finally, chromatin immunoprecipitation analysis revealed that Eto2 occupancy of the P4.2 promoter in MEL cells decreased with differentiation, in parallel with a decline in Eto2 protein abundance. These results identify Eto2 and Mtgr1 as authentic interaction partners of Tal1 and suggest they act as heteromeric corepressors of this bHLH transcription factor during erythroid differentiation.

  6. Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response.

    Science.gov (United States)

    Richardson, Chanté L; Delehanty, Lorrie L; Bullock, Grant C; Rival, Claudia M; Tung, Kenneth S; Kimpel, Donald L; Gardenghi, Sara; Rivella, Stefano; Goldfarb, Adam N

    2013-08-01

    The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

  7. Natural killer cells recognize friend retrovirus-infected erythroid progenitor cells through NKG2D-RAE-1 interactions In Vivo.

    Science.gov (United States)

    Ogawa, Tatsuya; Tsuji-Kawahara, Sachiyo; Yuasa, Takae; Kinoshita, Saori; Chikaishi, Tomomi; Takamura, Shiki; Matsumura, Haruo; Seya, Tsukasa; Saga, Toshihiko; Miyazawa, Masaaki

    2011-06-01

    Natural killer (NK) cells function as early effector cells in the innate immune defense against viral infections and also participate in the regulation of normal and malignant hematopoiesis. NK cell activities have been associated with early clearance of viremia in experimental simian immunodeficiency virus and clinical human immunodeficiency virus type 1 (HIV-1) infections. We have previously shown that NK cells function as major cytotoxic effector cells in vaccine-induced immune protection against Friend virus (FV)-induced leukemia, and NK cell depletion totally abrogates the above protective immunity. However, how NK cells recognize retrovirus-infected cells remains largely unclear. The present study demonstrates a correlation between the expression of the products of retinoic acid early transcript-1 (RAE-1) genes in target cells and their susceptibility to killing by NK cells isolated from FV-infected animals. This killing was abrogated by antibodies blocking the NKG2D receptor in vitro. Further, the expression of RAE-1 proteins on erythroblast surfaces increased early after FV inoculation, and administration of an RAE-1-blocking antibody resulted in increased spleen infectious centers and exaggerated pathology, indicating that FV-infected erythroid cells are recognized by NK cells mainly through the NKG2D-RAE-1 interactions in vivo. Enhanced retroviral replication due to host gene-targeting resulted in markedly increased RAE-1 expression in the absence of massive erythroid cell proliferation, indicating a direct role of retroviral replication in RAE-1 upregulation.

  8. In vitro and in vivo expression of human erythrocyte pyruvate kinase in erythroid cells: a gene therapy approach.

    Science.gov (United States)

    Meza, N W; Quintana-Bustamante, O; Puyet, A; Rio, P; Navarro, S; Diez, A; Bueren, J A; Bautista, J M; Segovia, J C

    2007-06-01

    Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

  9. Regulation of erythroid differentiation by miR-376a and its targets

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Jia Yu; Gui-Hua Yang; Xiao-Shuang Wang; Jun-Wu Zhang

    2011-01-01

    Lineage differentiation is a continuous process during which fated progenitor cells execute specific programs to produce mature counterparts. This lineage-restricted pathway can be controlled by particular regulators, which are usually exclusively expressed in certain cell types or at specific differentiation stages. Here we report that miR376a participates in the regulation of the early stages of human erythropoiesis by targeting cyclin-dependent kinase 2 (CDK2) and Argonaute 2 (Ago2). Among various human leukemia cell lines, miR-376a was only detected in K562 cells which originated from a progenitor common to the erythroid and megakaryotic lineages. Enforced expression of miR-376a or silencing of CDK2 and Ago2 by RNAi inhibits erythroid differentiation of K562 cells. Hematopoietic progenitor cells transduced with miR-376a showed a significant reduction of their erythroid clonogenic capacity. MiR-376a is relatively abundant in erythroid progenitor cells, where it reduces expression of CDK2 and maintains a low level of differentiation due to cell cycle arrest and decreased cell growth. Following erythroid induction, miR376a is significantly down-regulated and CDK2 is released from miR-376a inhibition, thereby facilitating the escape of progenitor cells from the quiescent state into erythroid differentiation. Moreover, our results establish a functional link between miR-376a and Ago2, a key factor in miRNA biogenesis and silencing pathways with novel roles in human hematopoiesis.

  10. Effect of human neural progenitor cells on injured spinal cord

    Institute of Scientific and Technical Information of China (English)

    XU Guang-hui; BAI Jin-zhu; CAI Qin-lin; LI Xiao-xia; LI Ling-song; SHEN Li

    2005-01-01

    Objective: To study whether human neural progenitor cells can differentiate into neural cells in vivo and improve the recovery of injured spinal cord in rats.Methods: Human neural progenitor cells were transplanted into the injured spinal cord and the functional recovery of the rats with spinal cord contusion injury was evaluated with Basso-Beattie-Bresnahan (BBB) locomotor scale and motor evoked potentials. Additionally, the differentiation of human neural progenitor cells was shown by immunocytochemistry.Results: Human neural progenitor cells developed into functional cells in the injured spinal cord and improved the recovery of injured spinal cord in both locomotor scores and electrophysiological parameters in rats.Conclusions: Human neural progenitor cells can treat injured spinal cord, which may provide a new cell source for research of clinical application.

  11. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells.

    Science.gov (United States)

    Griffin, J D; Linch, D; Sabbath, K; Larcom, P; Schlossman, S F

    1984-01-01

    Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.

  12. Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker

    Institute of Scientific and Technical Information of China (English)

    Parveen Nyamath; Ayesha AM; Aejaz Habeeb; Sanjeev Khosla; Aleem A Khan; CM Habibullah

    2007-01-01

    AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker.METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation.RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture.CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.

  13. Trichothecene toxicity on human megakaryocyte progenitors (CFU-MK).

    Science.gov (United States)

    Froquet, R; Sibiril, Y; Parent-Massin, D

    2001-02-01

    Trichothecenes are mycotoxins produced by various species of fungi, which can occur on various agricultural products. Among these compounds, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) are the most naturally encountered and the most potent trichothecenes. Consumption of trichothecene contaminated foods by farm animals and humans leads to mycotoxicosis. Trichothecenes are known to induce haematological disorders such as neutropenia, aplastic anemia and thrombocytopenia in humans and animals. Four trichothecenes, T-2 toxin, HT-2 toxin, DAS and DON have been tested on human platelet progenitors (CFU-MK) using a culture model of CFU-MK optimized for toxicological studies. Trichothecenes cause, at low concentrations, cytotoxic effects in megakaryocyte progenitors, which could induce thrombocytopenia. Sensitivity of human CFU-MK is compared to respective sensitivities of human red blood cell progenitors (BFU-E) and white blood cell progenitors (CF-U-GM) that were described in previous works.

  14. Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells from Patients with Sickle Cell Disease.

    Science.gov (United States)

    Uchida, Naoya; Haro-Mora, Juan J; Fujita, Atsushi; Lee, Duck-Yeon; Winkler, Thomas; Hsieh, Matthew M; Tisdale, John F

    2017-03-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent an ideal source for in vitro modeling of erythropoiesis and a potential alternative source for red blood cell transfusions. However, iPS cell-derived erythroid cells predominantly produce ε- and γ-globin without β-globin production. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production. In this study, we generated several iPS cell lines derived from bone marrow stromal cells (MSCs) and peripheral blood erythroid progenitors (EPs) from sickle cell disease patients, and evaluated hematopoietic stem/progenitor cell (HSPC) generation after iPS sac induction as well as subsequent erythroid differentiation. MSC-derived iPS sacs yielded greater amounts of immature hematopoietic progenitors (VEGFR2 + GPA-), definitive HSPCs (CD34 + CD45+), and megakaryoerythroid progenitors (GPA + CD41a+), as compared to EP-derived iPS sacs. Erythroid differentiation from MSC-derived iPS sacs resulted in greater amounts of erythroid cells (GPA+) and higher β-globin (and βS-globin) expression, comparable to ES sac-derived cells. These data demonstrate that human MSC-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, likely due to heightened emergence of immature progenitors. Our findings should be important for iPS cell-derived erythroid cell generation. Stem Cells 2017;35:586-596.

  15. Harmine stimulates proliferation of human neural progenitors

    Science.gov (United States)

    Dakic, Vanja; Maciel, Renata de Moraes; Drummond, Hannah; Nascimento, Juliana M.; Trindade, Pablo

    2016-01-01

    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. PMID:27957390

  16. 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.

  17. Hepatic progenitor cells in human liver tumor development

    Institute of Scientific and Technical Information of China (English)

    Louis Libbrecht

    2006-01-01

    In recent years, the results of several studies suggest that human liver tumors can be derived from hepatic progenitor cells rather than from mature cell types.The available data indeed strongly suggest that most combined hepatocellular-cholangiocarcinomas arise from hepatic progenitor cells that retained their potential to differentiate into the hepatocytic and biliary lineages.Hepatic progenitor cells could also be the basis for some hepatocellular carcinomas and hepatocellular adenomas, although it is very difficult to determine the origin of an individual hepatocellular carcinoma. There is currently not enough data to make statements regarding a hepatic progenitor cell origin of cholangiocarcinoma.The presence of hepatic progenitor cell markers and the presence and extent of the cholangiocellular component are factors that are related to the prognosis of hepatocellular carcinomas and combined hepatocellularcholangiocarcinomas, respectively.

  18. Human pancreatic islet progenitor cells demonstrate phenotypic plasticity in vitro

    Indian Academy of Sciences (India)

    Maithili P Dalvi; Malati R Umrani; Mugdha V Joglekar; Anandwardhan A Hardikar

    2009-10-01

    Phenotypic plasticity is a phenomenon that describes the occurrence of 2 or more distinct phenotypes under diverse conditions. This article discusses the work carried out over the past few years in understanding the potential of human pancreatic islet-derived progenitors for cell replacement therapy in diabetes. The phenotypic plasticity exhibited by pancreatic progenitors during reversible epithelial-to-mesenchymal transition (EMT) and possible role of microRNAs in regulation of this process is also presented herein.

  19. Synergistic Effect of Sodium Butyrate and Thalidomide in the Induction of Fetal Hemoglobin Expression in Erythroid Progenitors Derived from Cord Blood CD133 + Cells

    Directory of Open Access Journals (Sweden)

    Ali Dehghanifard

    2012-07-01

    Full Text Available Background: The use of drugs with the ability to induce production of fetal hemoglobin as a novel therapeutic approach in treating β-Hemoglobinopathies is considered. γ-globin gene expression inducer drugs including sodium butyrate and thalidomide can reduce additional α-globin chains accumulation in erythroid precursors. Materials and Methods: In this experimental study, MACS kit was used to isolate CD133+ cells of umbilical cord blood. Further, the effect of two drugs of thalidomide and sodium butyrate were separately and combined studied on the induction of quantitative expression of β-globin and γ-globin genes in erythroid precursor cells derived from CD133+ stem cells in-vitro. For this purpose, the technique SYBR green Real-time PCR was used.Results: Flow cytometry results showed that approximately 95% of purified cells were CD133+. Real-time PCR results also showed the increased levels of γ-globin mRNA in the cell groups treated with thalidomide, sodium butyrate and combination of drugs as 2.6 and 1.2 and 3.5 times respectively, and for β-globin gene, it is respectively 1.4 and 1.3 and 1.6 times compared with the control group (p<0.05.Conclusion: The study results showed that the mentioned drug combination can act as a pharmaceutical composition affecting the induction of fetal hemoglobin expression in erythroid precursor cells derived from CD133 + cells.

  20. Assessment of human multi-potent hematopoietic stem/progenitor cell potential using a single in vitro screening system.

    Directory of Open Access Journals (Sweden)

    Julien Calvo

    Full Text Available Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34(+CD38(-/low(CD45RA(-CD90(+ cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.

  1. Loss of IKKβ but Not NF-κB p65 Skews Differentiation towards Myeloid over Erythroid Commitment and Increases Myeloid Progenitor Self-Renewal and Functional Long-Term Hematopoietic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.

  2. Senegenin promotes in vitro proliferation of human neural progenitor cells

    Institute of Scientific and Technical Information of China (English)

    Fang Shi; Zhigang Liang; Zixuan Guo; Ran Li; Fen Yu; Zhanjun Zhang; Xuan Wang; Xiaomin Wang

    2011-01-01

    Senegenin, an effective component of Polygala tenuifolia root extract, promotes proliferation and differentiation of neural progenitor cells in the hippocampus.However, the effects of senegenin on mesencephalon-derived neural progenitor cells remain poorly understood.Cells from a ventral mesencephalon neural progenitor cell line (ReNcell VM) were utilized as models for pharmaceutical screening.The effects of various senegenin concentrations on cell proliferation were analyzed,demonstrating that high senegenin concentrations (5, 10, 50, and 100 pmo/L), particularly 50 pmol/L, significantly promoted proliferation of ReNcell VM cells.In the mitogen-activated protein kinase signal transduction pathway, senegenin significantly increased phosphorylation levels of extracellular signal-regulated kinases.Moreover, cell proliferation was suppressed by extracellular signal-regulated kinase inhibitors.Results suggested that senegenin contributed to in vitro proliferation of human neural progenitor cells by upregulating phosphorylation of extracellular signal-regulated kinase.

  3. Differentiation Potential of O Bombay Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells into Fetal Erythroid-Like Cells

    OpenAIRE

    2015-01-01

    Objective: There is constant difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of "universal" red blood cells. Advances in somatic cell reprogramming of human-induced pluripotent stem cells (hiPSCs) have provided a valuable alternative source to differentiate into any desired cell type as a therapeutic promise to cure many human disease. Materials and Methods: In this experimental study, we examined the erythroid differentiation potential of n...

  4. Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

    Science.gov (United States)

    Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

    2011-02-25

    Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature

  5. Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

    Directory of Open Access Journals (Sweden)

    Laurie Chicha

    Full Text Available BACKGROUND: Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC and induced pluripotent stem cells (iPSC into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. METHODOLOGY/PRINCIPAL FINDINGS: ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+ cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB CD34(+ cells. ESC-derived CD34(+ cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(- cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. CONCLUSIONS/SIGNIFICANCE: Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and i

  6. Intracellular regulation of the production and release of human erythroid-directed lymphokines.

    OpenAIRE

    Dainiak, N; Sorba, S

    1991-01-01

    Erythroid burst-promoting activity (BPA) is released from B lymphocytes in soluble (sBPA) and membrane-bound (mBPA) forms. To study intracellular processes involved in production of these physically separable factors, we measured their time course release into serum-free medium from B cells that were pulse-exposed for 5-240 min to nonmitogenic base medium or inhibitors of energy-dependent metabolism (2,4-dinitrophenol, sodium azide, and 2-deoxy-D-glucose), transcription and translation (actin...

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Ex vivo expansion of human peripheral blood progenitors.

    Science.gov (United States)

    Chabannon, C; Herrera-Rodriguez, D; Bardin, F; Mouren, M; Novakovitch, G; Blaise, D; Maraninchi, D; Mannoni, P

    1995-01-01

    Culture of human hematopoietic progenitors on a large scale could lead to several clinical applications within the near future, including the production of differentiated and functional cells, the increase in the number of early progenitors, especially stem cells, with such use as gene transfer, or the improvement of grafts used to limit the hematological toxicity associated with high-dose chemotherapy. In this case, one can still distinguish different objectives: improvement of grafts that contain low numbers of progenitors because of prior chemotherapies or because of marrow involvement for example, and qualitative changes in the graft content that would allow to envision the disappearance, or the further reduction, in the duration of absolute neutropenia that follows delivery of high dose chemotherapy ("nadir rescue"), despite substitution of mobilized blood cells to marrow cells and the in vivo use of hematopoietic growth factors. Additional advantages may be related to tumor purging in autologous expanded cells, and to the change in the ratio between hematopoietic progenitors and immunocompetent cells in allogeneic expanded populations. Therefore it appears that in vitro expansion currently raises two types of questions: the first ones are related to the definition of clinical or biological endpoints to be achieved, the second ones are related to "bioengineering", and deal with the efficiency and safety of progenitor cell cultures to be used for clinical applications. We here present preliminary results preparing future pilot clinical studies with ex vivo cultured human hematopoietic cells.

  9. 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.

  10. H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner.

    Directory of Open Access Journals (Sweden)

    Soichiro Sakamoto

    Full Text Available Ferritin is an iron-storage protein composed of different ratios of 24 light (L and heavy (H subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

  11. H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

    Science.gov (United States)

    Sakamoto, Soichiro; Kawabata, Hiroshi; Masuda, Taro; Uchiyama, Tatsuki; Mizumoto, Chisaki; Ohmori, Katsuyuki; Koeffler, H. Phillip; Kadowaki, Norimitsu; Takaori-Kondo, Akifumi

    2015-01-01

    Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin. PMID:26441243

  12. Generation and Characterization of Erythroid Cells from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells: An Overview

    Directory of Open Access Journals (Sweden)

    Kai-Hsin Chang

    2011-01-01

    Full Text Available Because of the imbalance in the supply and demand of red blood cells (RBCs, especially for alloimmunized patients or patients with rare blood phenotypes, extensive research has been done to generate therapeutic quantities of mature RBCs from hematopoietic stem cells of various sources, such as bone marrow, peripheral blood, and cord blood. Since human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs can be maintained indefinitely in vitro, they represent potentially inexhaustible sources of donor-free RBCs. In contrast to other ex vivo stem-cell-derived cellular therapeutics, tumorigenesis is not a concern, as RBCs can be irradiated without marked adverse effects on in vivo function. Here, we provide a comprehensive review of the recent publications relevant to the generation and characterization of hESC- and iPSC-derived erythroid cells and discuss challenges to be met before the eventual realization of clinical usage of these cells.

  13. Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies.

    Science.gov (United States)

    Araghi, Atefeh; Nassiri, Seyed Mahdi; Atyabi, Nahid; Rahbarghazi, Reza; Mohammadi, Elham

    2014-04-01

    There is a paucity of species-specific antibodies available for feline haematopoietic conditions. The purpose of this study was to broaden the panel of antibodies available for use in the immunophenotypic characterisation of feline haematopoietic cells by testing clones of anti-human monoclonal antibodies (mAbs) on normal, neoplastic and cultured feline haematopoietic progenitors to determine cross-reactivity to feline counterparts. In this study, 24 clones of anti-human mAbs were tested on normal or neoplastic feline bone marrow and peripheral blood cells. Six of these mAbs, including anti-cluster of differentiation (CD)61, anti-CD18, anti-CD14, anti-CD235a, anti-CD41 and anti-CD29, cross-reacted with normal feline bone marrow cells, whereas anti-CD33 and anti-CD117 cross-reacted with the blast cells in the bone marrow of two cats with myelodysplastic syndrome, and anti-CD71, anti-235a, anti-41 and anti-42 cross-reacted with immature erythroid cells in a cat with erythroleukaemia. In a feline immunodeficiency virus-positive cat, bone marrow cells were labelled with anti-CD33, anti-14 and anti-45. Anti-CD18, anti-CD14, anti-CD41 and anti-CD61 also reacted with the peripheral blood cells of the healthy cats. The feline haematopoietic progenitors formed colonies in the methylcellulose-based semisolid medium with significant enrichment of colony-forming unit-granulocyte, monocyte and burst-forming unit-erythroid. A panel of six anti-feline mAbs (anti-CD21-like, anti-T lymphocytes, anti-CD172a, anti-granulocyte, anti-CD45-like and anti-CD18) and eight anti-human antibodies (anti-CD71, anti-CD33, anti-CD235a, anti-CD41, anti-CD61, anti-CD117, anti-CD38 and anti-CD34) were used for the immunophenotypic characterisation of the feline bone marrow progenitors. CD45, CD33, CD235a and CD18 were expressed by the feline haematopoietic progenitor cells, with the highest expression level for CD45.

  14. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Akiyoshi Uezumi

    2016-08-01

    Full Text Available Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

  15. Generation of multipotent early lymphoid progenitors from human embryonic stem cells.

    Science.gov (United States)

    Larbi, Aniya; Mitjavila-Garcia, Maria Teresa; Flamant, Stéphane; Valogne, Yannick; Clay, Denis; Usunier, Benoît; l'Homme, Bruno; Féraud, Olivier; Casal, Ibrahim; Gobbo, Emilie; Divers, Dominique; Chapel, Alain; Turhan, Ali G; Bennaceur-Griscelli, Annelise; Haddad, Rima

    2014-12-15

    During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the initial steps of lymphopoiesis remains elusive. Using a two-step culture procedure, we identified two original populations of ESC-derived hematopoietic progenitor cells (HPCs) with CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) phenotypes. Bulk cultures and limiting dilution assays, culture with MS5 cells in the presence of Notch ligand Delta-like-1 (DL-1), and ex vivo colonization tests using fetal thymic organ cultures showed that although CD34(+)CD45RA(+)CD7(-) HPCs could generate cells of the three lymphoid lineages, their potential was skewed toward the B cell lineages. In contrast, CD34(+)CD45RA(+)CD7(+) HPCs predominantly exhibited a T/natural killer (NK) cell differentiation potential. Furthermore these cells could differentiate equivalently into cells of the granulo-macrophagic lineage and dendritic cells and lacked erythroid potential. Expression profiling of 18 markers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs express genes of the lymphoid specification and that CD34(+)CD45RA(+)CD7(-) cells express B-cell-associated genes, while CD34(+)CD45RA(+)CD7(+) HPCs display a T-cell molecular profile. Altogether, these findings indicate that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs correspond to candidate multipotent early lymphoid progenitors polarized toward either the B or T/NK lineage, respectively. This work should improve our understanding of the early steps of lymphopoiesis from pluripotent stem cells and pave the way for the production of lymphocytes for cell-based immunotherapy and lymphoid development studies.

  16. Intracellular regulation of the production and release of human erythroid-directed lymphokines.

    Science.gov (United States)

    Dainiak, N; Sorba, S

    1991-01-01

    Erythroid burst-promoting activity (BPA) is released from B lymphocytes in soluble (sBPA) and membrane-bound (mBPA) forms. To study intracellular processes involved in production of these physically separable factors, we measured their time course release into serum-free medium from B cells that were pulse-exposed for 5-240 min to nonmitogenic base medium or inhibitors of energy-dependent metabolism (2,4-dinitrophenol, sodium azide, and 2-deoxy-D-glucose), transcription and translation (actinomycin D and cycloheximide), replicative DNA synthesis (cytosine arabinoside), or posttranslational processing (monensin). mBPA and sBPA were initially detectable after 1 and 2 h, respectively. Maximum cumulative levels of 8 +/- 0.6 and 9 +/- 1.0 U/ml, respectively, were reached after 8 h. In contrast, cumulative mBPA and sBPA levels in medium prepared from cells treated with metabolic inhibitors were reduced by up to 90%. Both surface exfoliation and mBPA expression by intact plasma membranes were diminished. Whereas pulse-exposure to cytosine arabinoside had no effect, treatment with actinomycin D or cycloheximide abolished BPA expression. Exposure to monensin reduced mBPA and sBPA levels to zero in a concentration-and time-dependent fashion. We conclude that production and release of BPA is an energy-dependent process, requiring mRNA synthesis and translation and posttranslational remodeling of the protein but not replicative DNA synthesis.

  17. Effect of Reishi polysaccharides on human stem/progenitor cells.

    Science.gov (United States)

    Chen, Wan-Yu; Yang, Wen-Bin; Wong, Chi-Huey; Shih, Daniel Tzu-Bi

    2010-12-15

    The polysaccharide fraction of Ganoderma lucidum (F3) was found to benefit our health in many ways by influencing the activity of tissue stem/progenitor cells. In this study, F3 was found to promote the adipose tissue MSCs' aggregation and chondrosphere formation, with the increase of CAM (N-CAM, I-CAM) expressions and autokine (BMP-2, IL-11, and aggrecan) secretions, in an in vitro chondrogenesis assay. In a stem cell expansion culture, it possesses the thrombopoietin (TPO) and GM-CSF like functions to enhance the survival/renewal abilities of primitive hematopoietic stem/progenitor cells (HSCs). F3 was found to promote the dendrite growth of blood mononuclear cells (MNCs) and the expression of cell adhesion molecules in the formation of immature dendritic cells (DC). On the other hand, F3 exhibited inhibitory effects on blood endothelial progenitor (EPC) colony formation, with concomitant reduction of cell surface endoglin (CD105) and vascular endothelial growth factor receptor-3 (VEGFR-3) marker expressions, in the presence of angiogenic factors. A further cytokine array analysis revealed that F3 indeed inhibited the angiogenin synthesis and enhanced IL-1, MCP-1, MIP-1, RANTES, and GRO productions in the blood EPC derivation culture. Collectively, we have demonstrated that the polysaccharide fraction of G. lucidum F3 exhibits cytokine and chemokine like functions which are beneficial to human tissue stem/progenitor cells by modulating their CAM expressions and biological activities. These findings provide us a better the observation that F3 glycopolysaccharides indeed possesses anti-angiogenic and immune-modulating functions and promotes hematopoietic stem/progenitor cell homing for better human tissue protection, reducing disease progression and health.

  18. Autophagy Proteins ATG5 and ATG7 Are Essential for the Maintenance of Human CD34(+) Hematopoietic Stem-Progenitor Cells.

    Science.gov (United States)

    Gomez-Puerto, Maria Catalina; Folkerts, Hendrik; Wierenga, Albertus T J; Schepers, Koen; Schuringa, Jan Jacob; Coffer, Paul J; Vellenga, Edo

    2016-06-01

    Autophagy is a highly regulated catabolic process that involves sequestration and lysosomal degradation of cytosolic components such as damaged organelles and misfolded proteins. While autophagy can be considered to be a general cellular housekeeping process, it has become clear that it may also play cell type-dependent functional roles. In this study, we analyzed the functional importance of autophagy in human hematopoietic stem/progenitor cells (HSPCs), and how this is regulated during differentiation. Western blot-based analysis of LC3-II and p62 levels, as well as flow cytometry-based autophagic vesicle quantification, demonstrated that umbilical cord blood-derived CD34(+) /CD38(-) immature hematopoietic progenitors show a higher autophagic flux than CD34(+) /CD38(+) progenitors and more differentiated myeloid and erythroid cells. This high autophagic flux was critical for maintaining stem and progenitor function since knockdown of autophagy genes ATG5 or ATG7 resulted in reduced HSPC frequencies in vitro as well as in vivo. The reduction in HSPCs was not due to impaired differentiation, but at least in part due to reduced cell cycle progression and increased apoptosis. This is accompanied by increased expression of p53, proapoptotic genes BAX and PUMA, and the cell cycle inhibitor p21, as well as increased levels of cleaved caspase-3 and reactive oxygen species. Taken together, our data demonstrate that autophagy is an important regulatory mechanism for human HSCs and their progeny, reducing cellular stress and promoting survival. Stem Cells 2016;34:1651-1663.

  19. Dynamics of alpha-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34(+) cells in culture.

    Science.gov (United States)

    Mahajan, Milind C; Karmakar, Subhradip; Newburger, Peter E; Krause, Diane S; Weissman, Sherman M

    2009-10-01

    The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron. A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis. Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway. Our results

  20. A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

    Science.gov (United States)

    Scoville, Steven D; Mundy-Bosse, Bethany L; Zhang, Michael H; Chen, Li; Zhang, Xiaoli; Keller, Karen A; Hughes, Tiffany; Chen, Luxi; Cheng, Stephanie; Bergin, Stephen M; Mao, Hsiaoyin C; McClory, Susan; Yu, Jianhua; Carson, William E; Caligiuri, Michael A; Freud, Aharon G

    2016-05-17

    The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

  1. Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

    Science.gov (United States)

    Banda, Erin; Grabel, Laura

    2016-01-01

    A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

  2. Phorbol ester-treated human acute myeloid leukemia cells secrete G-CSF, GM-CSF and erythroid differentiation factor into serum-free media in primary culture.

    Science.gov (United States)

    Scher, W; Eto, Y; Ejima, D; Den, T; Svet-Moldavsky, I A

    1990-12-10

    Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.

  3. FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1

    NARCIS (Netherlands)

    Bakker, WJ; Blazquez-Domingo, M; Kolbus, A; Besooyen, J; Steinlein, P; Beug, H; Coffer, PJ; Lowenberg, B; von Lindern, M; van Dijk, TB

    2004-01-01

    Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor-induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Fo

  4. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    OpenAIRE

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex...

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

    Science.gov (United States)

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

    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 hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

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

    Science.gov (United States)

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

    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 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. Derivation of multipotent progenitors from human circulating CD14+ monocytes.

    Science.gov (United States)

    Seta, Noriyuki; Kuwana, Masataka

    2010-07-01

    Circulating CD14(+) monocytes are originated from hematopoietic stem cells in the bone marrow and believed to be committed precursors for phagocytes, such as macrophages. Recently, we have reported a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which has a fibroblast-like morphology in culture and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from circulating CD14(+) monocytes, but circulating precursors for MOMCs still remain undetermined. Comparative analysis of gene expression profiles of MOMCs and other monocyte-derived cells has revealed that embryonic stem cell markers, Nanog and Oct-4, are specifically expressed by MOMCs. In vitro generation of MOMCs requires binding to fibronectin and exposure to soluble factors derived from activated platelets. MOMCs contain progenitors with capacity to differentiate into a variety of nonphagocytes, including bone, cartilage, fat, skeletal and cardiac muscle, neuron, and endothelium, indicating that circulating monocytes are more multipotent than previously thought. In addition, MOMCs are capable of promoting ex vivo expansion of human hematopoietic progenitor cells through direct cell-to-cell contact and secretion of a variety of hematopoietic growth factors. These findings obtained from the research on MOMCs indicate that CD14(+) monocytes in circulation are involved in a variety of physiologic functions other than innate and acquired immune responses, such as repair and regeneration of the damaged tissue.

  8. Initial function analysis of a novel erythroid differentiation related gene EDRF1

    Institute of Scientific and Technical Information of China (English)

    WANG; Duncheng(

    2001-01-01

    [1]Migliaccio, A. R, Vannucchi, A. M., Migliaccio, G., Molecular control of erythroid differentiation, International Journal of Hematology, 1996, 64(1): 1-29.[2]Migliaccio, A. R., Migliaccio, G., The making of an erythroid cell, Biotherapy, 1998, 10(2): 251-268.[3]Higgs, D. R., Sharpe, J. A., Wood, W. G., Understanding α-globin gene expression a step towards effective gene therapy,Seminars in Hematology, 1998, 35(1): 93-104.[4]Crosstey, M., Merika, M., Orkin, S. H., Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains, Mol. Cell Biol., 1992, 15: 2448-2456.[5]Wang. X., Chen, S. P., Xue, S. R, Preparation and determination of monoclonal antibodies against the proteins related to erythroid differentiation, Acta Anatomica Sinica, 1997, 28(2): 187-191.[6]Wang. X., Liu, P. X., Zhang, J. B. et al., Appearance of some novel proteins binding enhancer element of globin genes (HS2) during erythroid terminal differentiation, Acta Anatomica Sinica, 1994, 25(4): 379-384.[7]Wang. X.. Wang, D. C., Chen, X. et al., cDNA cloning and function analysis of two novel erythroid differentiation related genes. Science in China, Ser. C, 2001, 44(1): 99-105.[8]Wu, H., Liu, X.. Jaenisch, R. et al., Generation of committed erythroid BFU-E and CFR-E progenitors does not require erythropoietin or the erythropoietin receptor, Cell, 1995, 83 (1): 59-64.[9]Partington, G. A., Patient, R. K., Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells, Nucleic Acids Res., 1999, 27(4): 1168-1175.[10].Canelles, M., Delgado, M. D., Hyland, K. M. et al., Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells, Oncogene, 1997, 14(11): 1315- 1127.Acknowledgements This work was supported by National High Technology Programs of China (Grant No.102-08-01-03) and Natural Science Fund

  9. Human neural progenitor cells promote photoreceptor survival in retinal explants.

    Science.gov (United States)

    Englund-Johansson, Ulrica; Mohlin, Camilla; Liljekvist-Soltic, Ingela; Ekström, Per; Johansson, Kjell

    2010-02-01

    Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of

  10. TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

    Science.gov (United States)

    Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

    2015-05-01

    The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

  11. Adult human liver mesenchymal progenitor cells express phenylalanine hydroxylase.

    Science.gov (United States)

    Baruteau, Julien; Nyabi, Omar; Najimi, Mustapha; Fauvart, Maarten; Sokal, Etienne

    2014-09-01

    Phenylketonuria (PKU) is one of the most prevalent inherited metabolic diseases and is accountable for a severe encephalopathy by progressive intoxication of the brain by phenylalanine. This results from an ineffective L-phenylalanine hydroxylase enzyme (PAH) due to a mutated phenylalanine hydroxylase (PAH) gene. Neonatal screening programs allow an early dietetic treatment with restrictive phenylalanine intake. This diet prevents most of the neuropsychological disabilities but remains challenging for lifelong compliance. Adult-derived human liver progenitor cells (ADHLPC) are a pool of precursors that can differentiate into hepatocytes. We aim to study PAH expression and PAH activity in a differenciated ADHLPC. ADHLPC were isolated from human hepatocyte primary culture of two different donors and differenciated under specific culture conditions. We demonstrated the high expression of PAH and a large increase of PAH activity in differenciated LPC. The age of the donor, the cellular viability after liver digestion and cryopreservation affects PAH activity. ADHLPC might therefore be considered as a suitable source for cell therapy in PKU.

  12. The exosome complex establishes a barricade to erythroid maturation

    Science.gov (United States)

    McIver, Skye C.; Kang, Yoon-A; DeVilbiss, Andrew W.; O’Driscoll, Chelsea A.; Ouellette, Jonathan N.; Pope, Nathaniel J.; Camprecios, Genis; Chang, Chan-Jung; Yang, David; Bouhassira, Eric E.; Ghaffari, Saghi

    2014-01-01

    Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1–mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program. PMID:25115889

  13. Ultrastructure of human neural stem/progenitor cells and neurospheres

    Institute of Scientific and Technical Information of China (English)

    Yaodong Zhao; Tianyi Zhang; Qiang Huang; Aidong Wang; Jun Dong; Qing Lan; Zhenghong Qin

    2009-01-01

    BACKGROUND: Biological and morphological characteristics of neural stern/progenitor cells (NSPCs) have been widely investigated.OBJECTIVE: To explore the ultrastructure of human embryo-derived NSPCs and neurospheres cultivated in vitro using electron microscopy.DESIGN, TIME AND SETTING: A cell biology experiment was performed at the Brain Tumor Laboratory of Soochow University, and Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University between August 2007 and April 2008.MATERIALS: Human fetal brain tissue was obtained from an 8-week-old aborted fetus; serum-free Dulbecco's modified Eagle's medium/F12 culture medium was provided by Gibco, USA; scanning electron microscope was provided by Hitachi instruments, Japan; transmission electron microscope was provided by JEOL, Japan.METHODS: NSPCs were isolated from human fetal brain tissue and cultivated in serum-free Dulbecco's modified Eagle's medium/F12 culture medium. Cells were passaged every 5-7 days. After three passages, NSPCs were harvested and used for ultrastructural examination.MAIN OUTCOME MEASURES: Ultrastructural examination of human NSPCs and adjacent cells in neurospheres.RESULTS: Individual NSPCs were visible as spherical morphologies with rough surfaces under scanning electron microscope. Generally, they had large nuclei and little cytoplasm. Nuclei were frequently globular with large amounts of euchromatin and a small quantity of heterochromatin, and most NSPCs had only one nucleolus. The Golgi apparatus and endoplasmic reticulum were underdeveloped; however, autophagosomes were clearly visible. The neurospheres were made up of NSPCs and non-fixiform material inside. Between adjacent cells and at the cytoplasmic surface of apposed plasma membranes, there were vesicle-like structures. Some membrane boundaries with high permeabilities were observed between some contiguous NSPCs in neurospheres, possibly attributable to plasmalemmal fusion between adjacent cells.CONCLUSION: A large number

  14. Human fetal cardiac progenitors: The role of stem cells and progenitors in the fetal and adult heart.

    Science.gov (United States)

    Bulatovic, Ivana; Månsson-Broberg, Agneta; Sylvén, Christer; Grinnemo, Karl-Henrik

    2016-02-01

    The human fetal heart is formed early during embryogenesis as a result of cell migrations, differentiation, and formative blood flow. It begins to beat around gestation day 22. Progenitor cells are derived from mesoderm (endocardium and myocardium), proepicardium (epicardium and coronary vessels), and neural crest (heart valves, outflow tract septation, and parasympathetic innervation). A variety of molecular disturbances in the factors regulating the specification and differentiation of these cells can cause congenital heart disease. This review explores the contribution of different cardiac progenitors to the embryonic heart development; the pathways and transcription factors guiding their expansion, migration, and functional differentiation; and the endogenous regenerative capacity of the adult heart including the plasticity of cardiomyocytes. Unfolding these mechanisms will become the basis for understanding the dynamics of specific congenital heart disease as well as a means to develop therapy for fetal as well as postnatal cardiac defects and heart failure.

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

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    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.

  16. Characteristic of c-Kit+ progenitor cells in explanted human hearts

    OpenAIRE

    Matuszczak, Sybilla; Czapla, Justyna; Jarosz-Biej, Magdalena; Wiśniewska, Ewa; Cichoń, Tomasz; Smolarczyk, Ryszard; Kobusińska, Magdalena; Gajda, Karolina; Wilczek, Piotr; Śliwka, Joanna; Zembala, Michał; Zembala, Marian; Szala, Stanisław

    2014-01-01

    According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit+ progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit+ progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1 %) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum,...

  17. Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

    Science.gov (United States)

    Hakami, Nora Y.; Ranjan, Amaresh K.; Hardikar, Anandwardhan A.; Dusting, Greg J.; Peshavariya, Hitesh M.

    2017-01-01

    Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.

  18. Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle

    Science.gov (United States)

    Arrighi, N; Moratal, C; Clément, N; Giorgetti-Peraldi, S; Peraldi, P; Loubat, A; Kurzenne, J-Y; Dani, C; Chopard, A; Dechesne, C A

    2015-01-01

    A population of fibro/adipogenic but non-myogenic progenitors located between skeletal muscle fibers was recently discovered. The aim of this study was to determine the extent to which these progenitors differentiate into fully functional adipocytes. The characterization of muscle progenitor-derived adipocytes is a central issue in understanding muscle homeostasis. They are considered as being the cellular origin of intermuscular adipose tissue that develops in several pathophysiological situations. Here fibro/adipogenic progenitors were isolated from a panel of 15 human muscle biopsies on the basis of the specific cell-surface immunophenotype CD15+/PDGFRα+CD56−. This allowed investigations of their differentiation into adipocytes and the cellular functions of terminally differentiated adipocytes. Adipogenic differentiation was found to be regulated by the same effectors as those regulating differentiation of progenitors derived from white subcutaneous adipose tissue. Similarly, basic adipocyte functions, such as triglyceride synthesis and lipolysis occurred at levels similar to those observed with subcutaneous adipose tissue progenitor-derived adipocytes. However, muscle progenitor-derived adipocytes were found to be insensitive to insulin-induced glucose uptake, in association with the impairment of phosphorylation of key insulin-signaling effectors. Our findings indicate that muscle adipogenic progenitors give rise to bona fide white adipocytes that have the unexpected feature of being insulin-resistant. PMID:25906156

  19. Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke.

    Directory of Open Access Journals (Sweden)

    M Z Ratajczak

    2006-06-01

    Full Text Available The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day and 144 hrs (6th day since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM and erythroid (BFU-E-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.

  20. Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells

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    Frech Moritz J

    2010-12-01

    Full Text Available Abstract Background Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process. Results In this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis. Conclusion These results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.

  1. 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.

  2. Human Placenta: a Source of Progenitor/Stem Cells?

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    Parolini O

    2006-01-01

    Full Text Available Regenerative medicine based on cell therapy and tissue engineering methodologies is a newly emerging, multidisciplinary field involving biology, medicine, and genetic manipulation. This type of therapy is aimed at maintaining, restoring, or enhancing tissue and organ function, and is intended to assist in the treatment of a number of human conditions which range in severity from chronic to life threatening. In diseases where tissue or organ function is compromised, stem cell research holds great promise for providing an efficient avenue for regenerative therapy. However, the application of this type of research to the treatment of human disease will not be possible until much more is known about the biological properties of all types of stem cells. In the context of disease treatment, decisions will need to be made as to which is the best type of stem cell to use. Whether it is better to identify a stem cell able to differentiate into all tissue and organ types as opposed to using committed, lineage-specific stem cells, or perhaps embryonic cells are better than adult-derived stem cells, as well as the possible clinical applications of these cells. For some time, these questions have not only required significant scientific and medical consideration, but have also posed important social and ethical questions. There is no doubt that stem cells hold great therapeutic potential, however there is still much research required before their use can be accepted as a valuable tool in disease treatment. In particular, it is necessary to identify a source of stem cells that is easily accessible, provides a high cell yield and for which cell recovery does not provoke serious ethical debate. The aim of this review is to consider and discuss the findings regarding a new source of "adult" stem cells isolated from human term placenta tissue. So far, the placental tissue has generally been discarded post partum, but it is now becoming recognised as a potential

  3. Distribution and characterization of progenitor cells within the human filum terminale.

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    Lisa Arvidsson

    Full Text Available BACKGROUND: Filum terminale (FT is a structure that is intimately associated with conus medullaris, the most caudal part of the spinal cord. It is well documented that certain regions of the adult human central nervous system contains undifferentiated, progenitor cells or multipotent precursors. The primary objective of this study was to describe the distribution and progenitor features of this cell population in humans, and to confirm their ability to differentiate within the neuroectodermal lineage. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that neural stem/progenitor cells are present in FT obtained from patients treated for tethered cord. When human or rat FT-derived cells were cultured in defined medium, they proliferated and formed neurospheres in 13 out of 21 individuals. Cells expressing Sox2 and Musashi-1 were found to outline the central canal, and also to be distributed in islets throughout the whole FT. Following plating, the cells developed antigen profiles characteristic of astrocytes (GFAP and neurons (β-III-tubulin. Addition of PDGF-BB directed the cells towards a neuronal fate. Moreover, the cells obtained from young donors shows higher capacity for proliferation and are easier to expand than cells derived from older donors. CONCLUSION/SIGNIFICANCE: The identification of bona fide neural progenitor cells in FT suggests a possible role for progenitor cells in this extension of conus medullaris and may provide an additional source of such cells for possible therapeutic purposes. Filum terminale, human, progenitor cells, neuron, astrocytes, spinal cord.

  4. Down-regulation of Myc is essential for terminal erythroid maturation.

    Science.gov (United States)

    Jayapal, Senthil Raja; Lee, Kian Leong; Ji, Peng; Kaldis, Philipp; Lim, Bing; Lodish, Harvey F

    2010-12-17

    Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells.

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

    Science.gov (United States)

    Srisen, Kaemisa; Röhrl, Clemens; Meisslitzer-Ruppitsch, Claudia; Ranftler, Carmen; Ellinger, Adolf; Pavelka, Margit; Neumüller, Josef

    2013-01-01

    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 intraellular

  6. 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

  7. Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons

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    Parinya Noisa

    2015-01-01

    Full Text Available Human embryonic stem cells (hESCs are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs. hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

  8. 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.

  9. Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion.

    Science.gov (United States)

    Florio, Marta; Albert, Mareike; Taverna, Elena; Namba, Takashi; Brandl, Holger; Lewitus, Eric; Haffner, Christiane; Sykes, Alex; Wong, Fong Kuan; Peters, Jula; Guhr, Elaine; Klemroth, Sylvia; Prüfer, Kay; Kelso, Janet; Naumann, Ronald; Nüsslein, Ina; Dahl, Andreas; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B

    2015-03-27

    Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

  10. GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation.

    Science.gov (United States)

    Suzuki, Mikiko; Kobayashi-Osaki, Maki; Tsutsumi, Shuichi; Pan, Xiaoqing; Ohmori, Shin'ya; Takai, Jun; Moriguchi, Takashi; Ohneda, Osamu; Ohneda, Kinuko; Shimizu, Ritsuko; Kanki, Yasuharu; Kodama, Tatsuhiko; Aburatani, Hiroyuki; Yamamoto, Masayuki

    2013-11-01

    Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  11. Age-Related Dysfunction in Mechanotransduction Impairs Differentiation of Human Mammary Epithelial Progenitors

    Directory of Open Access Journals (Sweden)

    Fanny A. Pelissier

    2014-06-01

    Full Text Available Dysfunctional progenitor and luminal cells with acquired basal cell properties accumulate during human mammary epithelial aging for reasons not understood. Multipotent progenitors from women aged 55 years is unaffected by physiological stiffness changes. Efficient activation of Hippo pathway transducers YAP and TAZ is required for the modulus-dependent myoepithelial/basal bias in younger progenitors. In older progenitors, YAP and TAZ are activated only when stressed with extraphysiologically stiff matrices, which bias differentiation towards luminal-like phenotypes. In vivo YAP is primarily active in myoepithelia of younger breasts, but localization and activity increases in luminal cells with age. Thus, aging phenotypes of mammary epithelia may arise partly because alterations in Hippo pathway activation impair microenvironment-directed differentiation and lineage specificity.

  12. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

    Science.gov (United States)

    Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui

    2009-07-31

    The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  13. 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

    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...... 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...

  14. 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.

  15. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    Science.gov (United States)

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  16. Circulating human CD34(+) progenitor cells modulate neovascularization and inflammation in a nude mouse model

    NARCIS (Netherlands)

    van der Strate, B. W. A.; Popa, E. R.; Schipper, M.; Brouwer, L. A.; Hendriks, M.; Harmsen, M. C.; van Luyn, M. J. A.

    2007-01-01

    CD34(+) progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34(+) cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34(+) cells were incorporated in Matrigel,

  17. DJ-1 Modulates Nuclear Erythroid 2-Related Factor-2-Mediated Protection in Human Primary Alveolar Type II Cells in Smokers.

    Science.gov (United States)

    Bahmed, Karim; Messier, Elise M; Zhou, Wenbo; Tuder, Rubin M; Freed, Curt R; Chu, Hong Wei; Kelsen, Steven G; Bowler, Russell P; Mason, Robert J; Kosmider, Beata

    2016-09-01

    Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

  18. Characterization of hepatic progenitors from human fetal liver during second trimester

    Institute of Scientific and Technical Information of China (English)

    Mekala Subba Rao; Aleem Ahmed Khan; Nyamath Parveen; Mohammed Aejaz Habeeb; Chittor Mohammed Habibullah; Gopal Pande

    2008-01-01

    AIM: To enrich hepatic progenitors using epithelial cell adhesion molecule (EpCAH) as a marker from human fetal liver and investigate the expression of human leukocyte antigen (HLA) and their markers associated with hepatic progenitor cells.METHODS: EpCAM +ve cells were isolated usingmagnetic cell sorting (MACS) from human fetuses (n =10) at 15-25 wk gestation.Expression of markers for hepatic progenitors such as albumin,alpha-fetoprotein (AFP),CD29 (integrin β1),CD49f (integrin α6) and CD90 (Thy 1) was studied by using flow cytometry,immunocytochemistry and RT-PCR; HLA class Ⅰ (A,B,C) and class Ⅱ (DR) expression was studied by flow cytometry only.RESULTS: FACS analysis indicated that EpCAM +ve cells were positive for CD29,CD49f,CD90,CD34,HLA class Ⅰ,albumin and AFP but negative for HLA class Ⅱ (DR) and CD45.RT PCR showed that EpCAM +ve cells expressed liver epithelial markers (CK18),biliary specific marker (CK19) and hepatic markers (albumin,AFP).On immunocytochemical staining,EpCAH +ve cells were shown positive signals for CK18 and albumin.CONCLUSION: Our study suggests that these EpCAM +ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.

  19. Isolation and culture of human hematopoietic progenitors for studies of dendritic cell biology.

    Science.gov (United States)

    Svensson, Mattias

    2009-01-01

    Understanding the regulation of distinct dendritic cell (DC) function and differentiation pathways is important in many physiological and pathophysiological processes. This includes infectious and neoplastic diseases, vaccination and immunotherapy, allograft rejection, and the pathogenesis of autoimmune diseases. Isolation and culture of human hematopoietic progenitor cells provide a valuable model for studies on DC biology and may help uncover new means to manipulate DC differentiation and function in therapeutic settings. Here, a detailed protocol for the isolation of CD34+ hematopoietic progenitor cells from human cord blood is described. The isolated cell population consists of approximately 85% CD34+ CD45+ hematopoietic progenitor cells that in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor (TNF) expand and differentiate into CD11c+ HLA-DR+ DC-expressing CD1a.

  20. Pathogenesis of the erythroid failure in Diamond Blackfan anaemia.

    Science.gov (United States)

    Sieff, Colin A; Yang, Jing; Merida-Long, Lilia B; Lodish, Harvey F

    2010-02-01

    Diamond Blackfan anaemia (DBA) is a severe congenital failure of erythropoiesis. Despite mutations in one of several ribosome protein genes, including RPS19, the cause of the erythroid specificity is still a mystery. We hypothesized that, because the chromatin of late erythroid cells becomes condensed and transcriptionally inactive prior to enucleation, the rapidly proliferating immature cells require very high ribosome synthetic rates. RNA biogenesis was measured in primary mouse fetal liver erythroid progenitor cells; during the first 24 h, cell number increased three to fourfold while, remarkably, RNA content increased sixfold, suggesting an accumulation of an excess of ribosomes during early erythropoiesis. Retrovirus infected siRNA RPS19 knockdown cells showed reduced proliferation but normal differentiation, and cell cycle analysis showed a G1/S phase delay. p53 protein was increased in the knockdown cells, and the mRNA level for p21, a transcriptional target of p53, was increased. Furthermore, we show that RPS19 knockdown decreased MYB protein, and Kit mRNA was reduced, as was the amount of cell surface KIT protein. Thus, in this small hairpin RNA murine model of DBA, RPS19 insufficient erythroid cells may proliferate poorly because of p53-mediated cell cycle arrest, and also because of decreased expression of the key erythroid signalling protein KIT.

  1. Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Soh, Boon-Seng; Ng, Shi-Yan; Wu, Hao; Buac, Kristina; Park, Joo-Hye C; Lian, Xiaojun; Xu, Jiejia; Foo, Kylie S; Felldin, Ulrika; He, Xiaobing; Nichane, Massimo; Yang, Henry; Bu, Lei; Li, Ronald A; Lim, Bing; Chien, Kenneth R

    2016-03-08

    Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo.

  2. Simulated Microgravity Exerts an Age-Dependent Effect on the Differentiation of Cardiovascular Progenitors Isolated from the Human Heart.

    Directory of Open Access Journals (Sweden)

    Tania I Fuentes

    Full Text Available Microgravity has a profound effect on cardiovascular function, however, little is known about the impact of microgravity on progenitors that reside within the heart. We investigated the effect of simulated microgravity exposure on progenitors isolated from the neonatal and adult human heart by quantifying changes in functional parameters, gene expression and protein levels after 6-7 days of 2D clinorotation. Utilization of neonatal and adult cardiovascular progenitors in ground-based studies has provided novel insight into how microgravity may affect cells differently depending on age. Simulated microgravity exposure did not impact AKT or ERK phosphorylation levels and did not influence cell migration, but elevated transcripts for paracrine factors were identified in neonatal and adult cardiovascular progenitors. Age-dependent responses surfaced when comparing the impact of microgravity on differentiation. Endothelial cell tube formation was unchanged or increased in progenitors from adults whereas neonatal cardiovascular progenitors showed a decline in tube formation (p<0.05. Von Willebrand Factor, an endothelial differentiation marker, and MLC2v and Troponin T, markers for cardiomyogenic differentiation, were elevated in expression in adult progenitors after simulated microgravity. DNA repair genes and telomerase reverse transcriptase which are highly expressed in early stem cells were increased in expression in neonatal but not adult cardiac progenitors after growth under simulated microgravity conditions. Neonatal cardiac progenitors demonstrated higher levels of MESP1, OCT4, and brachyury, markers for early stem cells. MicroRNA profiling was used to further investigate the impact of simulated microgravity on cardiovascular progenitors. Fifteen microRNAs were significantly altered in expression, including microRNAs-99a and 100 (which play a critical role in cell dedifferentiation. These microRNAs were unchanged in adult cardiac progenitors

  3. Targeting oncogene expression to endothelial cells induces proliferation of the myelo-erythroid lineage by repressing the Notch pathway.

    Science.gov (United States)

    Alghisi, E; Distel, M; Malagola, M; Anelli, V; Santoriello, C; Herwig, L; Krudewig, A; Henkel, C V; Russo, D; Mione, M C

    2013-11-01

    Human oncogenes involved in the development of hematological malignancies have been widely used to model experimental leukemia. However, models of myeloid leukemia rarely reproduce the human disease in full, due to genetic complexity or to difficulties in targeting leukemia initiating cells. Here, we used a zebrafish genetic model to induce the expression of oncogenic RAS in endothelial cells, including the hemogenic endothelium of the dorsal aorta that generates hematopoietic cells, and observed the development of a myelo-erythroid proliferative disorder. In larvae, the phenotype is characterized by disruption of the vascular system and prominent expansion of the caudal hematopoietic tissue. In few surviving juveniles, increased number of immature hematopoietic cells and arrest of myeloid maturation was found in kidney marrow. Peripheral blood showed increased erythroblasts and myeloid progenitors. We found that the abnormal phenotype is associated with a downregulation of the Notch pathway, whereas overexpressing an activated form of Notch together with the oncogene prevents the expansion of the myelo-erythroid compartment. This study identifies the downregulation of the Notch pathway following an oncogenic event in the hemogenic endothelium as an important step in the pathogenesis of myelo-erythroid disorders and describes a number of potential effectors of this transformation.

  4. 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....

  5. Regulatory functions of TRAIL in hematopoietic progenitors: human umbilical cord blood and murine bone marrow transplantation.

    Science.gov (United States)

    Mizrahi, K; Stein, J; Pearl-Yafe, M; Kaplan, O; Yaniv, I; Askenasy, N

    2010-07-01

    The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling pathway has selective toxicity to malignant cells. The TRAIL receptors DR4 and DR5 are expressed at low levels in human umbilical cord blood cells (3-15%) and are upregulated by incubation with the cognate ligand, triggering apoptosis in 70-80% of receptor-positive cells (P<0.001). Apoptosis is not induced in hematopoietic progenitors, as determined from sustained severe combined immunodeficiency reconstituting potential and clonogenic activity. Furthermore, elimination of dead cells after incubation with TRAIL for 72 h results in a threefold enrichment in myeloid progenitors. Exposure to TRAIL in semisolid cultures showed synergistic activity of DR4 and granulocyte/macrophage colony-stimulating factor in recruiting lineage-negative (lin(-)) and CD34(+) progenitors and in promoting the formation of large colonies. In murine bone marrow, approximately 30% of lin(-) cells express TRAIL-R2 (the only murine receptor), and the receptor is upregulated after transplantation in cycling and differentiating donor cells that home to the host marrow. However, this receptor is almost ubiquitously expressed in the most primitive (lin(-)SCA-1(+)c-kit(+)) progenitors, and stimulates the clonogenic activity of lin(-) cells (P<0.001), suggesting a tropic function after transplantation. It is concluded that TRAIL does not trigger apoptosis in hematopoietic progenitors, and upregulation of its cognate receptors under stress conditions mediates tropic signaling that supports recovery from hypoplasia.

  6. High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker.

    Science.gov (United States)

    Choi, He Yun; Park, Ji Hye; Jang, Woong Bi; Ji, Seung Taek; Jung, Seok Yun; Kim, Da Yeon; Kang, Songhwa; Kim, Yeon Ju; Yun, Jisoo; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang-Mo

    2016-07-01

    Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes.

  7. Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept.

    Science.gov (United States)

    Boecker, Werner; Buerger, Horst

    2003-10-01

    Although experimental data clearly confirm the existence of self-renewing mammary stem cells, the characteristics of such progenitor cells have never been satisfactorily defined. Using a double immunofluorescence technique for simultaneous detection of the basal cytokeratin 5, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle actin (SMA), we were able to demonstrate the presence of CK5+ cells in human adult breast epithelium. These cells have the potential to differentiate to either glandular (CK8/18+) or myoepithelial cells (SMA+) through intermediary cells (CK5+ and CK8/18+ or SMA+). We therefore proceeded on the assumption that the CK5+ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of human breast epithelium. Furthermore, we furnish evidence that most of these progenitor cells are located in the luminal epithelium of the ductal lobular tree. Based on data obtained in extensive analyses of proliferative breast disease lesions, we have come to regard usual ductal hyperplasia as a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Double immunofluorescence experiments provide a new tool to characterize phenotypically progenitor (adult stem) cells and their progenies. This model has been shown to be of great value for a better understanding not only of normal tissue regeneration but also of proliferative breast disease. Furthermore, this model provides a new tool for unravelling further the regulatory mechanisms that govern normal and pathological cell growth.

  8. 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......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...... 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....

  9. In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

    Science.gov (United States)

    Cambot, Marie; Mazurier, Christelle; Canoui-Poitrine, Florence; Hebert, Nicolas; Picot, Julien; Clay, Denis; Picard, Véronique; Ripoche, Pierre; Douay, Luc; Dubart-Kupperschmitt, Anne; Cartron, Jean-Pierre

    2013-05-01

    Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer

    Directory of Open Access Journals (Sweden)

    Vêncio Ricardo Z

    2007-06-01

    Full Text Available Abstract Background Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer. Methods CD34+/CD133+ progenitor cells were purified from human umbilical cord blood and expanded in vitro. Correlated molecular changes were analyzed by gene expression profiling using microarrays covering up to 55,000 transcripts. Genes regulated during progenitor cell expansion were identified and functionally classified. Aberrant expression of such genes in cancer was indicated by in silico SAGE. Differential expression of selected genes was assessed by real-time PCR in hematopoietic cells from chronic myeloid leukemia patients and healthy individuals. Results Several genes and signaling pathways not previously associated with ex vivo expansion of CD133+/CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes was detected during conditions of enhanced progenitor cell expansion. Quantitative real-time PCR analysis confirmed down-regulation of several newly described cancer-associated genes in CD133+/CD34+ cells, including DOCK4 and SPARCL1 tumor suppressors, and parallel results were verified when comparing their expression in cells from chronic myeloid leukemia patients Conclusion Our findings reveal potential molecular targets for oncogenic transformation in CD133+/CD34+ cells and strengthen the link between deregulation of stem/progenitor cell expansion and the malignant process.

  12. Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate.

    Science.gov (United States)

    Zhao, Xiangshan; Malhotra, Gautam K; Lele, Subodh M; Lele, Manjiri S; West, William W; Eudy, James D; Band, Hamid; Band, Vimla

    2010-08-10

    There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

  13. LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages

    Science.gov (United States)

    Casero, David; Sandoval, Salemiz; Seet, Christopher S.; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M.

    2015-01-01

    To elucidate the transcriptional landscape that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitors spanning the earliest stages of B and T lymphoid specification. Over 3000 novel long non-coding RNA genes (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage-specific and more lineage-specific than protein coding patterns. Protein-coding genes co-expressed with neighboring lncRNA genes were enriched for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships between the earliest progenitors in the human bone marrow and thymus. PMID:26502406

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Improves Senescent Human Dermal Fibroblasts

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    Ji-Yong Jung

    2015-08-01

    Full Text Available Adult skin stem cells are recognized as potential therapeutics to rejuvenate aged skin. We previously demonstrated that human dermal stem/progenitor cells (hDSPCs with multipotent capacity could be enriched from human dermal fibroblasts using collagen type IV. However, the effects of hDSPCs on cellular senescence remain to be elucidated. In the present study, we investigated whether conditioned medium (CM collected from hDSPC cultures (hDSPC-CM exhibits beneficial effects on senescent fibroblasts. We found that hDSPC-CM promoted proliferation and decreased the expression level of senescence-associated β-galactosidase in senescent fibroblasts. In addition, p53 phosphorylation and p21 expression were significantly reduced in senescent fibroblasts treated with hDSPC-CM. hDSPC-CM restored the expression levels of collagen type I, collagen type III, and tissue inhibitor of metalloproteinase, and antagonized the increase of matrix metalloproteinase 1 expression. Finally, we demonstrated that hDSPC-CM significantly reduced reactive oxygen species levels by specifically up-regulating the expression level of superoxide dismutase 2. Taken together, these data suggest that hDSPC-CM can be applied as a potential therapeutic agent for improving human aged skin.

  16. Differential expression of CD150 (SLAM) family receptors by human hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Sintes, Jordi; Romero, Xavier; Marin, Pedro; Terhorst, Cox; Engel, Pablo

    2008-09-01

    Human hematopoietic stem cell (HSC)-containing grafts are most commonly used to treat various blood diseases, including leukemias and autoimmune disorders. CD150 (SLAM) family receptors have recently been shown to be differentially expressed by mouse HSC and progenitor cells. Members of the CD150 family are key regulators of leukocyte activation and differentiation. The goal of the present study is to analyze the expression patterns of the CD150 receptors CD48, CD84, CD150 (SLAM), CD229 (Ly9), and CD244 (2B4) on the different sources of human hematopoietic stem and progenitor cells. Expression of CD150 receptors was analyzed on human mobilized peripheral blood CD133(+)-isolated cells and CD34(+) bone marrow (BM) and umbilical cord blood (CB) cells using multicolor flow cytometry. CD244 was present on most CD133(+)Lin(-)-mobilized cells and CD34(+)Lin(-) BM and CB cells, including virtually all CD38(-)Lin(-) primitive progenitor cells. CD48 had a restricted expression pattern on CD133(+)Lin(-)CD38(-) cells, while its levels were significantly higher in CD34(+)Lin(-) BM and CB cells. In addition, CD84 was present on a significant number of CD133(+)Lin(-) cells, but only on a small fraction of CD133(+)Lin(-)CD38(-) peripheral blood mobilized cells. In contrast, CD84 was expressed on practically all CD34(+)Lin(-) BM cells. No CD150 expression was observed in mobilized peripheral blood CD133(+)Lin(-) or CD34(+)Lin(-) BM and CB cells. Furthermore, only a small fraction of CD34(+)Lin(-) BM and CB cells expressed CD229. Our results show that CD150 family molecules are present on human hematopoietic stem and progenitor cells and that their expression patterns differ between humans and mice.

  17. Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors.

    Science.gov (United States)

    Chin, Chee Jia; Cooper, Aaron R; Lill, Georgia R; Evseenko, Denis; Zhu, Yuhua; He, Chong Bin; Casero, David; Pellegrini, Matteo; Kohn, Donald B; Crooks, Gay M

    2016-05-01

    Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors. Stem Cells 2016;34:1239-1250.

  18. A human postnatal lymphoid progenitor capable of circulating and seeding the thymus.

    Science.gov (United States)

    Six, Emmanuelle M; Bonhomme, Delphine; Monteiro, Marta; Beldjord, Kheira; Jurkowska, Monika; Cordier-Garcia, Corinne; Garrigue, Alexandrine; Dal Cortivo, Liliane; Rocha, Benedita; Fischer, Alain; Cavazzana-Calvo, Marina; André-Schmutz, Isabelle

    2007-12-24

    Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34(+)CD10(+) progenitor population and which is distinct from B cell-committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin(-)CD34(+)CD10(+)CD24(-) progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor alpha, and CD3epsilon. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.

  19. Ultrastructural Evidence of Exosome Secretion by Progenitor Cells in Adult Mouse Myocardium and Adult Human Cardiospheres

    Directory of Open Access Journals (Sweden)

    Lucio Barile

    2012-01-01

    Full Text Available The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34+ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an “off-the-shelf” product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  20. The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene

    NARCIS (Netherlands)

    R. Tewari (Rita); N. Gillemans (Nynke); A. Harper; M.G.J.M. Wijgerde (Mark); G. Zafarana (Gaetano); D.D. Drabek (Dubravka); F.G. Grosveld (Frank); J.N.J. Philipsen (Sjaak)

    1996-01-01

    textabstractThe beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been

  1. The human β-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial β-galactosidase gene.

    NARCIS (Netherlands)

    R. Tewari (Rita); N. Gillemans (Nynke); A. Harper; M.G.J.M. Wijgerde (Mark); G. Zafarana (Gaetano); D.D. Drabek (Dubravka); F.G. Grosveld (Frank); J.N.J. Philipsen (Sjaak)

    1996-01-01

    textabstractThe beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the

  2. 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

  3. Human primordial germ cell-derived progenitors give rise to neurons and glia in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Yincheng [Department of Gynecology and Obstetrics, The 6th People' s Hospital, School of Medicine, Shanghai Jiao Tong University, 600 Yishan Road, Shanghai 200233 (China); Chen, Bin [Center for Developmental Biology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kong Jiang Road, Shanghai 200092 (China); Tao, Minfang, E-mail: Taomf@126.com [Department of Gynecology and Obstetrics, The 6th People' s Hospital, School of Medicine, Shanghai Jiao Tong University, 600 Yishan Road, Shanghai 200233 (China)

    2009-12-18

    We derived a cell population from cultured human primordial germ cells from early human embryos. The derivates, termed embryoid body-derived (EBD) cells, displayed an extensive capacity for proliferation and expressed a panel of markers in all three germ layers. Interestingly, EBD cells were also positive for markers of neural stem/progenitor cells, such as nestin and glial fibrillary acidic protein. When these cells were transplanted into the brain cavities of fetal sheep and postnatal NOD-SCID mice or nerve-degenerated tibialis anterior muscles, they readily gave rise to neurons or glial cells. To our knowledge, our data are the first to demonstrate that EBD cells can undergo further neurogenesis under suitable environments in vivo. Hence, with the abilities of extensive expansion, self-renewal, and differentiation, EBD cells may provide a useful donor source for neural stem/progenitor cells to be used in cell-replacement therapies for diseases of the nervous system.

  4. Brief Report: Efficient Generation of Hematopoietic Precursors and Progenitors from Human Pluripotent Stem Cell Lines

    Science.gov (United States)

    Woods, Niels-Bjarne; Parker, Aaron S.; Moraghebi, Roksana; Lutz, Margaret K.; Firth, Amy L.; Brennand, Kristen J.; Berggren, W. Travis; Raya, Angel; Izpisúa Belmonte, Juan Carlos; Gage, Fred H.; Verma, Inder M.

    2012-01-01

    By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD341 and CD45+/CD341/CD38− hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD341) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability. PMID:21544903

  5. Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines.

    Science.gov (United States)

    Woods, Niels-Bjarne; Parker, Aaron S; Moraghebi, Roksana; Lutz, Margaret K; Firth, Amy L; Brennand, Kristen J; Berggren, W Travis; Raya, Angel; Izpisúa Belmonte, Juan Carlos; Gage, Fred H; Verma, Inder M

    2011-07-01

    By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.

  6. Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue.

    Science.gov (United States)

    Ardehali, Reza; Ali, Shah R; Inlay, Matthew A; Abilez, Oscar J; Chen, Michael Q; Blauwkamp, Timothy A; Yazawa, Masayuki; Gong, Yongquan; Nusse, Roeland; Drukker, Micha; Weissman, Irving L

    2013-02-26

    A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

  7. 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

  8. Perivascular mesenchymal progenitors in human fetal and adult liver.

    Science.gov (United States)

    Gerlach, Jörg C; Over, Patrick; Turner, Morris E; Thompson, Robert L; Foka, Hubert G; Chen, William C W; Péault, Bruno; Gridelli, Bruno; Schmelzer, Eva

    2012-12-10

    The presence of mesenchymal stem cells (MSCs) has been described in various organs. Pericytes possess a multilineage differentiation potential and have been suggested to be one of the developmental sources for MSCs. In human liver, pericytes have not been defined. Here, we describe the identification, purification, and characterization of pericytes in human adult and fetal liver. Flow cytometry sorting revealed that human adult and fetal liver contains 0.56%±0.81% and 0.45%±0.39% of CD146(+)CD45(-)CD56(-)CD34(-) pericytes, respectively. Of these, 41% (adult) and 30% (fetal) were alkaline phosphatase-positive (ALP(+)). In situ, pericytes were localized around periportal blood vessels and were positive for NG2 and vimentin. Purified pericytes could be cultured extensively and had low population doubling times. Immunofluorescence of cultures demonstrated that cells were positive for pericyte and mesenchymal cell markers CD146, NG2, CD90, CD140b, and vimentin, and negative for endothelial, hematopoietic, stellate, muscle, or liver epithelial cell markers von Willebrand factor, CD31, CD34, CD45, CD144, CD326, CK19, albumin, α-fetoprotein, CYP3A7, glial fibrillary acid protein, MYF5, and Pax7 by gene expression; myogenin and alpha-smooth muscle actin expression were variable. Fluorescence-activated cell sorting analysis of cultures confirmed surface expression of CD146, CD73, CD90, CD10, CD13, CD44, CD105, and ALP and absence of human leukocyte antigen-DR. In vitro differentiation assays demonstrated that cells possessed robust osteogenic and myogenic, but low adipogenic and low chondrogenic differentiation potentials. In functional in vitro assays, cells had typical mesenchymal strong migratory and invasive activity. In conclusion, human adult and fetal livers harbor pericytes that are similar to those found in other organs and are distinct from hepatic stellate cells.

  9. DETECTION AND CHARACTERISTIC OF FLAT ERYTHROID COLONIES IN SEMISOLID CULTURAL MEDIUMS

    Directory of Open Access Journals (Sweden)

    M. D. Kuchma

    2014-06-01

    Full Text Available It have been shown that progenitor cells of cord blood, bone marrow and «mobilized» peripheral blood in semisolid mediums gave flat erythroid colonies. These colonies are able to form one or more red centers on the 14th day of cultivation and get a big size that evidence about high proliferative activity and resemble granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte colony-forming units. However 92% of the cells of flat colonies express CD235. It shows that the colonies are erythroid, although colony morphology differs from burstoforming erythroid units and erythroid colony forming units. Their occurrence probability in methylcellulose-containing medium is 2,5%±1%, that is significantly lower than in agar- containing medium (58%±4,8%. Thus, we suggested that flat colonies should be counted separately or they should be ascribed as BFU-E.

  10. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

    Science.gov (United States)

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young

    2016-09-01

    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

  11. Response of Human Fetal Liver Progenitor Cell Types to Temperature and pH Stresses In Vitro.

    Science.gov (United States)

    Schmelzer, Eva; Foka, Hubert G; Thompson, Robert L; Luca, Angelo; Gridelli, Bruno; Gerlach, Jörg C

    2017-09-11

    Prolonged physiological stresses including abnormal pH and temperature are deleterious. Yet, human hepatic progenitors have been shown to be quite tolerant of temporary temperature stress such as in cold ischemia. We aimed to identify how various stresses affect liver progenitors, and to determine whether distinct effects exist on different progenitor cells of the human liver. Total fetal liver cells were exposed to low (25°C), normal (37°C), or high (40°C) temperatures, or low (6.76), normal (7.35), or high (7.88) pH in vitro. Culture at 25°C increased cell numbers and percentages of proliferation marker Ki67 positive total cells. In total cell cultures, percentages of CD326+ hepatic progenitors co-expressing DLK1 (delta-like 1 homolog), SSEA4, or CD90 increased, as well as proliferation of SSEA4+ and CD235a+ progenitors. Analyses of pre-sorted hepatic progenitors revealed that culture at 25°C increased cell numbers of CD326+ hepatic stem/progenitor cells but not DLK+ hepatoblasts. The expressions of several mesenchymal genes were reduced, and distinct hepatic stem/progenitor cell colonies emerged. At 40°C, numbers of adherent hepatic cells decreased but those of hematopoietic non-adherent cells increased. High pH did not cause major effects. Acidic pH resulted in decreased total cell numbers and affected hematopoietic cells. Percentages of DLK1+ hepatoblasts were increased but those of hematopoietic mature CD45+ cells were decreased. In particular, proliferation of adherent hepatic CD326+, SSEA4+ progenitors, and hematopoietic CD45+ cells and CD235a+ erythroblasts were reduced. Conclusively, our data indicate that low-temperature stress stimulates hepatic progenitor and erythroblast proliferation, whereas acidic pH promotes hepatic maturation and reduces hematopoietic cells.

  12. Isolation of Human Fetal Liver Progenitors and Their Enhanced Proliferation by Three-Dimensional Coculture with Endothelial Cells

    Science.gov (United States)

    Xiong, Anming; Austin, Timothy W.; Lagasse, Eric; Uchida, Nobuko; Tamaki, Stanley; Bordier, Bruno B.; Weissman, Irving L.; Glenn, Jeffrey S.; Millan, Maria T.

    2008-01-01

    Liver progenitor cells, characterized by the coexpression of biliary and hepatocyte lineage markers and the ability to form colonies in culture, were isolated by flow cytometry from primary human fetal livers. These prospectively isolated liver progenitor cells supported hepatitis D virus infection, expressed, and produced albumin and α-fetoprotein, as tracked by albumin-and α-fetoprotein–driven lentiviral promoter reporter constructs and measured by ELISA, respectively. Coculture in three-dimensional (3D) fibrin gel with endothelial cells resulted in the formation of vascular structures by the endothelial cells and increased proliferation of liver progenitors. The enhanced proliferation of liver progenitors that was observed when liver progenitors and endothelial cells were cultured in direct contact was not achieved when liver progenitors and endothelial cells were cultured on adjacent but separate matrices and when they were cultured across transwell membranes. In conclusion, coculture of liver progenitors and endothelial cells in three-dimensional matrix resulted in enhanced liver progenitor proliferation and function. This coculture methodology offers a novel coculture system that could be applied for the development of engineered liver tissues. PMID:19230124

  13. 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.

  14. 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. PMID:26795421

  15. 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.

  16. 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

    2016-01-22

    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.

  17. The complexity of the calretinin-expressing progenitors in the human cerebral cortex

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    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.

  18. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

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    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.

  19. 脐血单个核细胞来源红系祖细胞的诱导扩增及保存的研究%The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    陈琳; 范增; 裴雪涛; 谢小燕; 习佳飞; 吕洋; 田宇; 刘大庆岳文; 李艳华; 南雪; 李思婷

    2016-01-01

    Objective To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells(EPCs)derived from umbilical cord blood(UCB)mononuclear cells(MNCs). Methods UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing. Results With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14-day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as(88.92±0.95)%. The percentages of cell surface markers were(86.77±9.11)%forCD71 and(64.47 ± 16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright-Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells(326.00 ± 97.96 vs 61.60 ± 20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10%DMSO were better than 5%DMSO. Additionally, 10%DMSO+2%HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more

  20. Intracellular Immunization of Human Fetal Cord Blood Stem/Progenitor Cells with a Ribozyme Against Human Immunodeficiency Virus Type 1

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    Yu, Mang; Leavitt, Mark C.; Maruyama, Midori; Yamada, Osamu; Young, Dennis; Ho, Anthony D.; Wong-Staal, Flossie

    1995-01-01

    Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34^+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

  1. Human progenitor cell recruitment via SDF-1α coacervate-laden PGS vascular grafts.

    Science.gov (United States)

    Lee, Kee-Won; Johnson, Noah R; Gao, Jin; Wang, Yadong

    2013-12-01

    Host cell recruitment is crucial for vascular graft remodeling and integration into the native blood vessel; it is especially important for cell-free strategies which rely on host remodeling. Controlled release of growth factors from vascular grafts may enhance host cell recruitment. Stromal cell-derived factor (SDF)-1α has been shown to induce host progenitor cell migration and recruitment; however, its potential in regenerative therapies is often limited due to its short half-life in vivo. This report describes a coacervate drug delivery system for enhancing progenitor cell recruitment into an elastomeric vascular graft by conferring protection of SDF-1α. Heparin and a synthetic polycation are used to form a coacervate, which is incorporated into poly(glycerol sebacate) (PGS) scaffolds. In addition to protecting SDF-1α, the coacervate facilitates uniform scaffold coating. Coacervate-laden scaffolds have high SDF-1α loading efficiency and provide sustained release under static and physiologically-relevant flow conditions with minimal initial burst release. In vitro assays showed that coacervate-laden scaffolds enhance migration and infiltration of human endothelial and mesenchymal progenitor cells by maintaining a stable SDF-1α gradient. These results suggest that SDF-1α coacervate-laden scaffolds show great promise for in situ vascular regeneration.

  2. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells.

    Science.gov (United States)

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.

  3. Dynamic Expression Patterns of Progenitor and Neuron Layer Markers in the Developing Human Dentate Gyrus and Fimbria.

    Science.gov (United States)

    Cipriani, Sara; Journiac, Nathalie; Nardelli, Jeannette; Verney, Catherine; Delezoide, Anne-Lise; Guimiot, Fabien; Gressens, Pierre; Adle-Biassette, Homa

    2017-01-01

    The molecular mechanisms that orchestrate the development of the human dentate gyrus are not known. In this study, we characterized the formation of human dentate and fimbrial progenitors and postmitotic neurons from 9 gestational weeks (GW9) to GW25. PAX6+ progenitor cells remained proliferative until GW16 in the dentate ventricular zone. By GW11, the secondary dentate matrix had developed in the intermediate zone, surrounding the dentate anlage and streaming toward the subpial layer. This secondary matrix contained proliferating PAX6+ and/or TBR2+ progenitors. In parallel, SOX2+ and PAX6+ fimbrial cells were detected approaching the dentate anlage, representing a possible source of extra-dentate progenitors. By GW16, when the granule cell layer could be delineated, a hilar matrix containing PAX6+ and some TBR2+ progenitors had become identifiable. By GW25, when the 2 limbs of the granule cell layer had formed, the secondary dentate matrix was reduced to a pool of progenitors at the fimbrio-dentate junction. Although human dentate development recapitulates key steps previously described in rodents, differences seemed to emerge in neuron layer markers expression. Further studies are necessary to better elucidate their role in dentate formation and connectivity. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. 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.

  5. Brief Report: Robo1 Regulates the Migration of Human Subventricular Zone Neural Progenitor Cells During Development.

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    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.

  6. Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage.

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    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

  7. SBDS expression and localization at the mitotic spindle in human myeloid progenitors.

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    Claudia Orelio

    Full Text Available BACKGROUND: Shwachman-Diamond Syndrome (SDS is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML. Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle. CONCLUSIONS/SIGNIFICANCE: SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34(+ hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when

  8. Growth Induction and Low-Oxygen Apoptosis Inhibition of Human CD34+ Progenitors in Collagen Gels

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    Daniele Avitabile

    2013-01-01

    Full Text Available Various reports have indicated low survival of injected progenitors into unfavorable environments such as the ischemic myocardium or lower limb tissues. This represents a major bottleneck in stem-cell-based cardiovascular regenerative medicine. Strategies to enhance survival of these cells in recipient tissues have been therefore sought to improve stem cell survival and ensure long-term engraftment. In the present contribution, we show that embedding human cord blood-derived CD34+ cells into a collagen I-based hydrogel containing cytokines is a suitable strategy to promote stem cell proliferation and protect these cells from anoxia-induced apoptosis.

  9. Transplantation of human oligodendrocyte progenitor cells in an animal model of diffuse traumatic axonal injury: survival and differentiation.

    Science.gov (United States)

    Xu, Leyan; Ryu, Jiwon; Hiel, Hakim; Menon, Adarsh; Aggarwal, Ayushi; Rha, Elizabeth; Mahairaki, Vasiliki; Cummings, Brian J; Koliatsos, Vassilis E

    2015-05-14

    Diffuse axonal injury is an extremely common type of traumatic brain injury encountered in motor vehicle crashes, sports injuries, and in combat. Although many cases of diffuse axonal injury result in chronic disability, there are no current treatments for this condition. Its basic lesion, traumatic axonal injury, has been aggressively modeled in primate and rodent animal models. The inexorable axonal and perikaryal degeneration and dysmyelination often encountered in traumatic axonal injury calls for regenerative therapies, including therapies based on stem cells and precursors. Here we explore the proof of concept that treatments based on transplants of human oligodendrocyte progenitor cells can replace or remodel myelin and, eventually, contribute to axonal regeneration in traumatic axonal injury. We derived human oligodendrocyte progenitor cells from the human embryonic stem cell line H9, purified and characterized them. We then transplanted these human oligodendrocyte progenitor cells into the deep sensorimotor cortex next to the corpus callosum of nude rats subjected to traumatic axonal injury based on the impact acceleration model of Marmarou. We explored the time course and spatial distribution of differentiation and structural integration of these cells in rat forebrain. At the time of transplantation, over 90 % of human oligodendrocyte progenitor cells expressed A2B5, PDGFR, NG2, O4, Olig2 and Sox10, a profile consistent with their progenitor or early oligodendrocyte status. After transplantation, these cells survived well and migrated massively via the corpus callosum in both injured and uninjured brains. Human oligodendrocyte progenitor cells displayed a striking preference for white matter tracts and were contained almost exclusively in the corpus callosum and external capsule, the striatopallidal striae, and cortical layer 6. Over 3 months, human oligodendrocyte progenitor cells progressively matured into myelin basic protein(+) and adenomatous

  10. Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth.

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    Degistirici, Ozer; Jaquiery, Claude; Schönebeck, Bodo; Siemonsmeier, Jürgen; Götz, Werner; Martin, Ivan; Thie, Michael

    2008-02-01

    The connective tissue of the human tooth arises from cells that are derived from the cranial neural crest and, thus, are termed as "ectomesenchymal cells." Here, cells being located in a pad-like tissue adjacent to the apex of the developing tooth, which we designated the third molar pad, were separated by the microexplant technique. When outgrowing from the explant, dental neural crest-derived progenitor cells (dNC-PCs) adhered to plastic, proliferated steadily, and displayed a fibroblast-like morphology. At the mRNA level, dNC-PCs expressed neural crest marker genes like Sox9, Snail1, Snail2, Twist1, Msx2, and Dlx6. Cytofluorometric analysis indicated that cells were positive for CD49d (alpha4 integrin), CD56 (NCAM), and PDGFRalpha, while negative for CD31, CD34, CD45, and STRO-1. dNC-PCs could be differentiated into neurogenic, chondrogenic, and osteogenic lineages and were shown to produce bone matrix in athymic mice. These results demonstrate that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells. As a rather large amount of dNC-PCs could be obtained from each single third molar, cells may be used to regenerate a wide range of tissues within the craniofacial region of humans.

  11. What is the potential of oligodendrocyte progenitor cells to successfully treat human spinal cord injury?

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    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.

  12. Dynamic Expression Patterns of Progenitor and Pyramidal Neuron Layer Markers in the Developing Human Hippocampus.

    Science.gov (United States)

    Cipriani, Sara; Nardelli, Jeannette; Verney, Catherine; Delezoide, Anne-Lise; Guimiot, Fabien; Gressens, Pierre; Adle-Biassette, Homa

    2016-03-01

    The molecular mechanisms underlying the formation of hippocampus are unknown in humans. To improve our knowledge of molecules that potentially regulate pyramidal neurogenesis and layering in various hippocampal fields, we investigated the expression of progenitor markers and cell fate molecules from gestational week (GW) 9 to GW 20. At GW 9, the progenitor cell compartment of the hippocampal formation mainly consisted of PAX6(+) cells in the ventricular zone. Between GW 9 and 11, a second germinal area, the subventricular zone (SVZ), was formed, as shown by TBR2 labeling. Postmitotic markers (TBR1, CTIP2, SATB2, and CUX1) might reflect the inside-out layering of the plate from GW 11 onwards. TBR1(+) neurons appeared in the deep plate, whereas CTIP2(+), SATB2(+), and CUX1(+) neurons occupied the upper layers. From GW 16, differences in layer segregation were observed between the ammonic and subicular plates. Moreover, an ammonic-to-subicular maturation gradient was observed in germinal/postmitotic areas. Taken together, these findings demonstrate for the first time the presence of an SVZ in the hippocampus of human fetuses and laminar differences in transcription factor expression in the pyramidal layer of the human ammonic and subicular plate, and provide new information to further investigate the connectivity of the hippocampal formation.

  13. Protection of visual functions by human neural progenitors in a rat model of retinal disease.

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    David M Gamm

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  14. Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience.

    Science.gov (United States)

    Menasché, Philippe; Vanneaux, Valérie; Fabreguettes, Jean-Roch; Bel, Alain; Tosca, Lucie; Garcia, Sylvie; Bellamy, Valérie; Farouz, Yohan; Pouly, Julia; Damour, Odile; Périer, Marie-Cécile; Desnos, Michel; Hagège, Albert; Agbulut, Onnik; Bruneval, Patrick; Tachdjian, Gérard; Trouvin, Jean-Hugues; Larghero, Jérôme

    2015-03-21

    There is now compelling evidence that cells committed to a cardiac lineage are most effective for improving the function of infarcted hearts. This has been confirmed by our pre-clinical studies entailing transplantation of human embryonic stem cell (hESC)-derived cardiac progenitors in rat and non-human primate models of myocardial infarction. These data have paved the way for a translational programme aimed at a phase I clinical trial. The main steps of this programme have included (i) the expansion of a clone of pluripotent hESC to generate a master cell bank under good manufacturing practice conditions (GMP); (ii) a growth factor-induced cardiac specification; (iii) the purification of committed cells by immunomagnetic sorting to yield a stage-specific embryonic antigen (SSEA)-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (iv) the incorporation of these cells into a fibrin scaffold; (v) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (vi) an extensive cytogenetic and viral testing; and (vii) the characterization of the final cell product and its release criteria. The data collected throughout this process have led to approval by the French regulatory authorities for a first-in-man clinical trial of transplantation of these SSEA-1(+) progenitors in patients with severely impaired cardiac function. Although several facets of this manufacturing process still need to be improved, these data may yet provide a useful platform for the production of hESC-derived cardiac progenitor cells under safe and cost-effective GMP conditions. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  15. Beta2-adrenergic signaling affects the phenotype of human cardiac progenitor cells through EMT modulation.

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    Pagano, Francesca; Angelini, Francesco; Siciliano, Camilla; Tasciotti, Julia; Mangino, Giorgio; De Falco, Elena; Carnevale, Roberto; Sciarretta, Sebastiano; Frati, Giacomo; Chimenti, Isotta

    2017-01-15

    Human cardiac progenitor cells (CPCs) offer great promises to cardiac cell therapy for heart failure. Many in vivo studies have shown their therapeutic benefits, paving the way for clinical translation. The 3D model of cardiospheres (CSs) represents a unique niche-like in vitro microenvironment, which includes CPCs and supporting cells. CSs have been shown to form through a process mediated by epithelial-to-mesenchymal transition (EMT). β2-Adrenergic signaling significantly affects stem/progenitor cells activation and mobilization in multiple tissues, and crosstalk between β2-adrenergic signaling and EMT processes has been reported. In the present study, we aimed at investigating the biological response of CSs to β2-adrenergic stimuli, focusing on EMT modulation in the 3D culture system of CSs. We treated human CSs and CS-derived cells (CDCs) with the β2-blocker butoxamine (BUT), using either untreated or β2 agonist (clenbuterol) treated CDCs as control. BUT-treated CS-forming cells displayed increased migration capacity and a significant increase in their CS-forming ability, consistently associated with increased expression of EMT-related genes, such as Snai1. Moreover, long-term BUT-treated CDCs contained a lower percentage of CD90+ cells, and this feature has been previously correlated with higher cardiogenic and therapeutic potential of the CDCs population. In addition, long-term BUT-treated CDCs had an increased ratio of collagen-III/collagen-I gene expression levels, and showed decreased release of inflammatory cytokines, overall supporting a less fibrosis-prone phenotype. In conclusion, β2 adrenergic receptor block positively affected the stemness vs commitment balance within CSs through the modulation of type1-EMT (so called "developmental"). These results further highlight type-1 EMT to be a key process affecting the features of resident cardiac progenitor cells, and mediating their response to the microenvironment.

  16. Derivation of Neural Progenitors and Retinal Pigment Epithelium from Common Marmoset and Human Pluripotent Stem Cells

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    Laughing Bear Torrez

    2012-01-01

    Full Text Available Embryonic and induced pluripotent stem cells (IPSCs derived from mammalian species are valuable tools for modeling human disease, including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs and retinal pigmented epithelium (RPE to the retina. Here we used transgenic common marmoset (Callithrix jacchus and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures, we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications.

  17. Knockdown of HPRT for selection of genetically modified human hematopoietic progenitor cells.

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    Rashmi Choudhary

    Full Text Available The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC using 6-thioguanine (6TG. Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45(+ cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection.

  18. A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

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    Moe, Morten C; Kolberg, Rebecca S; Sandberg, Cecilie; Vik-Mo, Einar; Olstorn, Havard; Varghese, Mercy; Langmoen, Iver A; Nicolaissen, Bjørn

    2009-01-01

    Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human

  19. Derivation of high purity neuronal progenitors from human embryonic stem cells.

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    Gabriel Nistor

    Full Text Available The availability of human neuronal progenitors (hNPs in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use.

  20. Direct and indirect effects of immune and central nervous system-resident cells on human oligodendrocyte progenitor cell differentiation.

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    Moore, Craig S; Cui, Qiao-Ling; Warsi, Nebras M; Durafourt, Bryce A; Zorko, Nika; Owen, David R; Antel, Jack P; Bar-Or, Amit

    2015-01-15

    In multiple sclerosis, successful remyelination within the injured CNS is largely dependent on the survival and differentiation of oligodendrocyte progenitor cells. During inflammatory injury, oligodendrocytes and oligodendrocyte progenitor cells within lesion sites are exposed to secreted products derived from both infiltrating immune cell subsets and CNS-resident cells. Such products may be considered either proinflammatory or anti-inflammatory and have the potential to contribute to both injury and repair processes. Within the CNS, astrocytes also contribute significantly to oligodendrocyte biology during development and following inflammatory injury. The overall objective of the current study was to determine how functionally distinct proinflammatory and anti-inflammatory human immune cell subsets, implicated in multiple sclerosis, can directly and/or indirectly (via astrocytes) impact human oligodendrocyte progenitor cell survival and differentiation. Proinflammatory T cell (Th1/Th17) and M1-polarized myeloid cell supernatants had a direct cytotoxic effect on human A2B5(+) neural progenitors, resulting in decreased O4(+) and GalC(+) oligodendrocyte lineage cells. Astrocyte-conditioned media collected from astrocytes pre-exposed to the same proinflammatory supernatants also resulted in decreased oligodendrocyte progenitor cell differentiation without an apparent increase in cell death and was mediated through astrocyte-derived CXCL10, yet this decrease in differentiation was not observed in the more differentiated oligodendrocytes. Th2 and M2 macrophage or microglia supernatants had neither a direct nor an indirect impact on oligodendrocyte progenitor cell differentiation. We conclude that proinflammatory immune cell responses can directly and indirectly (through astrocytes) impact the fate of immature oligodendrocyte-lineage cells, with oligodendrocyte progenitor cells more vulnerable to injury compared with mature oligodendrocytes.

  1. Is stage-specific embryonic antigen 4 a marker for human ductal stem/progenitor cells?

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    Afrikanova, Ivka; Kayali, Ayse; Lopez, Ana; Hayek, Alberto

    2012-08-01

    The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4(+) cells scattered in the ducts do not show a ductal cell phenotype. SSEA4(+)-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4(+) cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells.

  2. Is Stage-Specific Embryonic Antigen 4 a Marker for Human Ductal Stem/Progenitor Cells?

    Science.gov (United States)

    Kayali, Ayse; Lopez, Ana; Hayek, Alberto

    2012-01-01

    Abstract The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4+ cells scattered in the ducts do not show a ductal cell phenotype. SSEA4+-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4+ cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells. PMID:23515456

  3. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

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    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.

  4. 3D Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent VZV Infection

    Science.gov (United States)

    Goodwin, Thomas J.

    2013-01-01

    Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

  5. Xenotransplantation of Human Neural Progenitor Cells to the Subretinal Space of Nonimmunosuppressed Pigs

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    Karin Warfvinge

    2011-01-01

    Full Text Available 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 vessels appeared normal without signs of exudation, bleeding, or subretinal elevation. Eyes were harvested at 10–28 days. H&E consistently showed mild retinal vasculitis, depigmentation of the RPE, and marked mononuclear cell infiltrate in the choroid adjacent to the site of transplantation. Human-specific antibodies revealed donor cells in the subretinal space at 10–13 days and smaller numbers within the retina on days 12 and 13, with evidence suggesting a limited degree of morphological integration; however, no cells remained at 4 weeks. The strong mononuclear cell reaction and loss of donor cells indicate that modulation of host immunity is likely necessary for prolonged xenograft survival in this model.

  6. Transcriptomic profiling of human hippocampal progenitor cells treated with antidepressants and its application in drug repositioning

    Science.gov (United States)

    Powell, Timothy R; Murphy, Tytus; Lee, Sang H; Price, Jack; Thuret, Sandrine; Breen, Gerome

    2017-01-01

    Current pharmacological treatments for major depressive disorder (MDD) are ineffective in a significant proportion of patients, and the identification of new antidepressant compounds has been difficult. ‘Connectivity mapping’ is a method that can be used to identify drugs that elicit similar downstream effects on mRNA levels when compared to current treatments, and thus may point towards possible repositioning opportunities. We investigated genome-wide transcriptomic changes to human hippocampal progenitor cells treated with therapeutically relevant concentrations of a tricyclic antidepressant (nortriptyline) and a selective serotonin reuptake inhibitor (escitalopram). We identified mRNA changes common to both drugs to create an ‘antidepressant mRNA signature’. We used this signature to probe the Library of Integrated Network-based Cellular Signatures (LINCS) and to identify other compounds that elicit similar changes to mRNA in neural progenitor cells. Results from LINCS revealed that the tricyclic antidepressant clomipramine elicited mRNA changes most similar to our mRNA signature, and we identified W-7 and vorinostat as functionally relevant drug candidates, which may have repositioning potential. Our results are encouraging and represent the first attempt to use connectivity mapping for drug repositioning in MDD. PMID:28208023

  7. Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells

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    Hyland, Kendra A.; Olson, Erik R.; McIvor, R. Scott

    2015-01-01

    The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34+ HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon–chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications. PMID:26176276

  8. Using human neural crest-derived progenitor cells to investigate osteogenesis: an in vitro study.

    Science.gov (United States)

    Degistirici, Ozer; Grabellus, Florian; Irsen, Stephan; Schmid, Kurt Werner; Thie, Michael

    2010-04-01

    Human tooth contains a distinct population of neural crest-derived progenitor cells (dNC-PCs) which are known to give rise to specialized daughter cells of an osteogenic lineage. We hypothesised that dNC-PCs could develop into neural crest-derived bone in a self-propagating and extracorporal culture system. Thus, we examined the three-dimensional structure obtained from osteogenic-stimulated dNC-PCs by morphological, biochemical and spectroscopic methods. After the onset of stimulation, cells formed a multilayer with outer cells covering the surface and inner cells secreting a hyaline matrix. With prolonged culture, multilayers contracted and formed a three-dimensional construct which subsequently converted to a calcified mass. Differentiation of progenitor cells was associated with apoptosis. Cell types which survived were smooth muscle actin-positive cells and bone-like cells. The expression of osteoblastic markers and the secretion of a collagenous matrix indicate that the bone cells had acquired their functional phenotype. Furthermore, these cells produced and secreted membrane-bound vesicles into the newly forming matrix. Consequently, an early biomineralized extracellular matrix was found with calcium phosphate deposits being associated with the newly formed collagen matrix framework. The molar calcium-phosphorus-ratio of the mineralized collagen indicated that amorphous calcium phosphate was present within this matrix. The data suggest that stimulated cultures of dNC-PCs are able to recapitulate some processes of the early phase of osteogenesis.

  9. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

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    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.

  10. ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway

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    Vignudelli, Tatiana; Selmi, Tommaso; Martello, Andrea; Parenti, Sandra; Grande, Alexis; Gemelli, Claudia; Ferrari, Sergio

    2010-01-01

    ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3′-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by posttranscriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3′ untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, because cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders. PMID:20702587

  11. 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.

  12. Generation of neuronal progenitor cells in response to tumors in the human brain.

    Science.gov (United States)

    Macas, Jadranka; Ku, Min-Chi; Nern, Christian; Xu, Yuanzhi; Bühler, Helmut; Remke, Marc; Synowitz, Michael; Franz, Kea; Seifert, Volker; Plate, Karl H; Kettenmann, Helmut; Glass, Rainer; Momma, Stefan

    2014-01-01

    Data from transgenic mouse models show that neuronal progenitor cells (NPCs) migrate toward experimental brain tumors and modulate the course of pathology. However, the pathways whereby NPCs are attracted to CNS neoplasms are not fully understood and it is unexplored if NPCs migrate toward brain tumors (high-grade astrocytomas) in humans. We analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of (NPCs) and distinguished these physiological cells from the tumor mass. We observed that polysialic acid neural cell adhesion molecule-positive NPCs accumulate at the border of high-grade astrocytomas and display a marker profile consistent with immature migratory NPCs. Importantly, these high-grade astrocytoma-associated NPCs did not carry genetic aberrations that are indicative of the tumor. Additionally, we observed NPCs accumulating in CNS metastases. These metastatic tumors are distinguished from neural cells by defined sets of markers. Transplanting murine glioma cells embedded in a cell-impermeable hollow fiber capsule into the brains of nestin-gfp reporter mice showed that diffusible factors are sufficient to induce a neurogenic reaction. In vitro, vascular endothelial growth factor (VEGF) secreted from glioma cells increases the migratory and proliferative behavior of adult human brain-derived neural stem and progenitor cells via stimulation of VEGF receptor-2 (VEGFR-2). In vivo, inhibiting VEGFR-2 signaling with a function-blocking antibody led to a reduction in NPC migration toward tumors. Overall, our data reveal a mechanism by which NPCs are attracted to CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas.

  13. 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.

  14. Efficient in vitro generation of functional thymic epithelial progenitors from human embryonic stem cells.

    Science.gov (United States)

    Su, Min; Hu, Rong; Jin, Jingjun; Yan, Yuan; Song, Yinhong; Sullivan, Ryan; Lai, Laijun

    2015-06-05

    Thymic epithelial cells (TECs) are the major components of the thymic microenvironment for T cell development. TECs are derived from thymic epithelial progenitors (TEPs). It has been reported that human ESCs (hESCs) can be directed to differentiate into TEPs in vitro. However, the efficiency for the differentiation is low. Furthermore, transplantation of hESC-TEPs in mice only resulted in a very low level of human T cell development from co-transplanted human hematopoietic precursors. We show here that we have developed a novel protocol to efficiently induce the differentiation of hESCs into TEPs in vitro. When transplanted into mice, hESC-TEPs develop into TECs and form a thymic architecture. Most importantly, the hESC-TECs support the long-term development of functional mouse T cells or a higher level of human T cell development from co-transplanted human hematopoietic precursors. The hESC-TEPs may provide a new approach to prevent or treat patients with T cell immunodeficiency.

  15. [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.

  16. MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16

    NARCIS (Netherlands)

    Liu, Jia; van Mil, Alain; Aguor, Eissa N. E.; Siddiqi, Sailay; Vrijsen, Krijn; Jaksani, Sridevi; Metz, Corina; Zhao, Jiajun; Strijkers, Gustav J.; Doevendans, Pieter A.; Sluijter, Joost P. G.

    2012-01-01

    Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration vi

  17. Cytokeratin (CK5, CK8, CK14) expression and presence of progenitor stem cells in human fetal thymuses.

    Science.gov (United States)

    Gupta, Richa; Gupta, Tulika; Kaur, Harjeet; Sehgal, Shobha; Aggarwal, Anjali; Kapoor, Kanchan; Sharma, Anshu; Sahni, Daisy; Singla, Suhalika

    2016-09-01

    The aim of the current study was to observe the expression of cytokeratins in human fetal thymuses. Specific cytokeratin markers in adult humans and mice have been well described but there has been little similar work on human fetuses. We also aimed to see whether progenitor stem cells that could be harvested to treat various immunodeficiency disorders are present in fetal thymic tissue. Thymuses obtained from 30 aborted human fetuses (12 to 31 weeks) were examined immunohistochemically to investigate changes in cytokeratin expression in the epithelial cells (TEC) at various gestational ages. Before 16 weeks of gestation, cortical (cTEC) and medullary (mTEC) TEC exhibited homogenous staining for cytokeratins CK8 and CK5. After 16 weeks there was differential staining, with cTEC positive for CK8 and mTEC for CK5 and CK14. Interestingly, both CK5 + CK8+ progenitor stem cells were present in the fetal thymic cortex at all gestational ages, with a relatively high number from 12 to 16 weeks. Cytokeratin expression in fetal thymuses was quite different from that in the adult thymus owing to the presence of undifferentiated progenitor stem cells in fetal thymic stroma along with differentiated TEC. The best time to harvest these progenitor stem cells from fetal thymic stroma in order to treat various immune deficiency disorders appears to be 12-16 weeks. Clin. Anat. 29:711-717, 2016. © 2016 Wiley Periodicals, Inc.

  18. Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

    Science.gov (United States)

    Moon, Jisook; Schwarz, Sigrid C; Lee, Hyun-Seob; Kang, Jun Mo; Lee, Young-Eun; Kim, Bona; Sung, Mi-Young; Höglinger, Günter; Wegner, Florian; Kim, Jin Su; Chung, Hyung-Min; Chang, Sung Woon; Cha, Kwang Yul; Kim, Kwang-Soo; Schwarz, Johannes

    2016-09-02

    : We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening.

  19. Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

    Science.gov (United States)

    Eyckmans, Jeroen; Roberts, Scott J; Bolander, Johanna; Schrooten, Jan; Chen, Christopher S; Luyten, Frank P

    2013-06-01

    Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/collagen scaffolds (calcium phosphate rich matrix or CPRM), or on decalcified scaffolds (calcium phosphate depleted matrix or CPDM), followed by subcutaneous implantation in nude mice to trigger ectopic bone formation. In this system, osteoblast differentiation occurred in CPRM scaffolds, but not in CPDM scaffolds. Gene expression was assessed by human full-genome microarray at 20 h after seeding, and 2, 8 and 18 days after implantation. In both matrices, implantation of the cell constructs triggered a similar gene expression cascade, however, gene expression dynamics progressed faster in CPRM scaffolds than in CPDM scaffolds. The difference in gene expression dynamics was associated with differential activation of hub genes and molecular signaling pathways related to calcium signaling (CREB), inflammation (TNFα, NFkB, and IL6) and bone development (TGFβ, β-catenin, BMP, EGF, and ERK signaling). Starting from this set of pathways, a growth factor cocktail was developed that robustly enhanced osteogenesis in vitro and in vivo. Taken together, our data demonstrate that through the identification and subsequent stimulation of genes, proteins and signaling pathways associated with calcium phosphate mediated osteoinduction, a focused approach to develop targeted differentiation protocols in adult progenitor cells can be achieved.

  20. Human astrocytes derived from glial restricted progenitors support regeneration of the injured spinal cord.

    Science.gov (United States)

    Haas, Christopher; Fischer, Itzhak

    2013-06-15

    Cellular transplantation using neural stem cells and progenitors is a promising therapeutic strategy that has the potential to replace lost cells, modulate the injury environment, and create a permissive environment for the regeneration of injured host axons. Our research has focused on the use of human glial restricted progenitors (hGRP) and derived astrocytes. In the current study, we examined the morphological and phenotypic properties of hGRP prepared from the fetal central nervous system by clinically-approved protocols, compared with astrocytes derived from hGRP prepared by treatment with ciliary neurotrophic factor or bone morphogenetic protein 4. These differentiation protocols generated astrocytes that showed morphological differences and could be classified along an immature to mature spectrum, respectively. Despite these differences, the cells retained morphological and phenotypic plasticity upon a challenge with an alternate differentiation protocol. Importantly, when hGRP and derived astrocytes were transplanted acutely into a cervical dorsal column lesion, they survived and promoted regeneration of long ascending host sensory axons into the graft/lesion site, with no differences among the groups. Further, hGRP taken directly from frozen stocks behaved similarly and also supported regeneration of host axons into the lesion. Our results underscore the dynamic and permissive properties of human fetal astrocytes to promote axonal regeneration. They also suggest that a time-consuming process of pre-differentiation may not be necessary for therapeutic efficacy, and that the banking of large quantities of readily available hGRP can be an appropriate source of permissive cells for transplantation.

  1. Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy.

    Science.gov (United States)

    Johnsen, Erik O; Frøen, Rebecca C; Albert, Réka; Omdal, Bente K; Sarang, Zsolt; Berta, András; Nicolaissen, Bjørn; Petrovski, Goran; Moe, Morten C

    2012-05-01

    In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive

  2. Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

    Science.gov (United States)

    Fishman, Bettina; Segev, Hanna; Kopper, Oded; Nissenbaum, Jonathan; Schulman, Margarita; Benvenisty, Nissim; Itskovitz-Eldor, Joseph; Kitsberg, Danny

    2012-09-01

    New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

  3. High-level, erythroid specific, expression of the human α-globin gene in transgenic mice and the production of human haemoglobin in murine erythrocytes.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); M. Vidal; J. Kaeda; L. Luzzatto; D.R. Greaves (David); F.G. Grosveld (Frank)

    1989-01-01

    textabstractUsing the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of

  4. Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

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    Shimpei Gotoh

    2014-09-01

    Full Text Available No methods for isolating induced alveolar epithelial progenitor cells (AEPCs from human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs, we identified carboxypeptidase M (CPM as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

  5. A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

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    Xiangshan Zhao

    2011-01-01

    Full Text Available Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs immortalized with human telomerase reverse transcriptase (hTERT possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with g-radiation, share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

  6. Cell Therapy Using Human Induced Pluripotent Stem Cell-Derived Renal Progenitors Ameliorates Acute Kidney Injury in Mice

    Science.gov (United States)

    Toyohara, Takafumi; Mae, Shin-Ichi; Sueta, Shin-Ichi; Inoue, Tatsuyuki; Yamagishi, Yukiko; Kawamoto, Tatsuya; Kasahara, Tomoko; Hoshina, Azusa; Toyoda, Taro; Tanaka, Hiromi; Araoka, Toshikazu; Sato-Otsubo, Aiko; Takahashi, Kazutoshi; Sato, Yasunori; Yamaji, Noboru; Ogawa, Seishi; Yamanaka, Shinya

    2015-01-01

    Acute kidney injury (AKI) is defined as a rapid loss of renal function resulting from various etiologies, with a mortality rate exceeding 60% among intensive care patients. Because conventional treatments have failed to alleviate this condition, the development of regenerative therapies using human induced pluripotent stem cells (hiPSCs) presents a promising new therapeutic option for AKI. We describe our methodology for generating renal progenitors from hiPSCs that show potential in ameliorating AKI. We established a multistep differentiation protocol for inducing hiPSCs into OSR1+SIX2+ renal progenitors capable of reconstituting three-dimensional proximal renal tubule-like structures in vitro and in vivo. Moreover, we found that renal subcapsular transplantation of hiPSC-derived renal progenitors ameliorated the AKI in mice induced by ischemia/reperfusion injury, significantly suppressing the elevation of blood urea nitrogen and serum creatinine levels and attenuating histopathological changes, such as tubular necrosis, tubule dilatation with casts, and interstitial fibrosis. To our knowledge, few reports demonstrating the therapeutic efficacy of cell therapy with renal lineage cells generated from hiPSCs have been published. Our results suggest that regenerative medicine strategies for kidney diseases could be developed using hiPSC-derived renal cells. Significance This report is the first to demonstrate that the transplantation of renal progenitor cells differentiated from human induced pluripotent stem (iPS) cells has therapeutic effectiveness in mouse models of acute kidney injury induced by ischemia/reperfusion injury. In addition, this report clearly demonstrates that the therapeutic benefits come from trophic effects by the renal progenitor cells, and it identifies the renoprotective factors secreted by the progenitors. The results of this study indicate the feasibility of developing regenerative medicine strategy using iPS cells against renal diseases

  7. Unravelling the mystery of stem/progenitor cells in human breast milk.

    Directory of Open Access Journals (Sweden)

    Yiping Fan

    Full Text Available BACKGROUND: Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. METHODOLOGY/PRINCIPAL FINDINGS: We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6 ± 0.8% (mean ± SEM and 0.7 ± 0.2% of the whole cell population (WCP were found to be CD133+ and CD34+ respectively, 27.8 ± 9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR, and in 4.17 ± 0.2% and 0.9 ± 0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP (1.8 ± 0.4% of WCP as well as CD133+ cells (1.7 ± 0.5% of the WCP. Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM in the SP cells (50.6 ± 8.6 vs 18.1 ± 6.0, P-value  = 0.02. However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. CONCLUSIONS/SIGNIFICANCE: The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman.

  8. Human Muscle Progenitor Cells Displayed Immunosuppressive Effect through Galectin-1 and Semaphorin-3A

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    Séverine Lecourt

    2012-01-01

    Full Text Available In human skeletal muscle, myoblasts represent the main population of myogenic progenitors. We previously showed that, beside their myogenic differentiation capacities, myoblasts also differentiate towards osteogenic and chondrogenic lineages, some properties generally considered being hallmarks of mesenchymal stem cells (MSCs. MSCs are also characterized by their immunosuppressive potential, through cell-cell contacts and soluble factors, including prostaglandin E-2 (PGE-2, transforming growth factor-β1 (TGF-β1, interleukine-10, or indoleamine 2,3-dioxygenase. We and others also reported that Galectin-1 (Gal-1 and Semaphorin-3A (Sema-3A were involved in MSCs-mediated immunosuppression. Here, we show that human myoblasts induce a significant and dose-dependant proliferation inhibition, independently of PGE-2 and TGF-β1. Our experiments revealed that myoblasts, in culture or in situ in human muscles, expressed and secreted Gal-1 and Sema-3A. Furthermore, myoblasts immunosuppressive functions were reverted by using blocking antibodies against Gal-1 or Sema-3A. Together, these results demonstrate an unsuspected immunosuppressive effect of myoblasts that may open new therapeutic perspectives.

  9. Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

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    Wen Wang

    2015-05-01

    Full Text Available Mesenchymal stem cells (MSCs are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA. Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.

  10. Evolution of the clonogenic potential of human epidermal stem/progenitor cells with age.

    Science.gov (United States)

    Zobiri, Olivia; Deshayes, Nathalie; Rathman-Josserand, Michelle

    2012-01-01

    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 fatten 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.

  11. 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

  12. Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response.

    Science.gov (United States)

    Huang, Ke; Liu, PengFei; Li, Xiang; Chen, ShuBin; Wang, LiHui; Qin, Li; Su, ZhengHui; Huang, WenHao; Liu, Juli; Jia, Bei; Liu, Jie; Cai, JingLei; Pei, DuanQing; Pan, GuangJin

    2014-02-01

    The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However, whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study, we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs), we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However, a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore, no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3(+)CD8(-) T cells, CD3(+)CD8(+) T cells or CD3(-)CD56(+) NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants, and thus set a base for further preclinical evaluation of human iPSCs.

  13. 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

  14. 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....

  15. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  16. Development and application of human adult stem or progenitor cell organoids

    NARCIS (Netherlands)

    Rookmaaker, Maarten B; Schutgens, Frans; Verhaar, Marianne C; Clevers, Hans

    2015-01-01

    Adult stem or progenitor cell organoids are 3D adult-organ-derived epithelial structures that contain self-renewing and organ-specific stem or progenitor cells as well as differentiated cells. This organoid culture system was first established in murine intestine and subsequently developed for sever

  17. In Vitro Generation of Human XCR1(+) Dendritic Cells from CD34(+) Hematopoietic Progenitors.

    Science.gov (United States)

    Balan, Sreekumar; Dalod, Marc

    2016-01-01

    Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells which play a key role in orchestrating immune defenses. Most of the information gained on human DC biology was derived from studies conducted with DCs generated in vitro from peripheral blood CD14(+) monocytes (MoDCs) or from CD34(+) hematopoietic progenitors. Recent advances in the field revealed that these types of in vitro-derived DCs strikingly differ from the DC subsets that are naturally present in human lymphoid organs, in terms of global gene expression, of specialization in the sensing of different types of danger signals, and of the ability to polarize T lymphocytes toward different functions. Major efforts are being made to better characterize the biology and the functions of lymphoid organ-resident DC subsets in humans, as an essential step for designing innovative DC-based vaccines against infections or cancers. However, this line of research is hampered by the low frequency of certain DC subsets in most tissues, their fragility, and the complexity of the procedures necessary for their purification. Hence, there is a need for robust procedures allowing large-scale in vitro generation of human DC subsets, under conditions allowing their genetic or pharmacological manipulation, to decipher their functions and their molecular regulation. Human CD141(+)CLEC9A(+)XCR1(+) DCs constitute a very interesting DC subset for the design of immunotherapeutic treatments against infections by intracellular pathogens or against cancer, because these cells resemble mouse professional cross-presenting CD8α(+)Clec9a(+)Xcr1(+) DCs. Human XCR1(+) DCs have indeed been reported by several teams to be more efficient than other human DC subsets for cross-presentation, in particular of cell-associated antigens but also of soluble antigens especially when delivered into late endosomes or lysosomes. However, human XCR1(+) DCs are the rarest and perhaps the most fragile of the human DC

  18. Focal Transplantation of Human iPSC-Derived Glial-Rich Neural Progenitors Improves Lifespan of ALS Mice

    Directory of Open Access Journals (Sweden)

    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.

  19. Cocaine exposure impairs multilineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor [corrected].

    Science.gov (United States)

    Nixon, Christopher C; Schwartz, Brandon H; Dixit, Dhaval; Zack, Jerome A; Vatakis, Dimitrios N

    2015-03-02

    Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade.

  20. Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1

    Science.gov (United States)

    Samata, Bumpei; Doi, Daisuke; Nishimura, Kaneyasu; Kikuchi, Tetsuhiro; Watanabe, Akira; Sakamoto, Yoshimasa; Kakuta, Jungo; Ono, Yuichi; Takahashi, Jun

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients. PMID:27739432

  1. 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 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 < 0.05); this was inversely correlated with time from injury to surgery (r = -0.8, p < 0.05). These results indicate that severe focal human TBI results in OL death and increases in OPCs postinjury, which may influence white matter function following TBI.

  2. Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells

    Science.gov (United States)

    Humby, Monica S.

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four virus-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency has remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial chromosome (BAC)-derived clinical HCMV strain TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs but not the US28 wild-type protein. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3 in vitro latency model system and in primary ex vivo-cultured CD34+ HPCs. Our data suggest that US28 is required for latency as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naive fibroblasts. The other HCMV GPCRs are not required for this phenotype as a virus expressing only US28 but not the remaining virus-encoded GPCRs is phenotypically similar to that of wild-type latent infection. Finally, we found that US28 copurifies with mature virions and is expressed in HPCs upon virus entry although its expression at the time of infection does not complement the US28 deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells. IMPORTANCE Human cytomegalovirus (HCMV) is a widespread pathogen that, once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during

  3. A 3D in vitro bone organ model using human progenitor cells

    Directory of Open Access Journals (Sweden)

    A Papadimitropoulos

    2011-05-01

    Full Text Available Three-dimensional (3D organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0 ±0.3%, pre/osteoclastic (14.0 ±1.4% and mesenchymal/osteoblastic (44.0 ±8.4% phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+ osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides, resorption (by N-terminus collagen-I telopeptides and phosphate levels and osteoclastic activity (by TRAP-5b only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.

  4. Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways.

    Science.gov (United States)

    Zhang, Jing; Liu, Yangang; Beard, Caroline; Tuveson, David A; Jaenisch, Rudolf; Jacks, Tyler E; Lodish, Harvey F

    2007-06-15

    When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

  5. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Stopp, Sabine; Bornhäuser, Martin; Ugarte, Fernando; Wobus, Manja; Kuhn, Matthias; Brenner, Sebastian; Thieme, Sebastian

    2013-04-01

    The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

  6. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors.

    Science.gov (United States)

    Muschler, G F; Nitto, H; Boehm, C A; Easley, K A

    2001-01-01

    Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be induced to express a bone phenotype in vitro. The number of osteoblastic progenitors can be estimated by counting the colony-forming units which express alkaline phosphatase (CFU-APs). This study was undertaken to test the hypothesis that human aging is associated with a significant change in the number or prevalence of osteoblastic progenitors in the bone marrow. Four 2-ml bone marrow aspirates were harvested bilaterally from the anterior iliac crest of 57 patients, 31 men (age 15-83) and 26 women (age 13-79). A mean of 64 million nucleated cells was harvested per aspirate. The mean prevalence of CFU-APs was found to be 55 per million nucleated cells. These data revealed a significant age-related decline in the number of nucleated cells harvested per aspirate for both men and women (P = 0.002). The number of CFU-APs harvested per aspirate also decreased significantly with age for women (P = 0.02), but not for men (P = 0.3). These findings are relevant to the harvest of bone marrow derived connective tissue progenitors for bone grafting and other tissue engineering applications, and may also be relevant to the pathophysiology of age-related bone loss and post-menopausal osteoporosis.

  7. 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)

  8. Hepatic progenitor cells in human liver cirrhosis:Immunohistochemical,electron microscopic and immunofluorencence confocal microscopic findings

    Institute of Scientific and Technical Information of China (English)

    Jia-Cheng Xiao; Xiao-Long Jin; Peter Ruck; Anne Adam; Edwin Kaiserling

    2004-01-01

    AIM: To investigate whether hepatic progenitor cells (HPC),that reveal the features of oval cells in rodents and small epithelial cells (SEC) in certain human liver disease, were also found in human liver cirrhosis (HLC).METHODS: Surgical liver specimens from 20 cases of hepatitis B virus-positive HLC (15 cases containing hepatocellular carcinoma) were investigated by light microscopic immunohistochemistry (LM-IHC). Among them specimens from 15 cases were investigated by electron microscopy (EM)and those from 5 cases by immunofluorencence confocal laser scanning microscopy (ICLSM). Antibodies against cytokeratin 7 and albumin were used and single and/or double labelling were performed respectively.RESULTS: LM-IHC showed that at the margins of regenerating nodules and in the fibrous septae, a small number of cells in the proliferating bile ductules were positive for CK7 and albumin. At the EM level these HPC were morphologically similar to the SEC described previously, and also similar to the oval cells seen in experimental hepatocarcinogenesis.They were characterized by their small size, oval shape, a high nucleus/cytoplasm ratio, a low organelle content in cytoplasm, and existence of tonofilaments and intercellular junctions. ICLSM revealed that HPC expressed both cytokeratin 7 and albumin.CONCLUSION: HPC with ultrastructural and immunophenotypical features of oval cells, i.e., hepatic stem cell-like cells as noted in other liver diseases, were found in HLC. These findings further support the hypothesis that bipotent hepatic stem cells, that may give rise to biliary epithelial cells and hepatocytes, exist in human livers.

  9. Defining the Role of Oxygen Tension in Human Neural Progenitor Fate

    Directory of Open Access Journals (Sweden)

    Yuan Xie

    2014-11-01

    Full Text Available Hypoxia augments human embryonic stem cell (hESC self-renewal via hypoxia-inducible factor 2α-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined, these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here, we show that low O2 tension and hypoxia-inducible factor (HIF activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies, we implicate O2 tension as a modifier of a key cell fate decision, namely whether neural progenitors differentiate toward neurons or glia. Furthermore, our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC, a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage. We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types.

  10. Further assessment of neuropathology in retinal explants and neuroprotection by human neural progenitor cells

    Science.gov (United States)

    Mohlin, Camilla; Liljekvist-Soltic, Ingela; Johansson, Kjell

    2011-10-01

    Explanted rat retinas show progressive photoreceptor degeneration that appears to be caspase-12-dependent. Decrease in photoreceptor density eventually affects the inner retina, particularly in the bipolar cell population. Explantation and the induced photoreceptor degeneration are accompanied by activation of Müller and microglia cells. The goal of this study was to determine whether the presence of a feeder layer of human neural progenitor cells (hNPCs) could suppress the degenerative and reactive changes in the explants. Immunohistochemical analyses showed considerable sprouting of rod photoreceptor axon terminals into the inner retina and reduced densities of cone and rod bipolar cells. Both sprouting and bipolar cell degenerations were significantly lower in retinas cultured with feeder layer cells compared to cultured controls. A tendency toward reduced microglia activation in the retinal layers was also noted in the presence of feeder layer cells. These results indicate that hNPCs or factors produced by them can limit the loss of photoreceptors and secondary injuries in the inner retina. The latter may be a consequence of disrupted synaptic arrangement.

  11. Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies.

    Science.gov (United States)

    Chevalier, Fabien; Lavergne, Mélanie; Negroni, Elisa; Ferratge, Ségolène; Carpentier, Gilles; Gilbert-Sirieix, Marie; Siñeriz, Fernando; Uzan, Georges; Albanese, Patricia

    2014-05-01

    Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR(4131)] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR(4131)] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC.

  12. 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.

  13. Low oxygen alters mitochondrial function and response to oxidative stress in human neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Yury M. Lages

    2015-12-01

    Full Text Available Oxygen concentration should be carefully regulated in all living tissues, beginning at the early embryonic stages. Unbalances in oxygen regulation can lead to cell death and disease. However, to date, few studies have investigated the consequences of variations in oxygen levels for fetal-like cells. Therefore, in the present work, human neural progenitor cells (NPCs derived from pluripotent stem cells grown in 3% oxygen (v/v were compared with NPCs cultured in 21% (v/v oxygen. Low oxygen concentrations altered the mitochondrial content and oxidative functions of the cells, which led to improved ATP production, while reducing generation of reactive oxygen species (ROS. NPCs cultured in both conditions showed no differences in proliferation and glucose metabolism. Furthermore, antioxidant enzymatic activity was not altered in NPCs cultured in 3% oxygen under normal conditions, however, when exposed to external agents known to induce oxidative stress, greater susceptibility to DNA damage was observed. Our findings indicate that the management of oxygen levels should be considered for in vitro models of neuronal development and drug screening.

  14. Analysis of Pregnancy-Associated Plasma Protein A Production in Human Adult Cardiac Progenitor Cells

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    Piera D’Elia

    2013-01-01

    Full Text Available IGF-binding proteins (IGFBPs and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

  15. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

    Science.gov (United States)

    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.

  16. Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice

    OpenAIRE

    Vicente-Dueñas, Carolina; Campos-Sánchez, Elena; González, Marcos; Cobaleda, César; Abollo-Jiménez, Fernando; Martínez-Climent, José Ángel

    2012-01-01

    Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tum...

  17. Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors

    Directory of Open Access Journals (Sweden)

    Iverson Linda E

    2010-04-01

    Full Text Available Abstract Background Trisomic variants of human embryonic stem cells (hESCs arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting. Methods Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs, RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform. Results Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells. Conclusions Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem/progenitor

  18. Actions of estrogens and endocrine disrupting chemicals on human prostate stem/progenitor cells and prostate cancer risk.

    Science.gov (United States)

    Hu, Wen-Yang; Shi, Guang-Bin; Hu, Dan-Ping; Nelles, Jason L; Prins, Gail S

    2012-05-06

    Estrogen reprogramming of the prostate gland as a function of developmental exposures (aka developmental estrogenization) results in permanent alterations in structure and gene expression that lead to an increased incidence of prostatic lesions with aging. Endocrine disrupting chemicals (EDCs) with estrogenic activity have been similarly linked to an increased prostate cancer risk. Since it has been suggested that stem cells and cancer stem cells are potential targets of cancer initiation and disease management, it is highly possible that estrogens and EDCs influence the development and progression of prostate cancer through reprogramming and transforming the prostate stem and early stage progenitor cells. In this article, we review recent literature highlighting the effects of estrogens and EDCs on prostate cancer risk and discuss recent advances in prostate stem/progenitor cell research. Our laboratory has recently developed a novel prostasphere model using normal human prostate stem/progenitor cells and established that these cells express estrogen receptors (ERs) and are direct targets of estrogen action. Further, using a chimeric in vivo prostate model derived from these normal human prostate progenitor cells, we demonstrated for the first time that estrogens initiate and promote prostatic carcinogenesis in an androgen-supported environment. We herein discuss these findings and highlight new evidence using our in vitro human prostasphere assay for perturbations in human prostate stem cell self-renewal and differentiation by natural steroids as well as EDCs. These findings support the hypothesis that tissue stem cells may be direct EDC targets which may underlie life-long reprogramming as a consequence of developmental and/or transient adult exposures.

  19. Cultivation of human neural progenitor cells in a 3-dimensional self-assembling peptide hydrogel.

    Science.gov (United States)

    Liedmann, Andrea; Rolfs, Arndt; Frech, Moritz J

    2012-01-11

    technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells, however the concentration might be adapted to other cell types. The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

  20. Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice.

    Science.gov (United States)

    Vicente-Dueñas, Carolina; Fontán, Lorena; Gonzalez-Herrero, Ines; Romero-Camarero, Isabel; Segura, Victor; Aznar, M Angela; Alonso-Escudero, Esther; Campos-Sanchez, Elena; Ruiz-Roca, Lucía; Barajas-Diego, Marcos; Sagardoy, Ainara; Martinez-Ferrandis, Jose I; Abollo-Jimenez, Fernando; Bertolo, Cristina; Peñuelas, Ivan; Garcia-Criado, Francisco J; García-Cenador, María B; Tousseyn, Thomas; Agirre, Xabier; Prosper, Felipe; Garcia-Bragado, Federico; McPhail, Ellen D; Lossos, Izidore S; Du, Ming-Qing; Flores, Teresa; Hernandez-Rivas, Jesus M; Gonzalez, Marcos; Salar, Antonio; Bellosillo, Beatriz; Conde, Eulogio; Siebert, Reiner; Sagaert, Xavier; Cobaleda, Cesar; Sanchez-Garcia, Isidro; Martinez-Climent, Jose A

    2012-06-26

    Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.

  1. Stem/progenitor cells in the cerebral cortex of the human preterm: a resource for an endogenous regenerative neuronal medicine?

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    Laura Vinci

    2016-04-01

    Full Text Available The development of the central nervous system represents a very delicate period of embryogenesis. Premature interruption of neurogenesis in human preterm newborns can lead to motor deficits, including cerebral palsy, and significant cognitive, behavioral or sensory deficits in childhood. Preterm infants also have a higher risk of developing neurodegenerative diseases later in life. In the last decade, great importance has been given to stem/progenitor cells and their possible role in the development and treatment of several neurological disorders. Several studies, mainly carried out on experimental models, evidenced that immunohistochemistry may allow the identification of different neural and glial precursors inside the developing cerebral cortex. However, only a few studies have been performed on markers of human stem cells in the embryonic period.This review aims at illustrating the importance of stem/progenitor cells in cerebral cortex during pre- and post-natal life. Defining the immunohistochemical markers of stem/progenitor cells in the human cerebral cortex during development may be important to develop an “endogenous” target therapy in the perinatal period. Proceedings of the 2nd International Course on Perinatal Pathology (part of the 11th International Workshop on Neonatology · October 26th-31st, 2015 · Cagliari (Italy · October 31st, 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

  2. 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

    CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon gamma (IFN-gamma)-inducible protein 10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report the novel finding that CXCR3 is also...... expressed on CD34(+) hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34(+) progenitors. Freshly isolated CD34(+) progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up...... for the physiologic and pathophysiologic events of differentiation of CD34(+) hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34(+) hematopoietic progenitors. (Blood...

  3. Human Lyb-2 homolog CD72 is a marker for progenitor B-cell leukemias.

    Science.gov (United States)

    Schwarting, R; Castello, R; Moldenhauer, G; Pezzutto, A; von Hoegen, I; Ludwig, W D; Parnes, J R; Dörken, B

    1992-11-01

    S-HCL 2 is the prototype antibody of the recently defined CD72 cluster (human Lyb-2). Under nonreducing conditions, S-HCL 2 monoclonal antibody (mAb) precipitates a glycoprotein of 80-86 kDa. Under reducing conditions, a dimer of 43 and 39 kDa, with core proteins of 40 and 36 kDa, is precipitated. CD72 expression in normal and malignant tissues is different from expression of all other previously described human B-cell antigens. In peripheral blood and bone marrow, the antigen appears to be present on all B lymphocytes, with the exception of plasma cells. In tissue, immunohistochemical staining revealed positivity for all known B-cell compartments; however, pulpa macrophages of the spleen and von Kupffer cells exhibited distinct positivity for CD72 also. Among 83 malignant non-Hodgkin's lymphomas examined by immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase technique), all 54 B-cell lymphomas, including precursor B-cell lymphomas, Burkitt's lymphomas, germinal center lymphomas, chronic lymphocytic leukemias, and hairy cell leukemias, were CD72 positive, but no T-cell lymphomas were. Flow cytometry study of more than 80 mainly acute leukemias (52 B-cell leukemias) showed reactivity with S-HCL 2 mAb over the full range of B-cell differentiation. In particular, very early B cells in cytoplasmic Ig (cIg)-negative, CD19-positive pre-pre-B-cell leukemias and hybrid leukemias (mixed myeloid and B-cell type) were consistently positive for CD72 on the cell surface. Therefore, CD72 may become an important marker for progenitor B-cell leukemias.

  4. 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.

  5. Genome-Wide Analysis of Alpharetroviral Integration in Human Hematopoietic Stem/Progenitor Cells

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    Arianna Moiani

    2014-05-01

    Full Text Available Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses or the transcribed portion of expressed genes (lentiviruses, and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models. We performed a high-throughput analysis of the integration pattern of self-inactivating (SIN alpha-retroviral vectors in human CD34+ hematopoietic stem/progenitor cells (HSPCs and compared it to previously reported gamma-retroviral and lentiviral vectors integration profiles obtained in the same experimental setting. Compared to gamma-retroviral and lentiviral vectors, the SIN-ASLV vector maintains a preference for open chromatin regions, but shows no bias for transcriptional regulatory elements or transcription units, as defined by genomic annotations and epigenetic markers (H3K4me1 and H3K4me3 histone modifications. Importantly, SIN-ASLV integrations do not cluster in hot spots and target potentially dangerous genomic loci, such as the EVI2A/B, RUNX1 and LMO2 proto-oncogenes at a virtually random frequency. These characteristics predict a safer profile for ASLV-derived vectors for clinical applications.

  6. SSEA-4 and YKL-40 positive progenitor subtypes in the subventricular zone of developing human neocortex.

    Science.gov (United States)

    Brøchner, Christian B; Møllgård, Kjeld

    2016-01-01

    The glycosphingolipid SSEA-4 and the glycoprotein YKL-40 have both been associated with human embryonic and neural stem cell differentiation. We investigated the distribution of SSEA-4 and YKL-40 positive cells in proliferative zones of human fetal forebrain using immunohistochemistry and double-labeling immunofluorescence. A few small rounded SSEA-4 and YKL-40 labeled cells were present in the radial glial BLBP positive proliferative zones adjacent to the lateral ganglionic eminence from 12th week post conception. With increasing age, a similarly stained cell population appeared more widespread in the subventricular zone. At midgestation, the entire subventricular zone showed patches of SSEA-4, YKL-40, and BLBP positive cells. Co-labeling with markers for radial glial cells (RGCs) and neuronal, glial, and microglial markers tested the lineage identity of this subpopulation of radial glial descendants. Adjacent to the ventricular zone, a minor fraction showed overlap with GFAP but not with nestin, Olig2, NG2, or S100. No co-localization was found with neuronal markers NeuN, calbindin, DCX or with markers for microglial cells (Iba-1, CD68). Moreover, the SSEA-4 and YKL-40 positive cell population in subventricular zone was largely devoid of Tbr2, a marker for intermediate neuronal progenitor cells descending from RGCs. YKL-40 has recently been found in astrocytes in the neuron-free fimbria, and both SSEA-4 and YKL-40 are present in malignant astroglial brain tumors. We suggest that the population of cells characterized by immunohistochemical combination of antibodies against SSEA-4 and YKL-40 and devoid of neuronal and microglial markers represent a yet unexplored astrogenic lineage illustrating the complexity of astroglial development.

  7. Interleukin-10 inhibits burst-forming unit-erythroid growth by suppression of endogenous granulocyte-macrophage colony-stimulating factor production from T cells.

    Science.gov (United States)

    Oehler, L; Kollars, M; Bohle, B; Berer, A; Reiter, E; Lechner, K; Geissler, K

    1999-02-01

    Numerous cytokines released from accessory cells have been shown to exert either stimulatory or inhibitory growth signals on burst-forming unit-erythroid (BFU-E) growth. Because of its cytokine synthesis-inhibiting effects on T cells and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirectly affecting erythropoiesis. We investigated the effects of IL-10 on BFU-E growth from normal human peripheral blood mononuclear cells (PBMC) using a clonogenic progenitor cell assay. The addition of recombinant human IL-10 to cultures containing recombinant human erythropoietin suppressed BFU-E growth in a dose-dependent manner (by 55.2%, range 47.3-63.3%, p cultivating highly enriched CD34+ cells. BFU-E growth from PBMC also was markedly suppressed in the presence of a neutralizing anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (by 48.7%, range 32.9-61.2% inhibition,p < 0.01), but not by neutralizing antibodies against granulocyte colony-stimulating factor and interleukin-3. This suggests a stimulatory role of endogenously released GM-CSF on BFU-E formation. Also, the addition of exogenous GM-CSF completely restored IL-10-induced suppression of BFU-E growth. To determine the cellular source of GM-CSF production, we analyzed GM-CSF levels in suspension cultures containing PBMC that were either depleted of monocytes or T cells. Monocyte-depleted PBMC showed spontaneous production of increasing amounts of GM-CSF on days 3, 5, and 7, respectively, which could be suppressed by IL-10, whereas GM-CSF levels did not increase in cultures containing T-cell-depleted PBMC. Our data indicate that IL-10 inhibits the growth of erythroid progenitor cells in vitro, most likely by suppression of endogenous GM-CSF production from T cells.

  8. Stroma-conditioned media improve expansion of human primitive hematopoietic stem cells and progenitor cells.

    Science.gov (United States)

    Breems, D A; Blokland, E A; Ploemacher, R E

    1997-01-01

    It has been reported that stroma-dependent cultures support proliferation of hematopoietic stem cells (HSC). In order to investigate the effect of soluble stromal factors, we developed short-term serum-low liquid cultures in which the effect of stroma-conditioned media (SCM) from the murine FBMD-1, and human L87/4 and L88/5 cell lines was studied on the maintenance and expansion of various human HSC subsets in CD34-positive selected mobilized peripheral blood stem cells (PBSC) from autologous transplants of lymphoma and multiple myeloma patients. The human cobblestone area forming cell (CAFC) assay was employed to determine the frequencies of both the CAFC weeks 2 to 4 as tentative indicators of progenitor and transiently repopulating HSC, and the more primitive CAFC weeks 6 to 8 as indicators of long-term repopulating HSC. In 7-day liquid cultures containing interleukin-3 (IL-3), stem cell factor (SCF) and IL-6, we recovered 3.0-fold more colony-forming cells (CFC) and 1.7- to 1.9-fold more CAFC weeks 2 and 4. The absolute number of primitive CAFC weeks 6 and 8 were only maintained (1.1- to 1.4-fold) in these liquid cultures. This modest expansion was significantly improved by the addition of SCM from the FBMD-1, L87/4 or L88/5 cell lines. Output CFC numbers were 6.8-, 5.8- and 9.9-fold higher, respectively, than the input values, while absolute CAFC week 2 to 4 numbers were 4.5-, 10.2- and 10.2-fold expanded, respectively. The addition of SCM also improved expansion of the more primitive CAFC week 6 to 8 stem cell subsets by 2.2-, 4.5- and 4.9-fold, respectively. The addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), IL-1beta, IL-11 or macrophage inflammatory protein-1alpha to cultures containing IL-3, SCF and IL-6 could not explain the SCM effect and in all these combinations SCM addition further increased the recovery of HSC subsets. Similarly, addition of anti-cytokine antibodies (ie alpha-G-CSF, alpha-GM-CSF, alpha

  9. Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

    Science.gov (United States)

    Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

    2016-12-01

    : MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8(+) cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8(-)CD69(+) T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune

  10. Smooth Muscle Progenitor Cells Derived From Human Pluripotent Stem Cells Induce Histologic Changes in Injured Urethral Sphincter.

    Science.gov (United States)

    Li, Yanhui; Wen, Yan; Wang, Zhe; Wei, Yi; Wani, Prachi; Green, Morgaine; Swaminathan, Ganesh; Ramamurthi, Anand; Pera, Renee Reijo; Chen, Bertha

    2016-12-01

    : Data suggest that myoblasts from various sources, including bone marrow, skeletal muscle, and adipose tissue, can restore muscle function in patients with urinary incontinence. Animal data have indicated that these progenitor cells exert mostly a paracrine effect on the native tissues rather than cell regeneration. Limited knowledge is available on the in vivo effect of human stem cells or muscle progenitors on injured muscles. We examined in vivo integration of smooth muscle progenitor cells (pSMCs) derived from human pluripotent stem cells (hPSCs). pSMCs were derived from a human embryonic stem cell line (H9-ESCs) and two induced pluripotent stem cell (iPSC) lines. pSMCs were injected periurethrally into urethral injury rat models (2 × 10(6) cells per rat) or intramuscularly into severe combined immunodeficiency mice. Histologic and quantitative image analysis revealed that the urethras in pSMC-treated rats contained abundant elastic fibers and thicker muscle layers compared with the control rats. Western blot confirmed increased elastin/collagen III content in the urethra and bladder of the H9-pSMC-treated rats compared with controls. iPSC-pSMC treatment also showed similar trends in elastin and collagen III. Human elastin gene expression was not detectable in rodent tissues, suggesting that the extracellular matrix synthesis resulted from the native rodent tissues rather than from the implanted human cells. Immunofluorescence staining and in vivo bioluminescence imaging confirmed long-term engraftment of pSMCs into the host urethra and the persistence of the smooth muscle phenotype. Taken together, the data suggest that hPSC-derived pSMCs facilitate restoration of urethral sphincter function by direct smooth muscle cell regeneration and by inducing native tissue elastin/collagen III remodeling. The present study provides evidence that a pure population of human smooth muscle progenitor cells (pSMCs) derived from human pluripotent stem cells (hPSCs) (human

  11. Isolation of human umbilical cord blood aldehyde dehydrogenase-expressing progenitor cells that modulate vascular regenerative functions in vitro and in vivo.

    Science.gov (United States)

    Putman, David M; Hess, David A

    2013-01-01

    This unit describes the isolation and application of human umbilical cord blood progenitor cells to modulate vascular regenerative functions using in vitro co-culture systems and in vivo transplantation models. Using aldehyde dehydrogenase as a marker of stem cell function, blood-derived progenitors can be efficiently purified form human umbilical cord blood using flow cytometry. We describe in vitro approaches to measure cell-mediated effects on the survival, proliferation, and tube-forming function of endothelial cells using growth-rate assays and Matrigel tube-forming assays. Additionally, we provide a detailed protocol for inducing acute unilateral hindlimb ischemia in immune-deficient mice to assess progenitor cell-modulated effects on vascular regeneration by tracking the recovery of blood flow using noninvasive laser Doppler perfusion imaging. Collectively, we present combined in vitro and in vivo transplantation strategies for the pre-clinical assessment of human progenitor cell-based therapies to treat ischemic disease.

  12. Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination.

    Directory of Open Access Journals (Sweden)

    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.

  13. Isolation and characterization of connective tissue progenitor cells derived from human fracture-induced hemarthrosis in vitro.

    Science.gov (United States)

    Lee, Sang Yang; Miwa, Masahiko; Sakai, Yoshitada; Kuroda, Ryosuke; Oe, Keisuke; Niikura, Takahiro; Matsumoto, Tomoyuki; Fujioka, Hiroyuki; Doita, Minoru; Kurosaka, Masahiro

    2008-02-01

    In our search for alternative sources of connective tissue progenitor cells that can be obtained with minimal invasion, we studied human intraarticular fracture-induced hemarthrosis of the knee and attempted to isolate connective tissue progenitors from the hemarthrosis. Hemarthrosis was aspirated from the knee joints of 13 patients suffering from intraarticular osteochondral fractures of the knee. Mononuclear cells were isolated from the aspirated hemarthrosis by density gradient separation, and cultured. We were able to obtain fibroblastic adherent cells from the mononuclear cell fractions. Flow cytometry analysis after in vitro expansion on tissue culture plastic revealed that the fibroblastic cells were positive for CD29, CD44, CD105, and CD166, and negative for CD14, CD34, CD45, and CD133. These cells could differentiate in vitro into osteogenic, chondrogenic, and adipogenic cells in the presence of lineage-specific induction factors. These results demonstrate that human intraarticular fracture-induced knee hemarthrosis contains connective tissue progenitor cells with morphologic features, immunophenotypic markers, and differentiation potential that are similar to bone marrow stromal cells. This suggests that hemarthrosis, which is easy to harvest without unnecessary invasion to the patient, has possible future clinical applications such as in tissue-engineered therapies for severe osteochondral defects, posttraumatic osteoarthritis, and delayed fracture unions or nonunions.

  14. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation.

    Science.gov (United States)

    Nolbrant, Sara; Heuer, Andreas; Parmar, Malin; Kirkeby, Agnete

    2017-09-01

    Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. We show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts. By specifying how to perform quality control (QC), troubleshooting and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate reproducibility of experiments and enable shipping of cells between centers, we present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.

  15. Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells.

    Science.gov (United States)

    Butler, Jason M; Gars, Eric J; James, Daylon J; Nolan, Daniel J; Scandura, Joseph M; Rafii, Shahin

    2012-08-09

    Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.

  16. Separation of haemopoietic cells for biochemical investigation. Preparation of erythroid and myeloid cells from human and laboratory-animal bone marrow and the separation of erythroblasts according to their state of maturation.

    Science.gov (United States)

    Harrison, F L; Beswick, T M; Chesterton, C J

    1981-03-15

    The separation of haemopoietic bone-marrow cells by centrifugation through discontinuous density gradients of Percoll is described. This method was used to prepare fractions enriched in erythroblasts, myeloid blast cells or reticulocytes from bone marrow of anaemic and non-anaemic rabbits, from the marrow of other anaemic laboratory animals and from human samples. It is a simple, rapid, reproducible and inexpensive technique that can be readily adapted to suit individual requirements. Secondly, a convenient method is presented for the separation of large quantities of bone-marrow cells into fractions enriched in erythroblasts at different stages of maturation, by velocity sedimentation through a linear gradient of 1-2% sucrose at unit gravity. In vitro, erythroblasts adhere together strongly via a mechanism almost certainly involving a beta-galactoside-specific surface lectin termed erythroid developmental agglutinin. Since the efficiency of cell-separation techniques depends heavily on the maintenance of a single cell suspension in which each unit can move independently, the presence of an adhesive molecule at the cell surface is of considerable significance. The effect of washing the marrow with a lactose-containing medium, which has been shown to remove the agglutinin, was therefore investigated in relation to both methods. The separation on Percoll gradients is considerably enhanced by this treatment. In addition, the unit-gravity sedimentation gradient can be loaded with 5-10 times more cells after lactose extraction in comparison with intact marrow. Although enrichment is less, a useful fractionation according to maturation is still obtained.

  17. On the nucleolar size and density in human early granulocytic progenitors, myeloblasts

    Directory of Open Access Journals (Sweden)

    K Smetana

    2009-06-01

    Full Text Available Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.

  18. Gene Editing of Human Hematopoietic Stem and Progenitor Cells: Promise and Potential Hurdles.

    Science.gov (United States)

    Yu, Kyung-Rok; Natanson, Hannah; Dunbar, Cynthia E

    2016-08-02

    Hematopoietic stem and progenitor cells (HSPCs) have great therapeutic potential because of their ability to both self-renew and differentiate. It has been proposed that, given their unique properties, a small number of genetically modified HSPCs could accomplish lifelong, corrective reconstitution of the entire hematopoietic system in patients with various hematologic disorders. Scientists have demonstrated that gene addition therapies-targeted to HSPCs and using integrating retroviral vectors-possess clear clinical benefits in multiple diseases, among them immunodeficiencies, storage disorders, and hemoglobinopathies. Scientists attempting to develop clinically relevant gene therapy protocols have, however, encountered a number of unexpected hurdles because of their incomplete knowledge of target cells, genomic control, and gene transfer technologies. Targeted gene-editing technologies using engineered nucleases such as ZFN, TALEN, and/or CRISPR/Cas9 RGEN show great clinical promise, allowing for the site-specific correction of disease-causing mutations-a process with important applications in autosomal dominant or dominant-negative genetic disorders. The relative simplicity of the CRISPR/Cas9 system, in particular, has sparked an exponential increase in the scientific community's interest in and use of these gene-editing technologies. In this minireview, we discuss the specific applications of gene-editing technologies in human HSPCs, as informed by prior experience with gene addition strategies. HSPCs are desirable but challenging targets; the specific mechanisms these cells evolved to protect themselves from DNA damage render them potentially more susceptible to oncogenesis, especially given their ability to self-renew and their long-term proliferative potential. We further review scientists' experience with gene-editing technologies to date, focusing on strategies to move these techniques toward implementation in safe and effective clinical trials.

  19. Doxorubicin Regulates Autophagy Signals via Accumulation of Cytosolic Ca2+ in Human Cardiac Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Ji Hye Park

    2016-10-01

    Full Text Available Doxorubicin (DOXO is widely used to treat solid tumors. However, its clinical use is limited by side effects including serious cardiotoxicity due to cardiomyocyte damage. Resident cardiac progenitor cells (hCPCs act as key regulators of homeostasis in myocardial cells. However, little is known about the function of hCPCs in DOXO-induced cardiotoxicity. In this study, we found that DOXO-mediated hCPC toxicity is closely related to calcium-related autophagy signaling and was significantly attenuated by blocking mTOR signaling in human hCPCs. DOXO induced hCPC apoptosis with reduction of SMP30 (regucalcin and autophagosome marker LC3, as well as remarkable induction of the autophagy-related markers, Beclin-1, APG7, and P62/SQSTM1 and induction of calcium-related molecules, CaM (Calmodulin and CaMKII (Calmodulin kinase II. The results of an LC3 puncta assay further indicated that DOXO reduced autophagosome formation via accumulation of cytosolic Ca2+. Additionally, DOXO significantly induced mTOR expression in hCPCs, and inhibition of mTOR signaling by rapamycin, a specific inhibitor, rescued DOXO-mediated autophagosome depletion in hCPCs with significant reduction of DOXO-mediated cytosolic Ca2+ accumulation in hCPCs, and restored SMP30 and mTOR expression. Thus, DOXO-mediated hCPC toxicity is linked to Ca2+-related autophagy signaling, and inhibition of mTOR signaling may provide a cardio-protective effect against DOXO-mediated hCPC toxicity.

  20. Human neural progenitors derived from integration-free iPSCs for SCI therapy

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2017-03-01

    Full Text Available As a potentially unlimited autologous cell source, patient induced pluripotent stem cells (iPSCs provide great capability for tissue regeneration, particularly in spinal cord injury (SCI. However, despite significant progress made in translation of iPSC-derived neural progenitor cells (NPCs to clinical settings, a few hurdles remain. Among them, non-invasive approach to obtain source cells in a timely manner, safer integration-free delivery of reprogramming factors, and purification of NPCs before transplantation are top priorities to overcome. In this study, we developed a safe and cost-effective pipeline to generate clinically relevant NPCs. We first isolated cells from patients' urine and reprogrammed them into iPSCs by non-integrating Sendai viral vectors, and carried out experiments on neural differentiation. NPCs were purified by A2B5, an antibody specifically recognizing a glycoganglioside on the cell surface of neural lineage cells, via fluorescence activated cell sorting. Upon further in vitro induction, NPCs were able to give rise to neurons, oligodendrocytes and astrocytes. To test the functionality of the A2B5+ NPCs, we grafted them into the contused mouse thoracic spinal cord. Eight weeks after transplantation, the grafted cells survived, integrated into the injured spinal cord, and differentiated into neurons and glia. Our specific focus on cell source, reprogramming, differentiation and purification method purposely addresses timing and safety issues of transplantation to SCI models. It is our belief that this work takes one step closer on using human iPSC derivatives to SCI clinical settings.

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

    Science.gov (United States)

    Liu, Tuoen; Krysiak, Kilannin; Shirai, Cara Lunn; Kim, Sanghyun; Shao, Jin; Ndonwi, Matthew; Walter, Matthew J

    2017-01-01

    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.

  2. Doxorubicin Regulates Autophagy Signals via Accumulation of Cytosolic Ca2+ in Human Cardiac Progenitor Cells

    Science.gov (United States)

    Park, Ji Hye; Choi, Sung Hyun; Kim, Hyungtae; Ji, Seung Taek; Jang, Woong Bi; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang Mo

    2016-01-01

    Doxorubicin (DOXO) is widely used to treat solid tumors. However, its clinical use is limited by side effects including serious cardiotoxicity due to cardiomyocyte damage. Resident cardiac progenitor cells (hCPCs) act as key regulators of homeostasis in myocardial cells. However, little is known about the function of hCPCs in DOXO-induced cardiotoxicity. In this study, we found that DOXO-mediated hCPC toxicity is closely related to calcium-related autophagy signaling and was significantly attenuated by blocking mTOR signaling in human hCPCs. DOXO induced hCPC apoptosis with reduction of SMP30 (regucalcin) and autophagosome marker LC3, as well as remarkable induction of the autophagy-related markers, Beclin-1, APG7, and P62/SQSTM1 and induction of calcium-related molecules, CaM (Calmodulin) and CaMKII (Calmodulin kinase II). The results of an LC3 puncta assay further indicated that DOXO reduced autophagosome formation via accumulation of cytosolic Ca2+. Additionally, DOXO significantly induced mTOR expression in hCPCs, and inhibition of mTOR signaling by rapamycin, a specific inhibitor, rescued DOXO-mediated autophagosome depletion in hCPCs with significant reduction of DOXO-mediated cytosolic Ca2+ accumulation in hCPCs, and restored SMP30 and mTOR expression. Thus, DOXO-mediated hCPC toxicity is linked to Ca2+-related autophagy signaling, and inhibition of mTOR signaling may provide a cardio-protective effect against DOXO-mediated hCPC toxicity. PMID:27735842

  3. Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group

    Science.gov (United States)

    Westers, Theresia M.; Cremers, Eline M.P.; Oelschlaegel, Uta; Johansson, Ulrika; Bettelheim, Peter; Matarraz, Sergio; Orfao, Alberto; Moshaver, Bijan; Brodersen, Lisa Eidenschink; Loken, Michael R.; Wells, Denise A.; Subirá, Dolores; Cullen, Matthew; te Marvelde, Jeroen G.; van der Velden, Vincent H.J.; Preijers, Frank W.M.B.; Chu, Sung-Chao; Feuillard, Jean; Guérin, Estelle; Psarra, Katherina; Porwit, Anna; Saft, Leonie; Ireland, Robin; Milne, Timothy; Béné, Marie C.; Witte, Birgit I.; Della Porta, Matteo G.; Kern, Wolfgang; van de Loosdrecht, Arjan A.

    2017-01-01

    Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84–94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86–97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage. PMID:27758818

  4. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

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    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-06-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes.

  5. Long-term follow-up of myelodysplastic syndrome patients with moderate/severe anaemia receiving human recombinant erythropoietin + 13-cis-retinoic acid and dihydroxylated vitamin D3: independent positive impact of erythroid response on survival.

    Science.gov (United States)

    Crisà, Elena; Foli, Cristina; Passera, Roberto; Darbesio, Antonella; Garvey, Kimberly B; Boccadoro, Mario; Ferrero, Dario

    2012-07-01

    We previously reported a 60% erythroid response rate with recombinant erythropoietin + 13-cis retinoic acid + dihydroxylated vitamin D3 in 63 elderly myelodysplastic patients (median age 75 years) with unfavourable features for response to erythropoietin alone [70% transfusion-dependent, 35% refractory anaemia with ring sideroblasts/refractory anaemia with excess of blasts type 1 (RAEB1), 70% with International Prognostic Scoring System (IPSS) Intermediate-1 or -2]. This report updates that case study at a 7-year follow-up, and compared the impact on overall survival of erythroid response to known prognostic factors. The erythroid response duration (median 17 months; 22 in non-RAEB patients, with 20% patients in response after 6 years of therapy) was longer than in most studies with erythropoietin alone. Overall survival (median 55 months in non-RAEB, 15 in RAEB1 patients) was negatively affected by RAEB1 diagnosis, IPSS and WPSS intermediate scores and transfusion-dependence. In the multivariate analysis, erythroid response maintained an independent positive impact on survival, particularly in non-RAEB patients in the first 3 years from diagnosis (90% survival compared to 50% of non-responders). In conclusion, the long-term follow-up confirmed the achievement, by our combined treatment, of fairly long-lasting erythroid response in the majority of MDS patients with unfavourable prognostic features for response to erythropoietin: this translated in a survival benefit that was independent from other prognostic features.

  6. Interplay between human microglia and neural stem/progenitor cells in an allogeneic co-culture model.

    Science.gov (United States)

    Liu, Jia; Hjorth, Erik; Zhu, Mingqin; Calzarossa, Cinzia; Samuelsson, Eva-Britt; Schultzberg, Marianne; Åkesson, Elisabet

    2013-11-01

    Experimental neural cell therapies, including donor neural stem/progenitor cells (NPCs) have been reported to offer beneficial effects on the recovery after an injury and to counteract inflammatory and degenerative processes in the central nervous system (CNS). The interplay between donor neural cells and the host CNS still to a large degree remains unclear, in particular in human allogeneic conditions. Here, we focused our studies on the interaction of human NPCs and microglia utilizing a co-culture model. In co-cultures, both NPCs and microglia showed increased survival and proliferation compared with mono-cultures. In the presence of microglia, a larger subpopulation of NPCs expressed the progenitor cell marker nestin, whereas a smaller group of NPCs expressed the neural markers polysialylated neural cell adhesion molecule, A2B5 and glial fibrillary acidic protein compared with NPC mono-cultures. Microglia thus hindered differentiation of NPCs. The presence of human NPCs increased microglial phagocytosis of latex beads. Furthermore, we observed that the expression of CD200 molecules on NPCs and the CD200 receptor protein on microglia was enhanced in co-cultures, whereas the release of transforming growth factor-β was increased suggesting anti-inflammatory features of the co-cultures. To conclude, the interplay between human allogeneic NPCs and microglia, significantly affected their respective proliferation and phenotype. Neural cell therapy including human donor NPCs may in addition to offering cell replacement, modulate host microglial phenotypes and functions to benefit neuroprotection and repair.

  7. Treating Diet-Induced Diabetes and Obesity with Human Embryonic Stem Cell-Derived Pancreatic Progenitor Cells and Antidiabetic Drugs

    Directory of Open Access Journals (Sweden)

    Jennifer E. Bruin

    2015-04-01

    Full Text Available Human embryonic stem cell (hESC-derived pancreatic progenitor cells effectively reverse hyperglycemia in rodent models of type 1 diabetes, but their capacity to treat type 2 diabetes has not been reported. An immunodeficient model of type 2 diabetes was generated by high-fat diet (HFD feeding in SCID-beige mice. Exposure to HFDs did not impact the maturation of macroencapsulated pancreatic progenitor cells into glucose-responsive insulin-secreting cells following transplantation, and the cell therapy improved glucose tolerance in HFD-fed transplant recipients after 24 weeks. However, since diet-induced hyperglycemia and obesity were not fully ameliorated by transplantation alone, a second cohort of HFD-fed mice was treated with pancreatic progenitor cells combined with one of three antidiabetic drugs. All combination therapies rapidly improved body weight and co-treatment with either sitagliptin or metformin improved hyperglycemia after only 12 weeks. Therefore, a stem cell-based therapy may be effective for treating type 2 diabetes, particularly in combination with antidiabetic drugs.

  8. Complementary populations of human adipose CD34+ progenitor cells promote growth, angiogenesis, and metastasis of breast cancer.

    Science.gov (United States)

    Orecchioni, Stefania; Gregato, Giuliana; Martin-Padura, Ines; Reggiani, Francesca; Braidotti, Paola; Mancuso, Patrizia; Calleri, Angelica; Quarna, Jessica; Marighetti, Paola; Aldeni, Chiara; Pruneri, Giancarlo; Martella, Stefano; Manconi, Andrea; Petit, Jean-Yves; Rietjens, Mario; Bertolini, Francesco

    2013-10-01

    Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.

  9. Trichostatin A enhances vascular repair by injected human endothelial progenitors through increasing the expression of TAL1-dependent genes.

    Science.gov (United States)

    Palii, Carmen G; Vulesevic, Branka; Fraineau, Sylvain; Pranckeviciene, Erinija; Griffith, Alexander J; Chu, Alphonse; Faralli, Hervé; Li, Yuhua; McNeill, Brian; Sun, Jie; Perkins, Theodore J; Dilworth, F Jeffrey; Perez-Iratxeta, Carol; Suuronen, Erik J; Allan, David S; Brand, Marjorie

    2014-05-01

    A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential.

  10. VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures.

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    Lynda S Wright

    Full Text Available Three dimensional (3D culture techniques are frequently used for CNS tissue modeling and organoid production, including generation of retina-like tissues. A proposed advantage of these 3D systems is their potential to more closely approximate in vivo cellular microenvironments, which could translate into improved manufacture and/or maintenance of neuronal populations. Visual System Homeobox 2 (VSX2 labels all multipotent retinal progenitor cells (RPCs and is known to play important roles in retinal development. In contrast, the proneural transcription factor Acheate scute-like 1 (ASCL1 is expressed transiently in a subset of RPCs, but is required for the production of most retinal neurons. Therefore, we asked whether the presence of VSX2 and ASCL1 could gauge neurogenic potential in 3D retinal cultures derived from human prenatal tissue or ES cells (hESCs. Short term prenatal 3D retinal cultures displayed multiple characteristics of human RPCs (hRPCs found in situ, including robust expression of VSX2. Upon initiation of hRPC differentiation, there was a small increase in co-labeling of VSX2+ cells with ASCL1, along with a modest increase in the number of PKCα+ neurons. However, 3D prenatal retinal cultures lost expression of VSX2 and ASCL1 over time while concurrently becoming refractory to neuronal differentiation. Conversely, 3D optic vesicles derived from hESCs (hESC-OVs maintained a robust VSX2+ hRPC population that could spontaneously co-express ASCL1 and generate photoreceptors and other retinal neurons for an extended period of time. These results show that VSX2 and ASCL1 can serve as markers for neurogenic potential in cultured hRPCs. Furthermore, unlike hESC-OVs, maintenance of 3D structure does not independently convey an advantage in the culture of prenatal hRPCs, further illustrating differences in the survival and differentiation requirements of hRPCs extracted from native tissue vs. those generated entirely in vitro.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  12. Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers.

    Directory of Open Access Journals (Sweden)

    Wendy W Hwang-Verslues

    Full Text Available Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+/CD24(-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+/ESA(+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+/CD24(-/low, ESA(+, CD133(+, CXCR4(+ and PROCR(+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

  13. Characterization of human acute myeloid leukemia progenitor cells: growth factor responsiveness and membrane phenotype.

    NARCIS (Netherlands)

    H.R. Delwel (Ruud)

    1990-01-01

    textabstractBlood cell formation takes place Multipotential bone marrow stem cells primarily in the bone marrow. are able to undergo selfrenewal or produce progenitor cells which are predestinated to mature towards certain functional blood cells, e.g. erythrocytes, granulocytes,

  14. Neonatal CD71+ erythroid cells do not modify murine sepsis mortality

    Science.gov (United States)

    Wynn, James L.; Scumpia, Philip O.; Stocks, Blair T.; Romano-Keeler, Joann; Alrifai, Mhd Wael; Liu, Jin-Hua; Kim, Annette S.; Alford, Catherine E.; Matta, Pranathi; Weitkamp, Jörn-Hendrik; Moore, Daniel J.

    2015-01-01

    Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested murine neonatal host defense against infection could be compromised by immunosuppressive CD71+ erythroid splenocytes. We examined the impact of CD71+ erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71+ erythroid (CD235a+) cells in human neonates. Adoptive transfer or antibody-mediated reduction of neonatal CD71+ erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b+ cells was not limited to neonatal splenocytes as it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 antibody showed reduced splenic bacterial load following bacterial challenge compared to isotype-treated mice. However, adoptive transfer of enriched CD71+ erythroid splenocytes to CD71+-reduced animals did not reduce bacterial clearance. Human CD71+CD235a+ cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71+ erythroid splenocytes under these experimental conditions suggests the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 antibody treatment was likely responsible for the reported enhanced bacterial clearance, rather than a reduction of immunosuppressive CD71+ erythroid splenocytes. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests they may have a limited role in reducing inflammation secondary to microbial colonization. PMID:26101326

  15. Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    XuanWang; YanyanLu; HuanqingZhang; KunWang; QihuaHe; YueWang; XianyuLiu; LinsongLi; XiaominWang

    2005-01-01

    Neural progenitor cells have shown tile effectiveness in the treatment of Parkinson's disease, but tile therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity ofpre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50ng/ml fibroblast growth factor 8, 10ng/ml glial cell line-derived neurotrophic factor and 10μM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum ofparkinsonian rats, only pre-differentiated grafts resulted in an elevated production ofdopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intactprogenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance ofpre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.

  16. Grain and bean lysates improve function of endothelial progenitor cells from human peripheral blood: involvement of the endogenous antioxidant defenses.

    Directory of Open Access Journals (Sweden)

    Daniela Lucchesi

    Full Text Available Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs, the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG, a Triticum Sativum grain powder, and Lady Joy (LJ, a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2. Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2-like 2/ARE (antioxidant response element activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35-0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35-0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35-0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2 expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a

  17. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

    Science.gov (United States)

    Zalzman, Michal; Gupta, Sanjeev; Giri, Ranjit K.; Berkovich, Irina; Sappal, Baljit S.; Karnieli, Ohad; Zern, Mark A.; Fleischer, Norman; Efrat, Shimon

    2003-06-01

    Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

  18. Carbon monoxide induced erythroid differentiation of K562 cells mimics the central macrophage milieu in erythroblastic islands.

    Directory of Open Access Journals (Sweden)

    Shlomi Toobiak

    Full Text Available Growing evidence supports the role of erythroblastic islands (EI as microenvironmental niches within bone marrow (BM, where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule--CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage.Based on the above, the study hypothesized CO to have a role in erythroid maturation. Thus, the effect of CO gas as a potential erythroid differentiation inducer on the common model for erythroid progenitors, K562 cells, was explored. Cells were kept under oxygen lacking environment to mimic BM conditions. Nitrogen anaerobic atmosphere (N₂A served as control for CO atmosphere (COA. Under both atmospheres cells proliferation ceased: in N₂A due to cell death, while in COA as a result of erythroid differentiation. Maturation was evaluated by increased glycophorin A expression and Hb concentration. Addition of 1%CO only to N₂A, was adequate for maintaining cell viability. Yet, the average Hb concentration was low as compared to COA. This was validated to be the outcome of diversified maturation stages of the progenitor's population.In fact, the above scenario mimics the in vivo EI conditions, where at any given moment only a minute portion of the progenitors proceeds into terminal differentiation. Hence, this model might provide a basis for further molecular investigations of the EI structure/function relationship.

  19. Nestin-positive progenitor cells isolated from human fetal pancreas have phenotypic markers identical to mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Ling Zhang; Tian-Pei Hong; Jiang Hu; Yi-Nan Liu; Yong-Hua Wu; Ling-Song Li

    2005-01-01

    AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro.METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescenceactivated cell sorting (FACS) and radioimmunoassay (RIA)were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 timesin vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cellmarkers; (3) FACS analysis revealed that CD44, CD90and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreaticduodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intracellular insulin content was detected by RIA after the induction culture.CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulinproducing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.

  20. Trisomy 21 enhances human fetal erythro-megakaryocytic development

    Science.gov (United States)

    Chou, Stella T.; Opalinska, Joanna B.; Yao, Yu; Fernandes, Myriam A.; Kalota, Anna; Brooks, John S. J.; Choi, John K.; Gewirtz, Alan M.; Danet-Desnoyers, Gwenn-ael; Nemiroff, Richard L.

    2008-01-01

    Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes. PMID:18812473

  1. Recombinant human thrombopoietin augments mobilization of peripheral blood progenitor cells for autologous transplantation.

    Science.gov (United States)

    Linker, Charles; Anderlini, Paolo; Herzig, Roger; Christiansen, Neal; Somlo, George; Bensinger, William; Fay, Joseph; Lynch, Joseph P; Goodnough, Lawrence T; Ashby, Mark; Benyunes, Mark C; Jones, Dennie V; Yang, Timothy A; Miller, Langdon L; Weaver, Charles

    2003-06-01

    This study assessed the ability of various schedules of recombinant human thrombopoietin (rhTPO) to enhance mobilization of peripheral blood progenitor cells (PBPCs) in 134 patients with cancer undergoing high-dose chemotherapy and autologous PBPC transplantation. Patients received the study drug on days 1, 3, and 5 before initiation of granulocyte colony-stimulating factor (G-CSF) 10 microg/kg/day on day 5 and pheresis starting on day 9. Randomly assigned treatments on days 1, 3, and 5 were: group 1 (n=27) placebo, placebo, rhTPO 1.5 microg/kg; group 2 (n=27) rhTPO 1.5 microg/kg, placebo, placebo; groups 3 (n=28) and 4 (n=22) rhTPO 0.5 microg/kg on all 3 treatment days; and group 5 (n=30) placebo on all 3 treatment days. After high-dose chemotherapy and PBPC transplantation, groups 1 through 4 received rhTPO 1.5 microg/kg days 0, +2, +4, and +6 with either G-CSF 5 microg/kg/day (groups 1-3) or granulocyte-macrophage colony-stimulating factor 250 microg/m(2)/day (group 4). Group 5 received placebo plus G-CSF 5 microg/kg/day. The addition of rhTPO to G-CSF increased median CD34+ cell yield/pheresis in cohorts in which rhTPO was started before day 5, with higher yields in groups 2 (2.67 x 10(6)/kg) and groups 3 and 4 (3.10 x 10(6)/kg) than in group 1 (1.86 x 10(6)/kg) or group 5 (1.65 x 10(6)/kg) (P=.006 across groups). Comparing rhTPO to placebo, higher percentages of patients achieved the minimum yield of CD34+ > or =2 x 10(6)/kg (92% v 75%; P=.050) as well as the target yield of CD34+ > or =5 x 10(6)/kg (73% v 46%; P= .041). rhTPO-treated patients required fewer phereses to achieve minimum (P= .011) and target (P= .015) CD34+ cell values. rhTPO given after transplantation did not speed platelet recovery. No neutralizing antibodies were observed. We conclude that rhTPO can safely enhance mobilization of PBPC, reduce the number of leukapheresis, and allow more patients to meet minimal cell yield requirements to receive high-dose chemotherapy with PBPC

  2. GpIIb/IIIa+ subpopulation of rat megakaryocyte progenitor cells exhibits high responsiveness to human thrombopoietin.

    Science.gov (United States)

    Kato, T; Horie, K; Hagiwara, T; Maeda, E; Tsumura, H; Ohashi, H; Miyazaki, H

    1996-08-01

    The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIIb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa- population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/IIIa- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIIa+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

  3. Hypoxia Enhances Chondrogenic Differentiation of Human Cord Blood Multilineage Progenitor Cells Seeded on a Novel Scaffold of Freeze Dried Polycaprolactone

    DEFF Research Database (Denmark)

    Munir, Samir; Figueroa, Ryan Jude; Koch, Thomas Gadegaard

    Background Cartilage defects are common and causes osteoarthritis. Articular chondrocytes or bone marrow-derived stromal cells are presently the favoured cells for cartilage tissue engineering. Human umbilical cord blood multilineage progenitor cells (MLPCs) are easily harvested and have capability...... blue. Sulphated glycosaminoglycans (sGAG) and secreted CD-RAP were assessed as markers of cartilage anabolism. Results MLPCs pellets and scaffolds induced in 5% O2 showed increased cellularity and matrix deposition compared with induction in 21% O2. Matrix deposition in pellets was observed in a zonal...

  4. In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts

    Directory of Open Access Journals (Sweden)

    F Di Meglio

    2009-08-01

    Full Text Available The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (a-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively inclines toward an increase in both a-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

  5. Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach.

    Directory of Open Access Journals (Sweden)

    Fahimeh Mirakhori

    Full Text Available A number of studies generated induced neural progenitor cells (iNPCs from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach.Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo.These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.

  6. Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

    Directory of Open Access Journals (Sweden)

    Daisuke Doi

    2014-03-01

    Full Text Available Human induced pluripotent stem cells (iPSCs can provide a promising source of midbrain dopaminergic (DA neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

  7. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.

    Science.gov (United States)

    Månsson-Broberg, Agneta; Rodin, Sergey; Bulatovic, Ivana; Ibarra, Cristián; Löfling, Marie; Genead, Rami; Wärdell, Eva; Felldin, Ulrika; Granath, Carl; Alici, Evren; Le Blanc, Katarina; Smith, C I Edvard; Salašová, Alena; Westgren, Magnus; Sundström, Erik; Uhlén, Per; Arenas, Ernest; Sylvén, Christer; Tryggvason, Karl; Corbascio, Matthias; Simonson, Oscar E; Österholm, Cecilia; Grinnemo, Karl-Henrik

    2016-04-12

    The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

  8. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Agneta Månsson-Broberg

    2016-04-01

    Full Text Available The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

  9. Developing Animal Models for Optimizing the Musculoskeletal Repair Potential of Emerging Human Progenitor Cell Therapies

    Science.gov (United States)

    2014-06-01

    Implications for future research: .................................................................................................... 20 3. Tibial fracture...aspect of work that is derivative of project 1. 3.   Tibial  fracture  model   We initiated this project in the initial award as a way to...understand how the periosteal progenitor cells successfully heal a fracture as a way to better appreciate the why a segmental defect is either non- critical

  10. Calcium Signaling Is Required for Erythroid Enucleation.

    Science.gov (United States)

    Wölwer, Christina B; Pase, Luke B; Russell, Sarah M; Humbert, Patrick O

    2016-01-01

    Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.

  11. Calcium Signaling Is Required for Erythroid Enucleation.

    Directory of Open Access Journals (Sweden)

    Christina B Wölwer

    Full Text Available Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation.

  12. Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Shafiee, Abbas [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), Brisbane, QLD (Australia); Seyedjafari, Ehsan [Department of Biotechnology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Sadat Taherzadeh, Elham [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Dinarvand, Peyman [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO (United States); Soleimani, Masoud [Hematology Department, Faculty of Medical Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ai, Jafar, E-mail: jafar_ai@tums.ac.ir [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Brain and Spinal Injury Research Center, Imam Hospital, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2014-07-01

    Topographical cues can be exploited to regulate stem cell attachment, proliferation, differentiation and function in vitro and in vivo. In this study, we aimed to investigate the influence of different nanofibrous topographies on the chondrogenic differentiation potential of nasal septum derived progenitors (NSP) in vitro. Aligned and randomly oriented Ploy (L-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds were fabricated via electrospinning. First, scaffolds were fully characterized, and then NSP were seeded on them to study their capacity to support stem cell attachment, proliferation and chondrogenic differentiation. Compared to randomly oriented nanofibers, aligned scaffolds showed a high degree of nanofiber alignment with much better tensile strength properties. Both scaffolds supported NSP adhesion, proliferation and chondrogenic differentiation. Despite the higher rate of cell proliferation on random scaffolds, a better chondrogenic differentiation was observed on aligned nanofibers as deduced from higher expression of chondrogenic markers such as collagen type II and aggrecan on aligned scaffolds. These findings demonstrate that electrospun constructs maintain NSP proliferation and differentiation, and that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors. - Highlights: • Electrospun nanofiber scaffolds with different topographies were fabricated. • Aligned nanofiber scaffolds had better tensile strength properties. • Nasal septum derived progenitors were cultured on nanofibrous scaffolds. • Both topographies support proliferation and chondrogenic differentiation. • Better chondrogenic differentiation was observed on aligned nanofibers.

  13. Human parvovirus B19 in patients with beta thalassemia major from Tehran, Iran

    OpenAIRE

    Arabzadeh, Seyed Ali Mohammad; Alizadeh, Farideh; Tavakoli, Ahmad; Mollaei, Hamidreza; Bokharaei-Salim, Farah; Karimi, Gharib; Farahmand, Mohammad; Mortazavi, Helya Sadat; Monavari, Seyed Hamidreza

    2017-01-01

    Background Due to the tropism of human parvovirus B19 to erythroid progenitor cells, infection in patients with an underlying hemolytic disorder such as beta-thalassemia major leads to suppression of erythrocyte formation, referred to as transient aplasia crisis (TAC), which may be life-threatening. We investigated the prevalence of parvovirus B19 among patients with beta thalassemia major attending the Zafar Adult Thalassemia Clinic in Tehran, Iran. Methods This cross-sectional study was per...

  14. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  15. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  16. Myelotoxicity of trichothecenes and apoptosis: an in vitro study on human cord blood CD34+ hematopoietic progenitor.

    Science.gov (United States)

    Le Dréan, G; Auffret, M; Batina, P; Arnold, F; Sibiril, Y; Arzur, D; Parent-Massin, D

    2005-12-01

    Previous studies have revealed that hematological disorders associated with trichothecenes intoxication in humans could result from hematopoiesis inhibition. The most frequent and potent trichothecene mycotoxins are T-2 toxin and deoxynivalenol (DON), respectively. Apoptosis induction by these two toxins was investigated in vitro on human hematopoietic progenitors (CD34+ cells). Hoechst coloration, DNA fragmentation and annexin-V/PI labeling in flow cytometry showed that T-2 toxin, in contrast to DON, induced apoptosis in CD34+ cells. T-2 toxin effect was dose- and time-dependent with a significant increase of apoptotic cells as early as 3h after incubation at 10(-7) M and a maximum reached at 12 h. This observation evidenced the high sensitivity of hematopoietic progenitors to T-2 toxin. The inhibition of T-2 toxin-induced apoptosis by a pan-caspase inhibitor (Z-VAD-fmk) suggested the involvement of caspases. The proportional increase of caspase-3 specific activity (DEVDase) with T-2 toxin concentration confirmed its role in the process. After incubation of CD34+ cells with T-2 toxin, in conditions that induced apoptosis, clonal expansion of granulo-monocytes, erythrocytes and megakaryocytes precursors was dose-dependently inhibited. The hematological effects observed in T-2 toxin mycotoxicosis could then be assigned to hematopoiesis inhibition by apoptosis. Different mechanisms that need to be further elucidated are involved in DON myelotoxicity.

  17. Western Zika Virus in Human Fetal Neural Progenitors Persists Long Term with Partial Cytopathic and Limited Immunogenic Effects

    Directory of Open Access Journals (Sweden)

    Natasha W. Hanners

    2016-06-01

    Full Text Available The recent Zika virus (ZIKV outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs. In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.

  18. Growth factor-activated stem cell circuits and stromal signals cooperatively accelerate non-integrated iPSC reprogramming of human myeloid progenitors.

    Directory of Open Access Journals (Sweden)

    Tea Soon Park

    Full Text Available 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

  19. Three-dimensional normal human neural progenitor tissue-like assemblies: a model of persistent varicella-zoster virus infection.

    Directory of Open Access Journals (Sweden)

    Thomas J Goodwin

    Full Text Available Varicella-zoster virus (VZV is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP in tissue-like assemblies (TLAs, which can be successfully maintained for at least 180 days in three-dimensional (3D culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

  20. Three-dimensional normal human neural progenitor tissue-like assemblies: a model of persistent varicella-zoster virus infection.

    Science.gov (United States)

    Goodwin, Thomas J; McCarthy, Maureen; Osterrieder, Nikolaus; Cohrs, Randall J; Kaufer, Benedikt B

    2013-01-01

    Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

  1. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains

    Directory of Open Access Journals (Sweden)

    Kuypers Frans A

    2006-07-01

    Full Text Available Abstract Background The formation of acyl-CoA by the action of acyl-CoA synthetases plays a crucial role in membrane lipid turnover, including the plasma membrane of erythrocytes. In human, five Acyl-CoA Synthetase Long-chain (ACSL genes have been identified with as many as 3 different transcript variants for each. Results Acyl-CoA Synthetase Long-chain member 6 (ACSL6 is responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants were also isolated from brain and testis. We report the expression in reticulocytes of two new variants and of the one isolated from brain. All three represented different spliced variants of a mutually exclusive exon pair. They encode a slightly different short motif which contains a conserved structural domain, the fatty acid Gate domain. The motifs differ in the presence of either the aromatic residue phenylalanine (Phe or tyrosine (Tyr. Based on homology, two new isoforms for the closely related ACSL1 were predicted and characterized. One represented a switch of the Phe- to the Tyr-Gate domain motif, the other resulted from the exclusion of both. Swapping of this motif also appears to be common in all mammalian ACSL member 1 and 6 homologs. Conclusion We propose that a Phe to Tyr substitution or deletion of the Gate domain, is the structural reason for the conserved alternative splicing that affects these motifs. Our findings support our hypothesis that this region is structurally important to define the activity of these enzymes.

  2. Differential sensitivity to JAK inhibitory drugs by isogenic human erythroblasts and hematopoietic progenitors generated from patient-specific induced pluripotent stem cells.

    Science.gov (United States)

    Ye, Zhaohui; Liu, Cyndi F; Lanikova, Lucie; Dowey, Sarah N; He, Chaoxia; Huang, Xiaosong; Brodsky, Robert A; Spivak, Jerry L; Prchal, Josef T; Cheng, Linzhao

    2014-01-01

    Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD34+ hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.

  3. Comparative Effects of Human Neural Stem Cells and Oligodendrocyte Progenitor Cells on the Neurobehavioral Disorders of Experimental Autoimmune Encephalomyelitis Mice

    Directory of Open Access Journals (Sweden)

    Dae-Kwon Bae

    2016-01-01

    Full Text Available Since multiple sclerosis (MS is featured with widespread demyelination caused by autoimmune response, we investigated the recovery effects of F3.olig2 progenitors, established by transducing human neural stem cells (F3 NSCs with Olig2 transcription factor, in myelin oligodendrocyte glycoprotein- (MOG- induced experimental autoimmune encephalomyelitis (EAE model mice. Six days after EAE induction, F3 or F3.olig2 cells (1 × 106/mouse were intravenously transplanted. MOG-injected mice displayed severe neurobehavioral deficits which were remarkably attenuated and restored by cell transplantation, in which F3.olig2 cells were superior to its parental F3 cells. Transplanted cells migrated to the injured spinal cord, matured to oligodendrocytes, and produced myelin basic proteins (MBP. The F3.olig2 cells expressed growth and neurotrophic factors including brain-derived neurotrophic factor (BDNF, nerve growth factor (NGF, ciliary neurotrophic factor (CNTF, and leukemia inhibitory factor (LIF. In addition, the transplanted cells markedly attenuated inflammatory cell infiltration, reduced cytokine levels in the spinal cord and lymph nodes, and protected host myelins. The results indicate that F3.olig2 cells restore neurobehavioral symptoms of EAE mice by regulating autoimmune inflammatory responses as well as by stimulating remyelination and that F3.olig2 progenitors could be a candidate for the cell therapy of demyelinating diseases including MS.

  4. Human Bone Marrow Mesenchymal Progenitors: Perspectives on an Optimized In Vitro Manipulation

    Directory of Open Access Journals (Sweden)

    Eric eCordeiro-Spinetti

    2014-03-01

    Full Text Available When it comes to regenerative medicine, mesenchymal stem cells (MSCs are considered one of the most promising cell types for use in many cell therapies and bioengineering protocols. The International Society of Cellular Therapy recommended minimal criteria for defining multipotential MSC is based on adhesion and multipotency in vitro, and the presence or absence of select surface markers. Though these criteria help minimize discrepancies and allow some comparisons of data generated in different laboratories, the conditions in which cells are isolated and expanded are often not considered. Herein, we propose and recommend a few procedures to be followed to facilitate the establishment of quality control standards when working with mesenchymal progenitors isolation and expansion. Following these procedures, the classic Colony-Forming Unit-Fibroblast (CFU-f assay is revisited and three major topics are considered to define conditions and to assist on protocol optimization and data interpretation. We envision that the creation of a guideline will help in the identification and isolation of long-term stem cells and short-term progenitors to better explore their regenerative potential for multiple therapeutic purposes.

  5. Cystogenic potential of CD133+ progenitor cells of human polycystic kidneys.

    Science.gov (United States)

    Carvalhosa, Raquel; Deambrosis, Ilaria; Carrera, Paola; Pasquino, Chiara; Rigo, Francesca; Ferrari, Maurizio; Lasaponara, Fedele; Ranghino, Andrea; Biancone, Luigi; Segoloni, Giuseppe; Bussolati, Benedetta; Camussi, Giovanni

    2011-09-01

    In autosomal dominant polycystic kidney disease, cysts arise focally and disrupt normal renal tissue leading to renal failure. In the present study, we show that cyst-lining cells express the stem cell marker CD133. CD133+ progenitor cells isolated from polycystic kidney, carrying mutations of PKD genes, showed a dedifferentiated phenotype similar to CD133+ progenitor cells from normal kidney. However, these cells were more proliferative and presented a defective epithelial differentiation phenotype with respect to normal renal CD133+ cells as they were not able to express all tubular epithelial cell markers when cultured in epithelial differentiation medium. Polycystic CD133+ cells, in contrast to normal renal CD133+ cells, formed cysts in vitro in a three-dimensional culture system and in vivo when injected subcutaneously within Matrigel in SCID mice. Rapamycin treatment reduced in vitro proliferation of polycystic CD133+ cells and decreased cystogenesis both in vitro and in vivo. The in vitro epithelial differentiation was only partially improved by rapamycin. These results indicate that polycystic CD133+ cells retain a dedifferentiated phenotype and the ability to generate cysts.

  6. Human bone marrow mesenchymal progenitors: perspectives on an optimized in vitro manipulation

    Science.gov (United States)

    Cordeiro-Spinetti, Eric; de Mello, Wallace; Trindade, Lucas Siqueira; Taub, Dennis D.; Taichman, Russell S.; Balduino, Alex

    2013-01-01

    When it comes to regenerative medicine, mesenchymal stem cells (MSCs) are considered one of the most promising cell types for use in many cell therapies and bioengineering protocols. The International Society of Cellular Therapy recommended minimal criteria for defining multipotential MSC is based on adhesion and multipotency in vitro, and the presence or absence of select surface markers. Though these criteria help minimize discrepancies and allow some comparisons of data generated in different laboratories, the conditions in which cells are isolated and expanded are often not considered. Herein, we propose and recommend a few procedures to be followed to facilitate the establishment of quality control standards when working with mesenchymal progenitors isolation and expansion. Following these procedures, the classic Colony-Forming Unit-Fibroblast (CFU-f) assay is revisited and three major topics are considered to define conditions and to assist on protocol optimization and data interpretation. We envision that the creation of a guideline will help in the identification and isolation of long-term stem cells and short-term progenitors to better explore their regenerative potential for multiple therapeutic purposes. PMID:25364715

  7. Human bone marrow mesenchymal progenitors: perspectives on an optimized in vitro manipulation

    Directory of Open Access Journals (Sweden)

    Eric Cordeiro-Spinetti

    2014-03-01

    Full Text Available When it comes to regenerative medicine, mesenchymal stem cells (MSCs are considered one of the most promising cell types for use in many cell therapies and bioengineering protocols. The International Society of Cellular Therapy recommended minimal criteria for defining multipotential MSC is based on adhesion and multipotency in vitro, and the presence or absence of select surface markers. Though these criteria help minimize discrepancies and allow some comparisons of data generated in different laboratories, the conditions in which cells are isolated and expanded are often not considered. Herein, we propose and recommend a few procedures to be followed to facilitate the establishment of quality control standards when working with mesenchymal progenitors isolation and expansion. Following these procedures, the classic Colony-Forming Unit-Fibroblast (CFU-f assay is revisited and three major topics are considered to define conditions and to assist on protocol optimization and data interpretation. We envision that the creation of a guideline will help in the identification and isolation of long-term stem cells and short-term progenitors to better explore their regenerative potential for multiple therapeutic purposes.

  8. Silencing BRE expression in human umbilical cord perivascular (HUCPV progenitor cells accelerates osteogenic and chondrogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Elve Chen

    Full Text Available BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-β/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.

  9. TLR expression profile of human gingival margin-derived stem progenitor cells

    Science.gov (United States)

    Mekhemar, Mohamed; Adam-Klages, Sabine; Kabelitz, Dietrich; Dörfer, Christof

    2016-01-01

    Background Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. Material and Methods Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs’ incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated. Results G-MSCs showed all stem/progenitor cells’ characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test). Conclusions The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions. Key words:Stem cells, TLR, gingiva, polymerase chain reaction, FACS. PMID:26615501

  10. A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation.

    Science.gov (United States)

    Wontakal, Sandeep N; Guo, Xingyi; Smith, Cameron; MacCarthy, Thomas; Bresnick, Emery H; Bergman, Aviv; Snyder, Michael P; Weissman, Sherman M; Zheng, Deyou; Skoultchi, Arthur I

    2012-03-06

    Two mechanisms that play important roles in cell fate decisions are control of a "core transcriptional network" and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.

  11. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    WANG; Xin; (王鑫); WANG; Duncheng; (王敦成); CHEN; Xing; (陈兴),; HU; Meiru; (胡美茹); WANG; Jian'an; (王建安); LI; Yan; (黎燕); GUO; Ning; (郭宁); SHEN; Beifen; (沈倍奋)

    2001-01-01

    Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  12. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Our previous studies showed that some nuclear proteins that wereexpressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  13. Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

    OpenAIRE

    2012-01-01

    Umbilical cord blood (UCB) transplantation has emerged as promising therapy, but is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag’s effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, ...

  14. Cryopreserved hepatic progenitor cells derived from human embryonic stem cells can arrest progression of liver fibrosis in rats.

    Science.gov (United States)

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-10-01

    Hepatocytes generated from human embryonic stem cells (hESCs) are considered to be an excellent candidate for restoring the liver function deficiencies. We have earlier standardized a three-step differentiation protocol to generate functional hepatocyte-like cells (HLCs) from hESCs, which expressed the major hepatic markers. We have also found that the HLCs remain stable and functional even after extended period of in vitro culture and cryopreservation. In the present study, we have aimed to investigate the therapeutic potential of cryopreserved-thawed hESC-derived hepatic progenitor cells following transplantation in carbon tetrachloride-induced fibrotic rat livers. Significant therapeutic effects, including improved hepatic histology and normal serum biochemistry of hepatic enzymes along with increased survival rate, were observed in the cell transplanted rats. This result is an encouraging indication to develop methods for clinical application of hESC-derived hepatic lineage cells.

  15. Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss.

    Science.gov (United States)

    Wyatt, Tanya J; Rossi, Sharyn L; Siegenthaler, Monica M; Frame, Jennifer; Robles, Rockelle; Nistor, Gabriel; Keirstead, Hans S

    2011-01-01

    Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs) and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN), ALS (SOD1 G93A), and spinal cord injury (SCI). The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factors in vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss.

  16. Potential for cell therapy in Parkinson's disease using genetically programmed human embryonic stem cell-derived neural progenitor cells.

    Science.gov (United States)

    Ambasudhan, Rajesh; Dolatabadi, Nima; Nutter, Anthony; Masliah, Eliezer; Mckercher, Scott R; Lipton, Stuart A

    2014-08-15

    Neural transplantation is a promising strategy for restoring dopaminergic dysfunction and modifying disease progression in Parkinson's disease (PD). Human embryonic stem cells (hESCs) are a potential resource in this regard because of their ability to provide a virtually limitless supply of homogenous dopaminergic progenitors and neurons of appropriate lineage. The recent advances in developing robust cell culture protocols for directed differentiation of hESCs to near pure populations of ventral mesencephalic (A9-type) dopaminergic neurons has heightened the prospects for PD cell therapy. Here, we focus our review on current state-of-the-art techniques for harnessing hESC-based strategies toward development of a stem cell therapeutic for PD. Importantly, we also briefly describe a novel genetic-programming approach that may address many of the key challenges that remain in the field and that may hasten clinical translation. © 2014 Wiley Periodicals, Inc.

  17. OPTIMAL ERYTHROID CELL PRODUCTION DURING ERYTHROPOIETIN TREATMENT OF MICE OCCURS BY EXPLOITING THE SPLENIC MICROENVIRONMENT

    NARCIS (Netherlands)

    NIJHOF, W; GORIS, H; DONTJE, B; DRESZ, J; LOEFFLER, M

    1993-01-01

    In this study, quantitative effects on erythroid cell production by a prolonged recombinant human erythropoietin (rhEpo) treatment of mice are presented. Epo treatments, given subcutaneously (s.c.) twice per day in doses of 0.5 to 500 U per day, were performed under steady-state production condition

  18. High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

    NARCIS (Netherlands)

    Ros, J.E.; Libbrecht, L; Geuken, M; Jansen, PLM; Roskams, TAD

    An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on

  19. A Bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors.

    Science.gov (United States)

    Bouyer, Charlène; Chen, Pu; Güven, Sinan; Demirtaş, Tuğrul Tolga; Nieland, Thomas J F; Padilla, Frédéric; Demirci, Utkan

    2016-01-06

    A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.

  20. Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hideki Uosaki

    Full Text Available RATIONALE: Pluripotent stem cell-derived cardiac progenitor cells (CPCs have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations. OBJECTIVE: Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells. METHOD AND RESULT: To test the hypothesis, we cocultured mouse embryonic stem (ES cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1(+ PDGFRa(+ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5(+ and Isl1(+ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR(+ PDGFRa(+ CPCs from human ES cells. CONCLUSIONS: Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.

  1. Progenitor cell therapy for sacral pressure sore: a pilot study with a novel human chronic wound model.

    Science.gov (United States)

    Wettstein, Reto; Savic, Miodrag; Pierer, Gerhard; Scheufler, Oliver; Haug, Martin; Halter, Jörg; Gratwohl, Alois; Baumberger, Michael; Schaefer, Dirk Johannes; Kalbermatten, Daniel Felix

    2014-01-29

    Chronic wounds are a major health-care issue, but research is limited by the complexity and heterogeneity in terms of wound etiology as well as patient-related factors. A suitable animal model that replicates the situation in humans is not available. Therefore, the aim of the present work is to present a standardized human wound model and the data of a pilot study of topically applied progenitor cells in a sacral pressure sore. Three patients underwent cell harvest from the iliac crest at the time of the initial debridement. Forty-eight hours after bone marrow harvest and debridement, the CD34+ selected cell suspension was injected into the wound. With the aid of a laser scanner, three-dimensional analyses of wound morphometry were performed until the defect was reconstructed with a local flap 3 weeks after debridement. Decreases in volume to 60%±6% of baseline on the sham side and to 52%±3% of baseline on the cell side were measured. Histologic work-up revealed no signs of metaplastic, dysplastic, or neoplastic proliferation/differentiation after progenitor cell treatment. CD34+ cells were detected in the biopsies of day 0. The pressure sore wound model allows investigation of the initial 3 weeks after cell-based therapy. Objective outcome analysis in terms of wound volume and histology can be performed without, or with, minimal additional morbidity, and the anatomy of the sacral area allows a control and study side in the same patient. Therefore, this model can serve as a standard for wound-healing studies. ClinicalTrials.gov NCT00535548.

  2. csrnp1a is necessary for the development of primitive hematopoiesis progenitors in zebrafish.

    Directory of Open Access Journals (Sweden)

    Jaime Espina

    Full Text Available The CSRNP (cystein-serine-rich nuclear protein transcription factors are conserved from Drosophila to human. Functional studies in mice, through knockout for each of their paralogs, have resulted insufficient to elucidate the function of this family of proteins in vertebrate development. Previously, we described the function of the zebrafish ortholog, Csnrp1/Axud1, showing its essential role in the survival and proliferation of cephalic progenitors. To extend our understanding of this family, we have studied the function of its paralog csrnp1a. Our results show that csrnp1a is expressed from 0 hpf, until larval stages, particularly in cephalic territories and in the intermediate cell mass (ICM. Using morpholinos in wild type and transgenic lines we observed that Csrnp1a knockdown generates a mild reduction in head size and a depletion of blood cells in circulation. This was combined with in situ hybridizations to analyze the expression of different mesodermal and primitive hematopoiesis markers. Morphant embryos have impaired blood formation without disruption of mesoderm specification, angiogenesis or heart development. The reduction of circulating blood cells occurs at the hematopoietic progenitor level, affecting both the erythroid and myeloid lineages. In addition, cell proliferation was also altered in hematopoietic anterior sites, specifically in spi1 expression domain. These and previous observations suggest an important role of Csnrps transcription factors in progenitor biology, both in the neural and hematopoietic linages.

  3. Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

    Science.gov (United States)

    Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

    2015-10-01

    Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology.

  4. Erythroid-specific Expression of β-globin by Sleeping Beauty Transposon for Sickle Cell Disease

    Science.gov (United States)

    Zhu, Jianhui; Kren, Betsy T.; Park, Chang Won; Bilgim, Rasim; Wong, Phillip Y-P.; Steer, Clifford J.

    2013-01-01

    Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild type β- or γ-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel non-viral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild type β-globin expression cassette into the human genome for sustained expression of β-globin. We initially constructed a β-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer: chicken β-actin promoter (CAGGS) and full length β-globin cDNA, as well as truncated forms lacking either the 3′ or 5′ untranslated regions (UTRs), to optimize efficient expression of β-globin. β-globin with its 5′ UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-β-globin plasmids using a minimal β-globin gene driven by the hybrid promoters; IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human αLCR, ankyrin-1 promoter); IHβp (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human αLCR, β-globin promoter;) or HS3βp (HS3 core element from human βLCR, β-globin promoter) to establish erythroid-specific expression of β-globin. Stable genomic insertion of the minimal gene and expression of the β-globin transgene for > 5 months at a level comparable to the endogenous γ-globin gene were achieved using a SB-Tn β-globin cis construct. Interestingly, erythroid-specific expression of β-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable

  5. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Krüger Jan

    2012-03-01

    Full Text Available Abstract Background Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP. Methods CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND, osteoarthritis (OA and rheumatoid arthritis donors (RA without transforming growth factor beta 3. Results CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA. Conclusion These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.

  6. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells

    Science.gov (United States)

    Hoban, Megan D.; Cost, Gregory J.; Mendel, Matthew C.; Romero, Zulema; Kaufman, Michael L.; Joglekar, Alok V.; Ho, Michelle; Lumaquin, Dianne; Gray, David; Lill, Georgia R.; Cooper, Aaron R.; Urbinati, Fabrizia; Senadheera, Shantha; Zhu, Allen; Liu, Pei-Qi; Paschon, David E.; Zhang, Lei; Rebar, Edward J.; Wilber, Andrew; Wang, Xiaoyan; Gregory, Philip D.; Holmes, Michael C.; Reik, Andreas; Hollis, Roger P.

    2015-01-01

    Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By codelivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34+ hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγnull mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34+ cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers. PMID:25733580

  7. A bioartificial renal tubule device embedding human renal stem/progenitor cells.

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    Anna Giovanna Sciancalepore

    Full Text Available We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na(+K(+ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (20±5% and (13±5%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative "lab-on-a-chip" platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses.

  8. Sex Differences of Human Cardiac Progenitor Cells in the Biological Response to TNF-α Treatment

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    Elisabetta Straface

    2017-01-01

    Full Text Available Adult cardiac progenitor cells (CPCs, isolated as cardiosphere-derived cells (CDCs, represent promising candidates for cardiac regenerative therapy. CDCs can be expanded in vitro manyfolds without losing their differentiation potential, reaching numbers that are appropriate for clinical applications. Since mechanisms of successful CDC survival and engraftment in the damaged myocardium are still critical and unresolved issues, we aimed at deciphering possible key factors capable of bolstering CDC function. In particular, the response and the phenotype of CDCs exposed to low concentrations of the multifunctional cytokine tumor necrosis factor α (TNF-α, known to be capable of activating cell survival pathways, have been investigated. Furthermore, differential biological responses of CDCs from male and female donors, in terms of cell cycle progression and cell spreading, have also been assessed. The results obtained indicate that (i the intracellular signaling activated in our experimental conditions is most likely due to the prosurvival and proliferative signaling of TNF-α receptor 2 and that (ii cells from female patients appear more responsive to TNF-α treatment in terms of cell cycle progression and migration ability. In conclusion, the present report highlights the hypothesis that TNF-stimulated CDCs isolated from females may represent a promising candidate for cardiac regenerative therapy applications.

  9. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

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    Florian Wegner

    Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  10. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Science.gov (United States)

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

    2012-01-01

    Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  11. Integrin α6β4 identifies human distal lung epithelial progenitor cells with potential as a cell-based therapy for cystic fibrosis lung disease.

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    Xiaopeng Li

    Full Text Available To develop stem/progenitor cell-based therapy for cystic fibrosis (CF lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4(+ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4(+ epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4(- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4(+ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs serotypes, AAV2 and AAV8, capable of transducing α6β4(+ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4(+ epithelial cells significantly rescued defects in Cl(- transport. Therefore, targeting the α6β4(+ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

  12. Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report.

    Science.gov (United States)

    Menasché, Philippe; Vanneaux, Valérie; Hagège, Albert; Bel, Alain; Cholley, Bernard; Cacciapuoti, Isabelle; Parouchev, Alexandre; Benhamouda, Nadine; Tachdjian, Gérard; Tosca, Lucie; Trouvin, Jean-Hugues; Fabreguettes, Jean-Roch; Bellamy, Valérie; Guillemain, Romain; Suberbielle Boissel, Caroline; Tartour, Eric; Desnos, Michel; Larghero, Jérôme

    2015-08-07

    Comparative studies suggest that stem cells committed to a cardiac lineage are more effective for improving heart function than those featuring an extra-cardiac phenotype. We have therefore developed a population of human embryonic stem cell (ESC)-derived cardiac progenitor cells. Undifferentiated human ESCs (I6 line) were amplified and cardiac-committed by exposure to bone morphogenetic protein-2 and a fibroblast growth factor receptor inhibitor. Cells responding to these cardio-instructive cues express the cardiac transcription factor Isl-1 and the stage-specific embryonic antigen SSEA-1 which was then used to purify them by immunomagnetic sorting. The Isl-1(+) SSEA-1(+) cells were then embedded into a fibrin scaffold which was surgically delivered onto the infarct area in a 68-year-old patient suffering from severe heart failure [New York Heart Association [NYHA] functional Class III; left ventricular ejection fraction (LVEF): 26%]. A coronary artery bypass was performed concomitantly in a non-infarcted area. The implanted cells featured a high degree of purity (99% were SSEA-1(+)), had lost the expression of Sox-2 and Nanog, taken as markers for pluripotency, and strongly expressed Isl-1. The intraoperative delivery of the patch was expeditious. The post-operative course was uncomplicated either. After 3 months, the patient is symptomatically improved (NYHA functional Class I; LVEF: 36%) and a new-onset contractility is echocardiographically evident in the previously akinetic cell/patch-treated, non-revascularized area. There have been no complications such as arrhythmias, tumour formation, or immunosuppression-related adverse events. This observation demonstrates the feasibility of generating a clinical-grade population of human ESC-derived cardiac progenitors and combining it within a tissue-engineered construct. While any conclusion pertaining to efficacy would be meaningless, the patient's functional outcome yet provides an encouraging hint. Beyond this

  13. Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood.

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    Van Zant, G; Rummel, S A; Koller, M R; Larson, D B; Drubachevsky, I; Palsson, M; Emerson, S G

    1994-01-01

    Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation. Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells. We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources. To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma. The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo). Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days. At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC. When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected. At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma. To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors. After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the

  14. Carbamylated low-density lipoprotein induces oxidative stress and accelerated senescence in human endothelial progenitor cells.

    Science.gov (United States)

    Carracedo, Julia; Merino, Ana; Briceño, Carolina; Soriano, Sagrario; Buendía, Paula; Calleros, Laura; Rodriguez, Mariano; Martín-Malo, Alejandro; Aljama, Pedro; Ramírez, Rafael

    2011-04-01

    Carbamylated low-density lipoprotein (cLDL) plays a role in atherosclerosis. In this study we evaluate the effect of uremia on LDL carbamylation and the effect of cLDL and oxidized LDL (oxLDL; 200 μg/ml) on number, function, and genomic stability of endothelial progenitor cells (EPCs) obtained from healthy volunteers. cLDL was generated after incubation of native LDL (nLDL) with uremic serum from patients with chronic kidney disease (CKD) stages 2-4. Oxidative stress was measured by flow cytometry and fluorescent microscopy, mitochondrial depolarization by flow cytometry, senescence by β-galactosidase activity and telomere length, and DNA damage by phosphorylated histone H2AX (γH2AX). The percentage of cLDL by uremic serum was related to the severity of CKD. Compared with nLDL, cLDL induced an increase in oxidative stress (62±5 vs. 8±3%, P<0.001) and cells with mitochondrial depolarization (73±7 vs. 9±5%, P<0.001), and a decrease in EPC proliferation and angiogenesis. cLDL also induced accelerated senescence (73±16 vs. 12±9%, P<0.001), which was associated with a decrease in the expression of γH2AX (62±9 vs. 5±3%, P<0.001). The degree of injury induced by cLDL was comparable to that observed with oxLDL. This study supports the hypothesis that cLDL triggers genomic damage in EPCs, resulting in premature senescence. We can, therefore, hypothesize that EPCs injury by cLDL contributes to an increase in atherosclerotic disease in CKD.

  15. Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

    Science.gov (United States)

    Casero, David; Sandoval, Salemiz; Seet, Christopher S; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M

    2015-12-01

    To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

  16. Isolation of neural progenitor cells from the human adult subventricular zone based on expression of the cell surface marker CD271.

    Science.gov (United States)

    van Strien, Miriam E; Sluijs, Jacqueline A; Reynolds, Brent A; Steindler, Dennis A; Aronica, Eleonora; Hol, Elly M

    2014-04-01

    Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271(+) cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.

  17. Rotator cuff tear state modulates self-renewal and differentiation capacity of human skeletal muscle progenitor cells.

    Science.gov (United States)

    Thomas, Kelsey A; Gibbons, Michael C; Lane, John G; Singh, Anshuman; Ward, Samuel R; Engler, Adam J

    2017-08-01

    Full thickness rotator cuff tendon (RCT) tears have long-term effects on RC muscle atrophy and fatty infiltration, with lasting damage even after surgical tendon repair. Skeletal muscle progenitor cells (SMPs) are critical for muscle repair in response to injury, but the inability of RC muscles to recover from chronic RCT tear indicates possible deficits in repair mechanisms. Here we investigated if muscle injury state was a crucial factor during human SMP expansion and differentiation ex vivo. SMPs were isolated from muscles in patients with no, partial-thickness (PT), or full-thickness (FT) RCT tears. Despite using growth factors, physiological niche stiffness, and muscle-mimetic extracellular matrix (ECM) proteins, we found that SMPs isolated from human RC muscle with RCT tears proliferated slower but fused into myosin heavy chain (MHC)-positive myotubes at higher rates than SMPs from untorn RCTs. Proteomic analysis of RC muscle tissue revealed shifts in muscle composition with pathology, as muscle from massive RCT tears had increased ECM deposition compared with no tear RC muscle. Together these data imply that the remodeled niche in a torn RCT primes SMPs not for expansion but for differentiation, thus limiting longer-term self-renewal necessary for regeneration after surgical repair. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1816-1823, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

    Science.gov (United States)

    Chen, Hongfeng; Titushkin, Igor; Stroscio, Michael; Cho, Michael

    2007-02-15

    Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

  19. Differentiation of Human Embryonic Stem Cells to Endothelial Progenitor Cells on Laminins in Defined and Xeno-free Systems

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    Mien T.X. Nguyen

    2016-10-01

    Full Text Available A major hurdle for in vitro culturing of primary endothelial cells (ECs is that they readily dedifferentiate, hampering their use for therapeutic applications. Human embryonic stem cells (hESCs may provide an unlimited cell source; however, most current protocols deriving endothelial progenitor cells (EPCs from hESCs use direct differentiation approaches albeit on undefined matrices, yet final yields are insufficient. We developed a method to culture monolayer hESCs on stem cell niche laminin (LN LN511 or LN521 matrix. Here, we report a chemically defined, xeno-free protocol for differentiation of hESCs to EPCs using LN521 as the main culture substrate. We were able to generate ∼95% functional EPCs defined as VEGFR2+CD34+CD31+VE-Cadherin+. RNA-sequencing analyses of hESCs, EPCs, and primary human umbilical vein endothelial cells showed differentiation-related EC expression signatures, regarding basement membrane composition, cell-matrix interactions, and changes in endothelial lineage markers. Our results may facilitate production of stable ECs for the treatment of vascular diseases and in vitro cell modeling.

  20. Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

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    Hiroko Shimada

    Full Text Available The common marmoset (Callithrix jacchus is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs derived from mouse and human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

  1. Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction

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    Creer Michael H

    2010-03-01

    Full Text Available Abstract Human stem cells from adult sources have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature. The mechanisms by which this is accomplished are, however, still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDHhiLin-, and ALDHloLin- cells following transplantation to NOD/SCID or NOD/SCID β2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDHhiLin- stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDHloLin- committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was, however, a significant increase in vascular density in the central infarct zone of ALDHhiLin- cell-treated mice, as compared to PBS and ALDHloLin- cell-treated mice. Conclusions Our data indicate that adult human stem cells do not become a significant part of the regenerating tissue, but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery.

  2. Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system.

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    Minyong Kang

    Full Text Available OBJECTIVES: Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney, stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources, pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However, little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs. In this study, we developed a novel protocol for differentiation of nephron progenitor cells (NPCs from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS: We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak, intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR, real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS: After modification of culture period and concentration of exogenous factors, hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1. Moreover, NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular, approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS: Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.

  3. Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

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    Silvia Berardis

    Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

  4. Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse.

    Science.gov (United States)

    Zhang, Ye; Sloan, Steven A; Clarke, Laura E; Caneda, Christine; Plaza, Colton A; Blumenthal, Paul D; Vogel, Hannes; Steinberg, Gary K; Edwards, Michael S B; Li, Gordon; Duncan, John A; Cheshier, Samuel H; Shuer, Lawrence M; Chang, Edward F; Grant, Gerald A; Gephart, Melanie G Hayden; Barres, Ben A

    2016-01-01

    The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here, we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains and to maintain these cells in serum-free cultures. We found that human astrocytes have abilities similar to those of murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. Next, we performed RNA sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells (available at http://www.brainrnaseq.org). With these profiles, we identified novel human-specific astrocyte genes and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell-type-specific molecular profiles of the healthy and diseased human brain.

  5. 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 d...

  6. CP2 binding to the promoter is essential for the enhanced transcription of globin genes in erythroid cells.

    Science.gov (United States)

    Chae, Ji Hyung; Kim, Chul Geun

    2003-02-28

    We have previously reported that the reduced level of CP2 suppresses the mouse alpha- and beta-globin gene expression and hemoglobin synthesis during terminal differentiation of mouse erythroleukemia (MEL) cells in vitro [Chae et al. (1999)]. As an extension of this study, we demonstrated that human alpha-, epsilon-, and gamma- globin genes were also suppressed by the reduced expression of CP2 in K562 cells. To address how much CP2 contributes in the regulation of globin gene expression, we measured transcriptional activities of the wild type alpha-globin promoter and its various factor-binding sites mutants in erythroid and nonerythroid cells. Interestingly, CP2 site dependent transcriptional activation occurred in an erythroid-cell specific manner, even though CP2 is ubiquitously expressed. In addition, CP2 site mutation within the alpha-promoter severely suppressed promoter activity in differentiated, but not in undifferentiated MEL cells, suggesting that the CP2 binding site is needed for the enhanced transcription of globin genes during erythroid differentiation. When the human beta-globin locus control region was linked to the alpha-promoter, suppression was more severe in the CP2 site mutant in differentiated MEL cells. Overall data indicate that CP2 is a major factor in the regulation of globin expression in human and mouse erythroid cells, and CP2 binding to the globin gene promoter is essential for the enhanced transcription of globin genes in erythroid differentiation.

  7. Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system.

    Science.gov (United States)

    Sumiyoshi, Teiko; Holt, Nathalia G; Hollis, Roger P; Ge, Shundi; Cannon, Paula M; Crooks, Gay M; Kohn, Donald B

    2009-12-01

    Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell

  8. PAX7 Targets, CD54, Integrin α9β1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors

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    Alessandro Magli

    2017-06-01

    Full Text Available Pluripotent stem (PS-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1, and Syndecan2 (SDC2 as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases.

  9. A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q- syndrome.

    Science.gov (United States)

    Barlow, Jillian L; Drynan, Lesley F; Hewett, Duncan R; Holmes, Luke R; Lorenzo-Abalde, Silvia; Lane, Alison L; Jolin, Helen E; Pannell, Richard; Middleton, Angela J; Wong, See Heng; Warren, Alan J; Wainscoat, James S; Boultwood, Jacqueline; McKenzie, Andrew N J

    2010-01-01

    The identification of the genes associated with chromosomal translocation breakpoints has fundamentally changed understanding of the molecular basis of hematological malignancies. By contrast, the study of chromosomal deletions has been hampered by the large number of genes deleted and the complexity of their analysis. We report the generation of a mouse model for human 5q- syndrome using large-scale chromosomal engineering. Haploinsufficiency of the Cd74-Nid67 interval (containing Rps14, encoding the ribosomal protein S14) caused macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow. These effects were associated with defective bone marrow progenitor development, the appearance of bone marrow cells expressing high amounts of the tumor suppressor p53 and increased bone marrow cell apoptosis. Notably, intercrossing with p53-deficient mice completely rescued the progenitor cell defect, restoring common myeloid progenitor and megakaryocytic-erythroid progenitor, granulocyte-monocyte progenitor and hematopoietic stem cell bone marrow populations. This mouse model suggests that a p53-dependent mechanism underlies the pathophysiology of the 5q- syndrome.

  10. SSEA-4 and YKL-40 positive progenitor subtypes in the subventricular zone of developing human neocortex

    DEFF Research Database (Denmark)

    Brøchner, Christian B; Møllgård, Kjeld

    2016-01-01

    The glycosphingolipid SSEA-4 and the glycoprotein YKL-40 have both been associated with human embryonic and neural stem cell differentiation. We investigated the distribution of SSEA-4 and YKL-40 positive cells in proliferative zones of human fetal forebrain using immunohistochemistry and double-...... characterized by immunohistochemical combination of antibodies against SSEA-4 and YKL-40 and devoid of neuronal and microglial markers represent a yet unexplored astrogenic lineage illustrating the complexity of astroglial development. GLIA 2015....

  11. Elevated IKKα accelerates the differentiation of human neuronal progenitor cells and induces MeCP2-dependent BDNF expression.

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    Ali Khoshnan

    Full Text Available The IκB kinase α (IKKα is implicated in the differentiation of epithelial and immune cells. We examined whether IKKα also plays a role in the differentiation and maturation of embryonic human neuronal progenitor cells (NPCs. We find that expression of an extra copy of IKKα (IKKα+ blocks self-renewal and accelerates the differentiation of NPCs. This coincides with reduced expression of the Repressor Element Silencing Transcription Factor/Neuron-Restrictive Silencing Factor (REST/NRSF, which is a prominent inhibitor of neurogenesis, and subsequent induction of the pro-differentiation non-coding RNA, miR-124a. However, the effects of IKKα on REST/NRSF and miR-124a expression are likely to be indirect. IKKα+ neurons display extensive neurite outgrowth and accumulate protein markers of neuronal maturation such as SCG10/stathmin-2, postsynaptic density 95 (PSD95, syntaxin, and methyl-CpG binding protein 2 (MeCP2. Interestingly, IKKα associates with MeCP2 in the nuclei of human neurons and can phosphorylate MeCP2 in vitro. Using chromatin immunoprecipitation assays, we find that IKKα is recruited to the exon-IV brain-derived neurotrophic factor (BDNF promoter, which is a well-characterized target of MeCP2 activity. Moreover, IKKα induces the transcription of BDNF and knockdown expression of MeCP2 interferes with this event. These studies highlight a role for IKKα in accelerating the differentiation of human NPCs and identify IKKα as a potential regulator of MeCP2 function and BDNF expression.

  12. Human embryonic stem cell-derived oligodendrocyte progenitors aid in functional recovery of sensory pathways following contusive spinal cord injury.

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    Angelo H All

    Full Text Available BACKGROUND: Transplantations of human stem cell derivatives have been widely investigated in rodent models for the potential restoration of function of neural pathways after spinal cord injury (SCI. Studies have already demonstrated cells survival following transplantation in SCI. We sought to evaluate survival and potential therapeutic effects of transplanted human embryonic stem (hES cell-derived oligodendrocyte progenitor cells (OPCs in a contusive injury in rats. Bioluminescence imaging was utilized to verify survivability of cells up to 4 weeks, and somatosensory evoked potential (SSEPs were recorded at the cortex to monitor function of sensory pathways throughout the 6-week recovery period. PRINCIPAL FINDINGS: hES cells were transduced with the firefly luciferase gene and differentiated into OPCs. OPCs were transplanted into the lesion epicenter of rat spinal cords 2 hours after inducing a moderate contusive SCI. The hES-treatment group showed improved SSEPs, including increased amplitude and decreased latencies, compared to the control group. The bioluminescence of transplanted OPCs decreased by 97% in the injured spinal cord compared to only 80% when injected into an uninjured spinal cord. Bioluminescence increased in both experimental groups such that by week 3, no statistical difference was detected, signifying that the cells survived and proliferated independent of injury. Post-mortem histology of the spinal cords showed integration of human cells expressing mature oligodendrocyte markers and myelin basic protein without the expression of markers for astrocytes (GFAP or pluripotent cells (OCT4. CONCLUSIONS: hES-derived OPCs transplanted 2 hours after contusive SCI survive and differentiate into OLs that produce MBP. Treated rats demonstrated functional improvements in SSEP amplitudes and latencies compared to controls as early as 1 week post-injury. Finally, the hostile injury microenvironment at 2 hours post-injury initially caused

  13. Polychlorinated biphenyls disturb differentiation of normal human neural progenitor cells: clue for involvement of thyroid hormone receptors.

    Science.gov (United States)

    Fritsche, Ellen; Cline, Jason E; Nguyen, Ngoc-Ha; Scanlan, Thomas S; Abel, Josef

    2005-07-01

    Polychlorinated biphenyls (PCBs) are ubiquitous environmental chemicals that accumulate in adipose tissues over the food chain. Epidemiologic studies have indicated that PCBs influence brain development. Children who are exposed to PCBs during development suffer from neuropsychologic deficits such as a lower full-scale IQ (intelligence quotient), reduced visual recognition memory, and attention and motor deficits. The mechanisms leading to these effects are not fully understood. It has been speculated that PCBs may affect brain development by interfering with thyroid hormone (TH) signaling. Because most of the data are from animal studies, we established a model using primary normal human neural progenitor (NHNP) cells to determine if PCBs interfere with TH-dependent neural differentiation. NHNP cells differentiate into neurons, astrocytes, and oligodendrocytes in culture, and they express a variety of drug metabolism enzymes and nuclear receptors. Like triiodothyronine (T3), treatment with the mono-ortho-substituted PCB-118 (2,3',4,4 ,5-pentachlorobiphenyl; 0.01-1 microM) leads to a dose-dependent increase of oligodendrocyte formation. This effect was congener specific, because the coplanar PCB-126 (3,3',4,4 ,5-pentachlorobiphenyl) had no effect. Similar to the T3 response, the PCB-mediated effect on oligodendrocyte formation was blocked by retinoic acid and the thyroid hormone receptor antagonist NH-3. These results suggest that PCB-118 mimics T3 action via the TH pathway.

  14. Human Motor Neuron Progenitor Transplantation Leads to Endogenous Neuronal Sparing in 3 Models of Motor Neuron Loss

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    Tanya J. Wyatt

    2011-01-01

    Full Text Available Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN, ALS (SOD1 G93A, and spinal cord injury (SCI. The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factors in vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss.

  15. Multipotent adult progenitor cells decrease cold ischemic injury in ex vivo perfused human lungs: an initial pilot and feasibility study.

    Science.gov (United States)

    La Francesca, Saverio; Ting, Anthony E; Sakamoto, Jason; Rhudy, Jessica; Bonenfant, Nicholas R; Borg, Zachary D; Cruz, Fernanda F; Goodwin, Meagan; Lehman, Nicholas A; Taggart, Jennifer M; Deans, Robert; Weiss, Daniel J

    2014-01-01

    Primary graft dysfunction (PGD) is a significant cause of early morbidity and mortality following lung transplantation. Improved organ preservation techniques will decrease ischemia-reperfusion injury (IRI) contributing to PGD. Adult bone marrow-derived adherent stem cells, including mesenchymal stromal (stem) cells (MSCs) and multipotent adult progenitor cells (MAPCs), have potent anti-inflammatory actions, and we thus postulated that intratracheal MAPC administration during donor lung processing would decrease IRI. The goal of the study was therefore to determine if intratracheal MAPC instillation would decrease lung injury and inflammation in an ex vivo human lung explant model of prolonged cold storage and subsequent reperfusion. Four donor lungs not utilized for transplant underwent 8 h of cold storage (4°C). Following rewarming for approximately 30 min, non-HLA-matched allogeneic MAPCs (1 × 10(7) MAPCs/lung) were bronchoscopically instilled into the left lower lobe (LLL) and vehicle comparably instilled into the right lower lobe (RLL). The lungs were then perfused and mechanically ventilated for 4 h and subsequently assessed for histologic injury and for inflammatory markers in bronchoalveolar lavage fluid (BALF) and lung tissue. All LLLs consistently demonstrated a significant decrease in histologic and BALF inflammation compared to vehicle-treated RLLs. These initial pilot studies suggest that use of non-HLA-matched allogeneic MAPCs during donor lung processing can decrease markers of cold ischemia-induced lung injury.

  16. Brown-like adipose progenitors derived from human induced pluripotent stem cells: Identification of critical pathways governing their adipogenic capacity

    Science.gov (United States)

    Hafner, Anne-Laure; Contet, Julian; Ravaud, Christophe; Yao, Xi; Villageois, Phi; Suknuntha, Kran; Annab, Karima; Peraldi, Pascal; Binetruy, Bernard; Slukvin, Igor I.; Ladoux, Annie; Dani, Christian

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFβ pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFβ pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity. PMID:27577850

  17. A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

    Science.gov (United States)

    O'Connor, Christine M.

    2012-01-01

    Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractable in vitro model system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractable in vitro model system with which to study HCMV latency and reactivation. PMID:22761372

  18. Fibroblasts isolated from human middle turbinate mucosa cause neural progenitor cells to differentiate into glial lineage cells.

    Directory of Open Access Journals (Sweden)

    Xingjia Wu

    Full Text Available Transplantation of olfactory ensheathing cells (OECs is a potential therapy for repair of spinal cord injury (SCI. Autologous transplantation of OECs has been reported in clinical trials. However, it is still controversial whether purified OECs or olfactory mucosa containing OECs, fibroblasts and other cells should be used for transplantation. OECs and fibroblasts were isolated from olfactory mucosa of the middle turbinate from seven patients. The percentage of OECs with p75(NTR+ and GFAP(+ ranged from 9.2% to 73.2%. Fibroblasts were purified and co-cultured with normal human neural progenitors (NHNPs. Based on immunocytochemical labeling, NHNPs were induced into glial lineage cells when they were co-cultured with the mucosal fibroblasts. These results demonstrate that OECs can be isolated from the mucosa of the middle turbinate bone as well as from the dorsal nasal septum and superior turbinates, which are the typical sites for harvesting OECs. Transplantation of olfactory mucosa containing fibroblasts into the central nervous system (CNS needs to be further investigated before translation to clinical application.

  19. Neuroprotective Effects of Transplanted Mesenchymal Stromal Cells-derived Human Umbilical Cord Blood Neural Progenitor Cells in EAE

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    Hassan Rafieemehr

    2015-11-01

    Full Text Available Multiple Sclerosis (MS is an autoimmune inflammatory demyelinating disease of the central nervous system. The aim of this study was to investigate the neuroprotective effects of transplanted human umbilical cord blood mesenchymal stromal cells (UCB-MSC derived neural progenitor cell (MDNPC in EAE, an experimental model of MS. To initiate neuronal differentiation of UCB-MSCs, the pre-induction medium was removed and replaced with induction media containing retinoic acid, b FGF, h EGF, NGF, IBMX and ascorbic acid for one week. The expression of neural genes was examined in comparison to control group by real-time PCR assay. Then, experimental autoimmune encephalitis (EAE was induced using myelin oligodendrocyte glycoprotein (MOG, 35-55 peptides in 24 C57BL/6 mice. After induction, the mice were divided in four groups (n=6 as follows: healthy, PBS, UCB-MSCs and MDNPC, respectively. At the end of the study, disease status in all the groups was analyzed using hematoxylin-eosin (H&E staining of brain sections. We found that UCB-MSCs exhibit neuronal differentiation potential in vitro and transplanted MDNPC lowered clinical score and reduced CNS leukocyte infiltration compared to untreated mice. Our results showed that MDNPC from UCB may be a proper candidate for regenerative therapy in MS and other neurodegenerative diseases. 

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

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    Sandhya S Buchanan

    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

  1. Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

    Science.gov (United States)

    Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

    2016-01-28

    Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function.

  2. Human umbilical cord-derived endothelial progenitor cells promote growth cytokines-mediated neorevascularization in rat myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    HU Cheng-heng; LI Zhi-ming; DU Zhi-min; ZHANG Ai-xia; YANG Da-ya; WU Gui-fu

    2009-01-01

    Background Cell-based vascular therapies of endothelial progenitor cells (EPCs) mediated neovascularization is still a novel but promising approach for the treatment of ischemic disease. The present study was designed to investigate the therapeutic potentials of human umbilical cord blood-derived EPCs (hUCB-EPCs) in rat with acute myocardial infarction.Methods Human umbilical cord blood (hUCB) mononuclear cells were isolated using density gradient centrifugation from the fresh human umbilical cord in healthy delivery woman, and cultured in M199 medium for 7 days. The EPCs were identified by double-positive staining with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholorate-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and fluorescein isothiocyanate-conjugated Ulex europaeus lectin (FITC-UEA-I). The rat acute myocardial infarction model was established by the ligation of the left anterior descending artery. The hUCB-EPCs were intramyocardially injected into the peri-infarct area. Four weeks later, left ventricular function was assessed by a pressure-volume catheter. The average capillary density (CAD) was evaluated by anti-VⅢ immunohistochemistry staining to reflect the development of neovascularization at the peri-infarct area. The graft cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibody,representing human origin of EPCs and vascular endothelium, respectively. Expressions of cytokines, proliferating cell nuclear angigen (PCNA), platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor (VEGF) were detected to investigate the underlying mechanisms of cell differentiation and revascularization.Results The donor EPCs were detectable and integrated into the host myocardium as confirmed by double-positive immunofluorescence staining with HNA and CD31. And the anti-VⅢ staining demonstrated a higher degree of microvessel formation in EPCs transplanted

  3. Roles of store-operated Ca2+ channels in regulating cell cycling and migration of human cardiac c-kit+ progenitor cells.

    Science.gov (United States)

    Che, Hui; Li, Gang; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-11-15

    Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.

  4. Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

    Science.gov (United States)

    Zhang, Ying-Ying; Li, Gang; Che, Hui; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-01-01

    Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa), a voltage-gated TTX-sensitive sodium current (INa.TTX), and an inward rectifier K+ current (IKir) were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.

  5. Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Ying-Ying Zhang

    Full Text Available Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa, a voltage-gated TTX-sensitive sodium current (INa.TTX, and an inward rectifier K+ current (IKir were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.

  6. Function of Jam-B/Jam-C interaction in homing and mobilization of human and mouse hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Arcangeli, Marie-Laure; Bardin, Florence; Frontera, Vincent; Bidaut, Ghislain; Obrados, Elodie; Adams, Ralf H; Chabannon, Christian; Aurrand-Lions, Michel

    2014-04-01

    The junctional adhesion molecules Jam-b and Jam-c interact together at interendothelial junctions and have been involved in the regulation of immune response, inflammation, and leukocyte migration. More recently, Jam-c has been found to be expressed by hematopoietic stem and progenitor cells (HSPC) in mouse. Conversely, we have reported that Jam-b is present on bone marrow stromal cells and that Jam-b-deficient mice have defects in the regulation of hematopoietic stem cell pool. In this study, we have addressed whether interaction between Jam-b and Jam-c participates to HSPC mobilization or hematopoietic reconstitution after irradiation. We show that a blocking monoclonal antibody directed against Jam-c inhibits hematopoietic reconstitution, progenitor homing to the bone marrow, and induces HSPC mobilization in a Jam-b dependent manner. In the latter setting, antibody treatment over a period of 3 days does not alter hematopoietic differentiation nor induce leukocytosis. Results are translated to human hematopoietic system in which a functional adhesive interaction between JAM-B and JAM-C is found between human HSPC and mesenchymal stem cells. Such an interaction does not occur between HSPC and human endothelial cells or osteoblasts. It is further shown that anti-JAM-C blocking antibody interferes with CD34(+) hematopoietic progenitor homing in mouse bone marrow suggesting that monoclonal antibodies inhibiting JAM-B/JAM-C interaction may represent valuable therapeutic tools to improve stem cell mobilization protocols.

  7. Accelerated Maturation of Human Stem Cell-Derived Pancreatic Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice

    Directory of Open Access Journals (Sweden)

    Jennifer E. Bruin

    2015-12-01

    Full Text Available Pluripotent human embryonic stem cells (hESCs are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19–21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature β cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation.

  8. Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development

    NARCIS (Netherlands)

    C. Robin (Catherine); K. Bollerot (Karine); S.C. Mendes (Sandra); E. Haak (Esther); M. Crisan (Mihaela); F. Cerisoli (Francesco); I. Lauw (Ivoune); P. Kaimakis (Polynikis); R.J.J. Jorna (Ruud); M. Vermeulen (Mark); M.H. Kayser (Manfred); R. van der Linden (Reinier); P. Imanirad (Parisa); M.M.A. Verstegen (Monique); H. Nawaz-Yousaf (Humaira); N. Papazian (Natalie); E.A.P. Steegers (Eric); T. Cupedo (Tom); E.A. Dzierzak (Elaine)

    2009-01-01

    textabstractHematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emerg

  9. 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

    vessels appeared normal without signs of exudation, bleeding, or subretinal elevation. Eyes were harvested at 10-28 days. H&E consistently showed mild retinal vasculitis, depigmentation of the RPE, and marked mononuclear cell infiltrate in the choroid adjacent to the site of transplantation. Human...

  10. DNA Replication Licensing and Progenitor Numbers Are Increased by Progesterone in Normal Human Breast

    NARCIS (Netherlands)

    Graham, J. Dinny; Mote, Patricia A.; Salagame, Usha; van Dijk, Jessica H.; Balleine, Rosemary L.; Huschtscha, Lily I.; Reddel, Roger R.; Clarke, Christine L.

    2009-01-01

    Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in

  11. Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells.

    NARCIS (Netherlands)

    Levetzow, G. von; Spanholtz, J.; Beckmann, J.; Fischer, J.; Kogler, G.; Wernet, P.; Punzel, M.; Giebel, B.

    2006-01-01

    The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within

  12. Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi-compartment 3D perfusion bioreactors for extra-corporeal liver support.

    Science.gov (United States)

    Schmelzer, Eva; Mutig, Kerim; Schrade, Petra; Bachmann, Sebastian; Gerlach, Jörg C; Zeilinger, Katrin

    2009-07-01

    Cultivation of primary human liver cells in innovative 3D perfusion multi-compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale-up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto- and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non-parenchymal cells co-cultivated in bioreactors were used for clinical extra-corporeal liver support to bridge to liver transplantation. 3D tissue re-structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular-like structures, expression of HGFA and c-Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto- and paracrine interactions between co-cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment. (c) 2009 Wiley Periodicals, Inc.

  13. The balance of positive and negative effects of TGF-β signaling regulates the development of hematopoietic and endothelial progenitors in human pluripotent stem cells.

    Science.gov (United States)

    Bai, Hao; Xie, Yin-Liang; Gao, Yong-Xing; Cheng, Tao; Wang, Zack Z

    2013-10-15

    Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with a high-efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34(+)CD31(+)VE-cadherin(+) (VEC(+)) cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presence of hematopoietic and endothelial growth factors, some of CD34(+)CD31(+)VEC(+) cells give rise to blast colony-forming cells (BL-CFCs), which have been used to characterize bipotential hemangioblasts. We found that the level of the transforming growth factor beta (TGF-β) 1 protein is increased during hPSC differentiation, and that TGF-β signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-β to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34(+)CD31(+)VEC(+) cells. An addition of TGF-β inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34(+)CD31(+)VEC(+) progenitor cells. However, inhibition of TGF-β signaling after mesoderm induction increases CD34(+)CD31(+)VEC(+) progenitors and BL-CFCs. These data provide evidence that a balance of positive and negative effects of TGF-β signaling at the appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.

  14. 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

  15. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun; Wang, Jing [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Chen, Guian [Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Reproductive Medical Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Fan, Dongsheng, E-mail: dsfan@yahoo.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Deng, Min, E-mail: dengmin1706@yahoo.com.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China)

    2011-01-14

    Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  16. Endothelial progenitor cells (EPCs as gene carrier system for rat model of human glioma.

    Directory of Open Access Journals (Sweden)

    Nadimpalli Ravi S Varma

    Full Text Available BACKGROUND: Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1 intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2 whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities. METHODS AND RESULTS: Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS. Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors. CONCLUSION: EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for

  17. Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

    Directory of Open Access Journals (Sweden)

    Devaveena Dey

    Full Text Available BACKGROUND: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. METHODOLOGY: Single cell suspensions derived from human breast 'organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. PRINCIPAL FINDINGS: We show that primary mammospheres contain a distinct side-population (SP that displays a CD24(low/CD44(low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high/CD24(low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1 mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. CONCLUSIONS: Thus, the self-renewal potential of human breast stem cells is

  18. Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

    Directory of Open Access Journals (Sweden)

    Salini Vincenzo

    2009-02-01

    Full Text Available Abstract Background Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells. Results The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2 within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium. Electron microscopy analysis evidenced the best cell growth on this latter surface. Conclusion The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.

  19. ISOLATION AND EXPANSION OF HUMAN EMBRYONIC NEURAL STEM/PROGENITOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective: To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method. Method:The cerebral cortex cells were isolated from aborted embryos (11~13 weeks)by mechanical dissociation, and cultured in DMEM/F12 culture medium supplemented with N2 and growth factors for proliferation. Upon passaging, the neurospheres were pipetted gentlely to separate them into several cell masses and then grown in growth medium. The cells were grown in DMEM/F12 medium with serum (without growth factors) to induce differentiation. The stem cell, neuron, astrocyte and oligodendrocyte were identified by immunocytochemistry with antibodies to vimentin, MAP2, GFAP and GalC, respectively. Results:The primary cells grew together and formed neurospheres at 5th~7th day. They were all vimentin positive and could be passaged for at least 8passages. After passaging, the cell masses grew up and formed new neurospheres rapidly. These cells could differentiated into MAP2 ( + ), GFAP( + ) or GalC( + ) cells. Conclusion: The neural stem cells from human embryonic cerebral cortex have the capacity of proliferation and multi - differentiation in vitro. The passaging methods we used in this experiment were practical and convenient.

  20. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  1. Human hemarthrosis-derived progenitor cells can differentiate into osteoblast-like cells in vitro.

    Science.gov (United States)

    Niikura, Takahiro; Miwa, Masahiko; Sakai, Yoshitada; Lee, Sang Yang; Kuroda, Ryosuke; Fujishiro, Takaaki; Kubo, Seiji; Doita, Minoru; Kurosaka, Masahiro

    2005-11-04

    We hypothesized that intraarticular osteochondral fracture-induced hemarthrosis could be a useful cell source for bone regeneration, as it is thought to contain osteoprogenitor cells derived from bone marrow. Therefore, we investigated whether human hemarthrosis-derived cells have the potential to differentiate into osteoblast-like cells in vitro. We aspirated hemarthrosis from patients suffering from osteochondral fractures of knee joints, and cultured hemarthrosis-derived cells in a medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid, or without them as control. The morphology of the treated cells appeared to be cuboidal shape, differing from spindle-like shape observed in the control. Matrix mineralization was observed only in the treated culture. Alkaline phosphatase activity and gene expression of alkaline phosphatase, parathyroid hormone receptor, osteopontin, and osteocalcin were up-regulated compared with the control. These studies demonstrate that human hemarthrosis-derived cells can differentiate into osteoblast-like cells, i.e., they contain osteoprogenitor cells and are a useful cell source for bone regeneration.

  2. Neurogenic potential of progenitors derived from human circulating CD14+ monocytes.

    Science.gov (United States)

    Kodama, Hiroaki; Inoue, Takafumi; Watanabe, Ryuichi; Yasutomi, Daisuke; Kawakami, Yutaka; Ogawa, Satoshi; Mikoshiba, Katsuhiko; Ikeda, Yasuo; Kuwana, Masataka

    2006-04-01

    We previously reported a primitive cell fraction derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMC), that can differentiate along mesenchymal lineages, including bone, cartilage, fat, skeletal muscle and cardiac muscle. In this study, we investigated whether MOMC can differentiate into the neuronal lineage. MOMC were fluorescently labelled and cocultivated with a primary culture of rat neurons for up to 4 weeks. The protein and gene expressions of neuron-specific markers in the human MOMC were evaluated over time using immunohistochemistry, in situ hybridization and reverse transcription followed by PCR. Shortly after cocultivation with rat neurons, nearly all the MOMC expressed early neuroectodermal markers, Mash1, Neurogenin2 and NeuroD, together with nestin, an intermediate filament expressed in neurogenesis. After 14 days of coculture, a subpopulation of MOMC displayed a multipolar morphology with elongated neurites and expressed mature neuron-specific markers, including neurofilament, microtubule-associated protein type 2, beta3-tubulin, NeuN and Hu. Transdifferentiation of monocytes into the neuroectodermal lineage was shown by the simultaneous expression of proneural markers and CD45/CD14 early in the differentiation process. The cocultivated MOMC retained their proliferative capacity for at least 16 days. Finally, the neuronal differentiation of MOMC was observed when they were cultured with neurons without cell-to-cell contact. The capacity of MOMC to differentiate into both mesodermal and neuroectodermal lineages suggests that circulating CD14+ monocytes are more multipotential than previously thought.

  3. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

    DEFF Research Database (Denmark)

    Ameri, Jacqueline; Borup, Rehannah; Prawiro, Christy

    2017-01-01

    cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin......-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our...... results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells....

  4. 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......Women older than 50 years account for 75% of new breast cancer diagnoses, and the majority of these tumors are of a luminal subtype. Although age-associated changes, including endocrine profiles and alterations within the breast microenvironment, increase cancer risk, an understanding....... 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...

  5. Engrafted human induced pluripotent stem cell-derived anterior specified neural progenitors protect the rat crushed optic nerve.

    Directory of Open Access Journals (Sweden)

    Leila Satarian

    Full Text Available BACKGROUND: Degeneration of retinal ganglion cells (RGCs is a common occurrence in several eye diseases. This study examined the functional improvement and protection of host RGCs in addition to the survival, integration and neuronal differentiation capabilities of anterior specified neural progenitors (NPs following intravitreal transplantation. METHODOLOGY/PRINCIPAL FINDINGS: NPs were produced under defined conditions from human induced pluripotent stem cells (hiPSCs and transplanted into rats whose optic nerves have been crushed (ONC. hiPSCs were induced to differentiate into anterior specified NPs by the use of Noggin and retinoic acid. The hiPSC-NPs were labeled by green fluorescent protein or a fluorescent tracer 1,1' -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI and injected two days after induction of ONC in hooded rats. Functional analysis according to visual evoked potential recordings showed significant amplitude recovery in animals transplanted with hiPSC-NPs. Retrograde labeling by an intra-collicular DiI injection showed significantly higher numbers of RGCs and spared axons in ONC rats treated with hiPSC-NPs or their conditioned medium (CM. The analysis of CM of hiPSC-NPs showed the secretion of ciliary neurotrophic factor, basic fibroblast growth factor, and insulin-like growth factor. Optic nerve of cell transplanted groups also had increased GAP43 immunoreactivity and myelin staining by FluoroMyelin™ which imply for protection of axons and myelin. At 60 days post-transplantation hiPSC-NPs were integrated into the ganglion cell layer of the retina and expressed neuronal markers. CONCLUSIONS/SIGNIFICANCE: The transplantation of anterior specified NPs may improve optic nerve injury through neuroprotection and differentiation into neuronal lineages. These NPs possibly provide a promising new therapeutic approach for traumatic optic nerve injuries and loss of RGCs caused by other diseases.

  6. Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord

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    Naimisha Beeravolu

    2016-05-01

    Full Text Available Human umbilical cord (hUC blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs. While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ, cord tissue (CT, and Wharton's jelly (WJ. Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.

  7. Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes

    Directory of Open Access Journals (Sweden)

    R d’Aquino

    2009-11-01

    Full Text Available In this study we used a biocomplex constructed from dental pulp stem/progenitor cells (DPCs and a collagen sponge scaffold for oro-maxillo-facial (OMF bone tissue repair in patients requiring extraction of their third molars. The experiments were carried out according to our Internal Ethical Committee Guidelines and written informed consent was obtained from the patients. The patients presented with bilateral bone reabsorption of the alveolar ridge distal to the second molar secondary to impaction of the third molar on the cortical alveolar lamina, producing a defect without walls, of at least 1.5 cm in height. This clinical condition does not permit spontaneous bone repair after extraction of the third molar, and eventually leads to loss also of the adjacent second molar. Maxillary third molars were extracted first for DPC isolation and expansion. The cells were then seeded onto a collagen sponge scaffold and the obtained biocomplex was used to fill in the injury site left by extraction of the mandibular third molars. Three months after autologous DPC grafting, alveolar bone of patients had optimal vertical repair and complete restoration of periodontal tissue back to the second molars, as assessed by clinical probing and X-rays. Histological observations clearly demonstrated the complete regeneration of bone at the injury site. Optimal bone regeneration was evident one year after grafting. This clinical study demonstrates that a DPC/collagen sponge biocomplex can completely restore human mandible bone defects and indicates that this cell population could be used for the repair and/or regeneration of tissues and organs.

  8. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Karina Needham

    2014-01-01

    Full Text Available In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps, with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31–56 days and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

  9. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Needham, Karina; Hyakumura, Tomoko; Gunewardene, Niliksha; Dottori, Mirella; Nayagam, Bryony A

    2014-01-01

    In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

  10. Molecular profile of clonal strains of human skeletal stem/progenitor cells with different potencies

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    Brian J. Sworder

    2015-05-01

    Full Text Available Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells are fibroblastic reticular cells, a subset of which is composed of multipotent skeletal stem cells (SSCs. SSCs/BMSCs are able to recreate a bone/marrow organ in vivo. To determine differences between clonogenic multipotent SSCs and similarly clonogenic but non-multipotent BMSCs, we established single colony-derived strains (SCDSs, initiated by individual Colony Forming Unit-Fibroblasts and determined their differentiation capacity by vivo transplantation. In this series of human SCDSs (N = 24, 20.8% formed fibrous tissue (F, 66.7% formed bone (B, and 12.5% formed a bone/marrow organ, and thus were multipotent (M. RNA isolated from 12 SCDSs just prior to transplantation was analyzed by microarray. Although highly similar, there was variability from one SCDS to another, and SCDSs did not strictly segregate into the three functional groups (F, B or M by unsupervised hierarchical clustering. We then compared 3 F-SCDSs to 3 M-SCDSs that did segregate. Genes associated with skeletogenesis, osteoblastogeneis, hematopoiesis, and extracellular matrix were over-represented in M-SCDSs compared with F-SCDSs. These results highlight the heterogeneity of SSCs/BMSCs, even between functionally similar SCDSs, but also indicate that differences can be detected that may shed light on the character of the SSC.

  11. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

    Directory of Open Access Journals (Sweden)

    Jacqueline Ameri

    2017-04-01

    Full Text Available Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2 as a specific cell surface marker for isolating pancreatic endoderm cells (PECs from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

  12. Mesangiogenic Progenitor Cells Derived from One Novel CD64brightCD31brightCD14neg Population in Human Adult Bone Marrow

    Science.gov (United States)

    Barachini, Serena; Montali, Marina; Carnicelli, Vittoria; Fazzi, Rita; Parchi, Paolo; Petrini, Mario

    2016-01-01

    Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we first named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage, while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. In this study, we describe the ex vivo isolation of one novel bone marrow subpopulation (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either fluorescence-activated cell sorting or magnetic-activated cell sorting were applied to characterize Pop#8 as CD64brightCD31brightCD14neg. We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in hematopoietic stem cell niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture-expanded MSCs could be the consequence of different culture conditions that select or promote particular subpopulations of precursors. PMID:26975798

  13. Adult human brain neural progenitor cells (NPCs and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

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    Thomas In-Hyeup Park

    Full Text Available The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia, and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs. These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting Ah

  14. ETV6-PDGFRB and FIP1L1-PDGFRA stimulate human hematopoietic progenitor cell proliferation and differentiation into eosinophils: the role of nuclear factor-κB

    Science.gov (United States)

    Montano-Almendras, Carmen P.; Essaghir, Ahmed; Schoemans, Hélène; Varis, Inci; Noël, Laura A.; Velghe, Amélie I.; Latinne, Dominique; Knoops, Laurent; Demoulin, Jean-Baptiste

    2012-01-01

    Background ETV6-PDGFRB (also called TEL-PDGFRB) and FIP1L1-PDGFRA are receptor-tyrosine kinase fusion genes that cause chronic myeloid malignancies associated with hypereosinophilia. The aim of this work was to gain insight into the mechanisms whereby fusion genes affect human hematopoietic cells and in particular the eosinophil lineage. Design and Methods We introduced ETV6-PDGFRB and FIP1L1-PDGFRA into human CD34+ hematopoietic progenitor and stem cells isolated from umbilical cord blood. Results Cells transduced with these oncogenes formed hematopoietic colonies even in the absence of cytokines. Both oncogenes also stimulated the proliferation of cells in liquid culture and their differentiation into eosinophils. This model thus recapitulated key features of the myeloid neoplasms induced by ETV6-PDGFRB and FIP1L1-PDGFRA. We next showed that both fusion genes activated the transcription factors STAT1, STAT3, STAT5 and nuclear factor-κB. Phosphatidylinositol-3 kinase inhibition blocked nuclear factor-κB activation in transduced progenitor cells and patients’ cells. Nuclear factor-κB was also activated in the human FIP1L1-PDGFRA-positive leukemia cell line EOL1, the proliferation of which was blocked by borte-zomib and the IκB kinase inhibitor BMS-345541. A mutant IκB that prevents nuclear translocation of nuclear factor-κB inhibited cell growth and the expression of eosinophil markers, such as the interleukin-5 receptor and eosinophil peroxidase, in progenitors transduced with ETV6-PDGFRB. In addition, several potential regulators of this process, including HES6, MYC and FOXO3 were identified using expression microarrays. Conclusions We show that human CD34+ cells expressing PDGFR fusion oncogenes proliferate autonomously and differentiate towards the eosinophil lineage in a process that requires nuclear factor-κB. These results suggest new treatment possibilities for imatinib-resistant myeloid neoplasms associated with PDGFR mutations. PMID:22271894

  15. Histone demethylase LSD1-mediated repression of GATA-2 is critical for erythroid differentiation

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    Guo Y

    2015-06-01

    Full Text Available Yidi Guo,1 Xueqi Fu,1,2 Yue Jin,1 Jing Sun,1 Ye Liu,1 Bo Huo,1 Xiang Li,1 Xin Hu1–31School of Life Sciences, Jilin University, 2Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 3National Engineering Laboratory of AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, People’s Republic of ChinaBackground: The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors.Methods: Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL or mouse embryonic stem cells together with virus particles, in the presence of Polybrene® at 4 µg/mL, for 24–48 hours followed by puromycin selection (1 µg/mL for 2 weeks. Real-time polymerase chain reaction (PCR-based quantitative chromatin immunoprecipitation (ChIP analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue® assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD 570 nm and OD 600 nm.Results: In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the

  16. The chemotactic activity of beta-carotene in endothelial cell progenitors and human umbilical vein endothelial cells: A microarray analysis

    NARCIS (Netherlands)

    Polus, A.; Kiec-wilk, B.; Hartwich, J.; Balwierz, A.; Stachura, J.; Dyduch, G.; Laidler, P.; Zagajewski, J.; Langman, T.; Schmitz, G.; Goralcsky, R.; Wertz, K.; Riss, G.; Keijer, J.; Dembinska-Kiec, A.

    2006-01-01

    Objectives: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect

  17. Negative selection by apoptosis enriches progenitors in naïve and expanded human umbilical cord blood grafts.

    Science.gov (United States)

    Mizrahi, K; Ash, S; Peled, T; Yaniv, I; Stein, J; Askenasy, N

    2014-07-01

    The influence of TNF-α and Fas-ligand (FasL) on viability and function was evaluated in fresh- and expanded-umbilical cord blood (UCB) cells. CD34(+) progenitors and T cells display outstanding survival, whereas ~30% and >50% B lymphocytes and myeloid cells undergo spontaneous apoptosis within 24 and 48 h, respectively. Although the impact of exposure to toxic doses of FasL and TNF-α was undetectable in measurements of apoptosis; removal of dead cells after 2 days of incubation with the ligands revealed a twofold increase in frequency of colony-forming cells (CFU). The sensitivity of progenitors to apoptosis was also unaffected by Fas cross-linking following TNF-induced upregulation of the receptor, increasing CFU frequency without impairing SCID repopulating cell (SRC) activity. Most significant enrichment in CD34(+) progenitors and corresponding increase in CFU frequency were observed when FasL was applied during the final week of ex vivo expansion under the influence of nicotinamide, without impairing SRC activity. These data emphasize differential sensitivities of UCB progenitors and lineage-positive cells to apoptotic signaling mediated by the Fas and TNF receptors, which might be useful in improving the efficiency of ex vivo expansion and improving UCB cell engraftment.

  18. Enhanced selectivity of hyperthermic purging of human progenitor cells using Goralatide, an inhibitor of cell cycle progression

    NARCIS (Netherlands)

    Wierenga, PK; Brenner, MK; Konings, AWT

    1998-01-01

    Recurrence of leukemia is a major problem after autologous stem cell transplantation. One potential means of reducing this risk is to purge the autologous transplant in vitro by hyperthermia, We have demonstrated that after a hyperthermic treatment of 120 min at 43 degrees C, the leukemic progenitor

  19. The presence of local mesenchymal progenitor cells in human degenerated intervertebral discs and possibilities to influence these in vitro: a descriptive study in humans.

    Science.gov (United States)

    Brisby, Helena; Papadimitriou, Nikolaos; Brantsing, Camilla; Bergh, Peter; Lindahl, Anders; Barreto Henriksson, Helena

    2013-03-01

    Low back pain is common and degenerated discs (DDs) are believed to be a major cause. In non-degenerated intervertebral discs (IVDs) presence of stem/progenitor cells was recently reported in different mammals (rabbit, rat, pig). Understanding processes of disc degeneration and regenerative mechanisms within DDs is important. The aim of the study was to examine the presence of local stem/progenitor cells in human DDs and if these cell populations could respond to paracrine stimulation in vitro. Tissue biopsies from the IVD region (L3-S1) were collected from 15 patients, age 34-69 years, undergoing surgery (spinal fusion) and mesenchymal stem cells (MSCs) (iliac crest) from 2 donors. Non-DD cells were collected from 1 donor (scoliosis) and chordoma tissue was obtained from (positive control, stem cell markers) 2 donors. The IVD biopsies were investigated for gene and protein expression of: OCT3/4, CD105, CD90, STRO-1, and NOTCH1. DD cell cultures (pellet mass) were performed with conditioned media from MSCs and non-degenerated IVD cells. Pellets were investigated after 7, 14, 28 days for the same stem cell markers as above. Gene expression of OCT3/4 and STRO-1 was detected in 13/15 patient samples, CD105 in 14/15 samples, and CD90 and NOTCH1 were detected 15/15 samples. Immunohistochemistry analysis supported findings on the protein level, in cells sparsely distributed in DDs tissues. DDs cell cultures displayed more undifferentiated appearance with increased expression of CD105, CD90, STRO-1, OCT3/4, NOTCH1, and JAGGED1, which was observed when cultured in conditioned cell culture media from MSCs compared to cell cultures cultured with conditioned media from non-DD cells. Expression of OCT3/4 (multipotency marker) and NOTCH1 (regulator of cell fate), MSC-markers, CD105, CD90, and STRO-1, indicate that primitive cell populations are present within DDs. Furthermore, the possibility to influence cells from DDs by paracrine signaling /soluble factors from MSCs and from

  20. Non-Lethal Ionizing Radiation Promotes Aging-Like Phenotypic Changes of Human Hematopoietic Stem and Progenitor Cells in Humanized Mice.

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    Changshan Wang

    Full Text Available Precise understanding of radiation effects is critical to develop new modalities for the prevention and treatment of radiation-induced damage. We previously reported that non-lethal doses of X-ray irradiation induce DNA damage in human hematopoietic stem and progenitor cells (HSPCs reconstituted in NOD/Shi-scid IL2rγnull (NOG immunodeficient mice and severely compromise their repopulating capacity. In this study, we analyzed in detail the functional changes in human HSPCs in NOG mice following non-lethal radiation. We transplanted cord blood CD34+ HSPCs into NOG mice. At 12 weeks post-transplantation, the recipients were irradiated with 0, 0.5, or 1.0 Gy. At 2 weeks post-irradiation, human CD34+ HSPCs recovered from the primary recipient mice were transplanted into secondary recipients. CD34+ HSPCs from irradiated mice showed severely impaired reconstitution capacity in the secondary recipient mice. Of interest, non-lethal radiation compromised contribution of HSPCs to the peripheral blood cells, particularly to CD19+ B lymphocytes, which resulted in myeloid-biased repopulation. Co-culture of limiting numbers of CD34+ HSPCs with stromal cells revealed that the frequency of B cell-producing CD34+ HSPCs at 2 weeks post-irradiation was reduced more than 10-fold. Furthermore, the key B-cell regulator genes such as IL-7R and EBF1 were downregulated in HSPCs upon 0.5 Gy irradiation. Given that compromised repopulating capacity and myeloid-biased differentiation are representative phenotypes of aged HSCs, our findings indicate that non-lethal ionizing radiation is one of the critical external stresses that promote aging of human HSPCs in the bone marrow niche.

  1. Human CD5+ Innate Lymphoid Cells Are Functionally Immature and Their Development from CD34+ Progenitor Cells Is Regulated by Id2

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    Maho Nagasawa

    2017-08-01

    Full Text Available Innate lymphoid cells (ILCs have emerged as a key cell type involved in surveillance and maintenance of mucosal tissues. Mouse ILCs rely on the transcriptional regulator Inhibitor of DNA-binding protein 2 (Id2 for their development. Here, we show that Id2 also drives development of human ILC because forced expression of Id2 in human thymic progenitors blocked T cell commitment, upregulated CD161 and promyelocytic leukemia zinc finger (PLZF, and maintained CD127 expression, markers that are characteristic for human ILCs. Surprisingly CD5 was also expressed on these in vitro generated ILCs. This was not an in vitro artifact because CD5 was also found on ex vivo isolated ILCs from thymus and from umbilical cord blood. CD5 was also expressed on small proportions of ILC2 and ILC3. CD5+ ILCs were functionally immature, but could further differentiate into mature CD5− cytokine-secreting ILCs. Our data show that Id2 governs human ILC development from thymic progenitor cells toward immature CD5+ ILCs.

  2. Effect of human cytomegalovirus on proliferation of hematopoietic progenitor cells of cord blood%人类巨细胞病毒感染对脐血造血祖细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    刘文君; 金润铭; 付晓冬; 刘斌; 郭渠莲; 邓正华

    2006-01-01

    目的探讨人类巨细胞病毒(HCMV)感染对脐血造血祖细胞(CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk)体外增殖的抑制作用及其机制.方法20例脐血标本收集于正常足月顺产新生儿.实验共分5组:(1)3个HCMV感染组,每个感染组分别加入0.1 mL的103、104及105空斑形成单位(PFU)HCMV-AD169病毒液于培养体系中;(2)灭活对照组,加入同体积灭活HCMV病毒液;(3)空白对照组,不加HCMV病毒液,代之以同体积的IMDM.采用造血祖细胞体外半固体培养技术,培养、观察、计数HCMV-AD169株对脐血CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落数、抑制率和集落维持时间;并用聚合酶链反应(PCR)技术检测集落细胞内HCMV-DNA.结果(1)在造血祖细胞培养体系中加入不同滴度的HCMV-AD169后,104和105PFU滴度感染对CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落形成均有显著的抑制作用,103PFU滴度感染对CFU-Mix及CFU- Mk集落形成有显著的抑制作用,与空白对照组和灭活对照组比较,差异有显著性(P<0.05).病毒滴度越高,抑制程度越明显(P<0.05).(2)104和105 PFU滴度感染组CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落维持时间较对照组明显缩短(P<0.01),103 PFU滴度感染组CFU-Mix和CFU-Mk集落维持时间较对照组明显缩短(P<0.01).(3)PCR显示3个感染组的CFU-GM、CFU-E、CFU-Mix及CFU-Mk集落细胞内均有HCMV-AD169 DNA存在.结论HCMV-AD169能直接感染CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk造血祖细胞,并抑制造血祖细胞的增殖,这可能与HCMV感染患儿出现粒细胞减少、血小板减少和贫血等造血功能紊乱有关.%Objective This study was designed to investigate the effect of human cytomegalovirus (HCMV) on the proliferation of colony forming unit granulocyte-macrophage ( CFU-GM ), CFU-erythroid ( CFU-E), burst forming uniterythroid (BFU-E), CFU-multipotential (CFU-Mix) and CFU-megakaryocytic (CFU-Mk) progenitor cells of cord blood in vitro as well as

  3. Quantification of erythroid and granulocytic precursor cells in plateletpheresis residues

    Energy Technology Data Exchange (ETDEWEB)

    Abboud, C.N.; Brennan, J.K.; Lichtman, M.A.; Nusbacher, J.

    1978-01-01

    Mononuclear cell fractions of human blood and plateletpheresis residues were compared for their content of hemopoietic precursor cells. Erythroid burst-forming units (BFU-E) averaged 560 +- 130 per ml of blood and granulocyte--monocyte colony forming units (CFU-C) averaged 240 +- 90 per ml blood. Estimates based on a blood volume of 7% of body weight indicate that the total blood pools of BFU-E and CFU-C are about 3.5 x 10/sup 6/ and 1.5 x 10/sup 6/ cells respectively. Sequential studies were performed over 3 days following one plateletpheresis in 4 donors. CFU-C and BFU-E approximately doubled between 48 and 72 hours after a plateletpheresis. During this time there was no significant alteration in the percent of null, T or B lymphocytes in blood. Thus, plateletpheresis appears to lead to a mobilization of precursor cells, which results in a transient increase in their concentration in blood. Therefore, pheresis 48 to 72 hours after an initial short-term procedure could harvest much larger numbers of precursor cells. Moreover, such techniques would put blood precursor cell content of plateletpheresis residues within reach of the precursor cell content in the volume of human marrow used for transplantation.

  4. Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo.

    Science.gov (United States)

    Ferreira, Lino S; Gerecht, Sharon; Shieh, Hester F; Watson, Nicki; Rupnick, Maria A; Dallabrida, Susan M; Vunjak-Novakovic, Gordana; Langer, Robert

    2007-08-03

    We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34+ cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34+ cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (alpha-SM actin, SM myosin heavy chain, calponin, caldesmon, SM alpha-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering.

  5. Grafted human embryonic progenitors expressing neurogenin-2 stimulate axonal sprouting and improve motor recovery after severe spinal cord injury.

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    Florence E Perrin

    Full Text Available BACKGROUND: Spinal cord injury (SCI is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs in spinal cord-lesioned rats. METHODS AND PRINCIPAL FINDINGS: With the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either naïve or engineered to express Neurogenin 2 (Ngn2. Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of naïve hENPs is detrimental to functional recovery. CONCLUSIONS AND SIGNIFICANCE: Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of naïve-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell

  6. Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease.

    Science.gov (United States)

    Zhu, Jianhui; Kren, Betsy T; Park, Chang Won; Bilgim, Rasim; Wong, Phillip Y-P; Steer, Clifford J

    2007-06-12

    Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient

  7. Extracorporeal Shock Wave Treatment (ESWT) enhances the in vitro-induced differentiation of human tendon-derived stem/progenitor cells (hTSPCs).

    Science.gov (United States)

    Leone, Laura; Raffa, Salvatore; Vetrano, Mario; Ranieri, Danilo; Malisan, Florence; Scrofani, Cristina; Vulpiani, Maria Chiara; Ferretti, Andrea; Torrisi, Maria Rosaria; Visco, Vincenzo

    2016-02-09

    Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury.

  8. Modulation of the Innate Immune Response by Human Neural Precursors Prevails over Oligodendrocyte Progenitor Remyelination to Rescue a Severe Model of Pelizaeus-Merzbacher Disease.

    Science.gov (United States)

    Marteyn, Antoine; Sarrazin, Nadège; Yan, Jun; Bachelin, Corinne; Deboux, Cyrille; Santin, Mathieu D; Gressens, Pierre; Zujovic, Violetta; Baron-Van Evercooren, Anne

    2016-04-01

    Pelizaeus-Merzbacher disease (PMD) results from an X-linked misexpression of proteolipid protein 1 (PLP1). This leukodystrophy causes severe hypomyelination with progressive inflammation, leading to neurological dysfunctions and shortened life expectancy. While no cure exists for PMD, experimental cell-based therapy in the dysmyelinated shiverer model suggested that human oligodendrocyte progenitor cells (hOPCs) or human neural precursor cells (hNPCs) are promising candidates to treat myelinopathies. However, the fate and restorative advantages of human NPCs/OPCs in a relevant model of PMD has not yet been addressed. Using a model of Plp1 overexpression, resulting in demyelination with progressive inflammation, we compared side-by-side the therapeutic benefits of intracerebrally grafted hNPCs and hOPCs. Our findings reveal equal integration of the donor cells within presumptive white matter tracks. While the onset of exogenous remyelination was earlier in hOPCs-grafted mice than in hNPC-grafted mice, extended lifespan occurred only in hNPCs-grafted animals. This improved survival was correlated with reduced neuroinflammation (microglial and astrocytosis loads) and microglia polarization toward M2-like phenotype followed by remyelination. Thus modulation of neuroinflammation combined with myelin restoration is crucial to prevent PMD pathology progression and ensure successful rescue of PMD mice. These findings should help to design novel therapeutic strategies combining immunomodulation and stem/progenitor cell-based therapy for disorders associating hypomyelination with inflammation as observed in PMD.

  9. Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

    Science.gov (United States)

    Jaako, P; Debnath, S; Olsson, K; Zhang, Y; Flygare, J; Lindström, M S; Bryder, D; Karlsson, S

    2015-11-01

    Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

  10. Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

    Science.gov (United States)

    Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

    2016-02-01

    Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic

  11. Reversal of endothelial progenitor cell dysfunction in patients with type 2 diabetes using a conditioned medium of human embryonic stem cell-derived endothelial cells.

    Science.gov (United States)

    Ho, Jenny C Y; Lai, Wing-Hon; Li, Ming-Fang; Au, Ka-Wing; Yip, Mei-Chu; Wong, Navy L Y; Ng, Ethel S K; Lam, Francis F Y; Siu, Chung-Wah; Tse, Hung-Fat

    2012-07-01

    The potential clinical application of bone marrow or peripheral blood-derived progenitor cells for cardiovascular regeneration in patients with diabetes mellitus (DM) is limited by their functional impairment. We sought to determine the mechanisms of impaired therapeutic efficacy of peripheral blood-derived progenitor cells in type 2 DM patients and evaluated the use of cell-free conditioned medium obtained from human embryonic stem cell-derived endothelial-like cells (ESC-ECs) to reverse their functional impairment. The angiogenic potential of late outgrowth endothelial cells (OECs) and cytokine profile of the conditional medium of proangiogenic cells (PACs) derived from peripheral blood-mononuclear cells of healthy control and DM patients and ESC-ECs was compared by in vitro tube formation assay and a multiplex bead-based immunoassay kit, respectively. The in vivo angiogenic potential of ESC-ECs derived conditioned medium in rescuing the functional impairment of PB-PACs in DM patients was investigated using a hindlimb ischemia model. Human ESC-ECs had similar functional and phenotypic characteristics as OECs in healthy controls. Cytokine profiling showed that vascular endothelial growth factor, stromal cell-derived factor 1 and placental growth factor were down-regulated in PACs from DM patients. Tube formation assay that revealed functional impairment of OECs from DM patients could be rescued by ESC-ECs conditioned medium. Administration of ESC-ECs conditioned medium restored the therapeutic efficacy of PB-PACs from DM patients in a mouse model of hindlimb ischemia. Our results showed that peripheral blood-derived progenitor cells from DM patients have impaired function because of defective secretion of angiogenic cytokines, which could be restored by supplementation of ESC-ECs conditioned medium. Copyright © 2012 John Wiley & Sons, Ltd.

  12. Enhanced propagation of adult human renal epithelial progenitor cells to improve cell sourcing for tissue-engineered therapeutic devices for renal diseases.

    Science.gov (United States)

    Westover, Angela J; Buffington, Deborah A; Humes, H D

    2012-08-01

    Renal cell therapy employing cells derived from adult renal epithelial cell (REC) progenitors promises to reduce the morbidity of patients with renal insufficiency due to acute renal failure and end stage renal disease. To this end, tissue engineered devices addressing the neglected biologic component of renal replacement therapy are being developed. Because human donor tissue is limited, novel enhanced progenitor cell propagation (EP) techniques have been developed and applied to adult human kidney transplant discards from six donors. Changes include more efficient digestion and the amplification of progenitors prior to terminal epithelial differentiation promoted by contact inhibition and the addition of retinoic acid. Differentiated morphology in EP populations was demonstrated by the ability to form polarized epithelium with tight junctions, apical central cilia and expression of brush border membrane enzymes. Evaluation of lipopolysaccharide stimulated interleukin-8 secretion and γ-glutamyl transpeptisade activity in EP derived cells was used to confirm therapeutic equivalence to REC obtained using published techniques, which have previously shown efficacy in large animal models and clinical trials. Yield exceeded 10(16) cells/gram cortex from the only kidney obtained due to an anatomical defect, while the average yield from diseased kidneys ranged from 1.1 × 10(9) to 8.8 × 10(11) cells/gram cortex, representing an increase of more than 10 doublings over standard methods. Application of the EP protocol to REC expansion has solved the problem of cell sourcing as the limiting factor to the manufacture of cell based therapies targeting renal diseases and may provide a method for autologous device fabrication from core kidney biopsies. Copyright © 2012 John Wiley & Sons, Ltd.

  13. Age-Related Changes in FGF-2, Fibroblast Growth Factor Receptors and β-Catenin Expression in Human Mesenchyme-Derived Progenitor Cells.

    Science.gov (United States)

    Hurley, Marja M; Gronowicz, Gloria; Zhu, Li; Kuhn, Liisa T; Rodner, Craig; Xiao, Liping

    2016-03-01

    FGF-2 stimulates preosteoblast replication, and knockout of the FGF-2 gene in mice resulted in osteopenia with age, associated with decreased Wnt-β-Catenin signaling. In addition, targeted expression of FGF-2 in osteoblast progenitors increased bone mass in mice via Wnt-β-Catenin signaling. We posited that diminution of the intrinsic proliferative capacity of human mesenchyme-derived progenitor cells (HMDPCs) with age is due in part to reduction in FGF-2. To test this hypothesis HMDPCs from young (27-38), middle aged (47-56), and old (65-76) female human subjects were isolated from bone discarded after orthopedic procedures. HMDPCs cultures were mostly homogeneous with greater than 90% mesenchymal progenitor cells, determined by fluorescence-activated cell sorting. There was a progressive decrease in FGF-2 and FGFR1 mRNA and protein in HMDPCs with age. Since FGF-2 activates β-catenin, which can enhance bone formation, we also assessed its age-related expression in HMDPCs. An age-related decrease in total-β-Catenin mRNA and protein expression was observed. However there were increased levels of p-β-Catenin and decreased levels of activated-β-Catenin in old HMDSCs. FGF-2 treatment increased FGFR1 and β-Catenin protein, reduced the level of p-β-Catenin and increased activated-β-Catenin in aged HMDPCs. In conclusion, reduction in FGF-2 expression could contribute to age-related impaired function of HMDPCs via modulation of Wnt-β-catenin signaling.

  14. A common signaling pathway is activated in erythroid cells expressing high levels of fetal hemoglobin: a potential role for cAMP-elevating agents in β-globin disorders

    Directory of Open Access Journals (Sweden)

    Ikuta T

    2013-12-01

    Full Text Available Tohru Ikuta,1 Yuichi Kuroyanagi,1 Nadine Odo,1 Siyang Liu21Department of Anesthesiology and Perioperative Medicine, 2Department of Physiology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USABackground: Although erythroid cells prepared from fetal liver, cord blood, or blood from β-thalassemia patients are known to express fetal hemoglobin at high levels, the underlying mechanisms remain elusive. We previously showed that cyclic nucleotides such as cAMP and cGMP induce fetal hemoglobin expression in primary erythroid cells. Here we report that cAMP signaling contributes to high-level fetal hemoglobin expression in erythroid cells prepared from cord blood and β-thalassemia.Methods: The status of the cAMP signaling pathway was investigated using primary erythroid cells prepared from cord blood and the mononuclear cells of patients with β-thalassemia; erythroid cells from adult bone marrow mononuclear cells served as the control.Results: We found that intracellular cAMP levels were higher in erythroid cells from cord blood and β-thalassemia than from adult bone marrow. Protein kinase A activity levels and cAMP-response element binding protein phosphorylation were higher in erythroid cells from cord blood or β-thalassemia than in adult bone marrow progenitors. Mitogen-activated protein kinase pathways, which play a role in fetal hemoglobin expression, were not consistently activated in cord blood or β-thalassemia erythroid cells. When cAMP signaling was activated in adult erythroid cells, fetal hemoglobin was induced at high levels and associated with reduced expression of BCL11A, a silencer of the β-globin gene.Conclusion: These results suggest that activated cAMP signaling may be a common mechanism among erythroid cells with high fetal hemoglobin levels, in part because of downregulation of BCL11A. Activation of the cAMP signaling pathway with cAMP-elevating agents may prove to be an important signaling mechanism to

  15. Accelerated generation of oligodendrocyte progenitor cells from human induced pluripotent stem cells by forced expression of Sox10 and Olig2.

    Science.gov (United States)

    Li, Pengyan; Li, Mo; Tang, Xihe; Wang, Shuyan; Zhang, Y Alex; Chen, Zhiguo

    2016-11-01

    Oligodendrocyte progenitor cells (OPCs) hold great promise for treatment of dysmyelinating disorders, such as multiple sclerosis and cerebral palsy. Recent studies on generation of human OPCs mainly use human embryonic stem cells (hESCs) or neural stem cells (NSCs) as starter cell sources for the differentiation process. However, NSCs are restricted in availability and the present method for generation of oligodendrocytes (OLs) from ESCs often requires a lengthy period of time. Here, we demonstrated a protocol to efficiently derive OPCs from human induced pluripotent stem cells (hiPSCs) by forced expression of two transcription factors (2TFs), Sox10 and Olig2. With this method, PDGFRα(+) OPCs can be obtained in 14 days and O4(+) OPCs in 56 days. Furthermore, OPCs may be able to differentiate to mature OLs that could ensheath axons when co-cultured with rat cortical neurons. The results have implications in the development of autologous cell therapies.

  16. Human dermal stem/progenitor cell-derived conditioned medium ameliorates ultraviolet a-induced damage of normal human dermal fibroblasts.

    Directory of Open Access Journals (Sweden)

    Joong Hyun Shim

    Full Text Available Adult skin stem cells are considered an attractive cell resource for therapeutic potential in aged skin. We previously reported that multipotent human dermal stem/progenitor cells (hDSPCs can be enriched from (normal human dermal fibroblasts (NHDFs using collagen type IV. However, the beneficial effects of hDSPCs on aged skin remain to be elucidated. In the present study, we analyzed the growth factors secreted from hDSPCs in conditioned medium (CM derived from hDSPCs (hDSPC-CM and found that hDSPCs secreted higher levels of bFGF, IGFBP-1, IGFBP-2, HGF, VEGF and IGF-1 compared with non-hDSPCs. We then investigated whether hDSPC-CM has an effect on ultraviolet A (UVA-irradiated NHDFs. Real-time RT-PCR analysis revealed that the treatment of UVA-irradiated NHDFs with hDSPC-CM significantly antagonized the UVA-induced up-regulation of the MMP1 and the UVA-induced down-regulation of the collagen types I, IV and V and TIMP1 mRNA expressions. Furthermore, a scratch wound healing assay showed that hDSPC-CM enhanced the migratory properties of UVA-irradiated NHDFs. hDSPC-CM also significantly reduced the number of the early and late apoptotic cell population in UVA-irradiated NHDFs. Taken together, these data suggest that hDSPC-CM can exert some beneficial effects on aged skin and may be used as a therapeutic agent to improve skin regeneration and wound healing.

  17. Impact of the Autism-Associated Long Noncoding RNA MSNP1AS on Neuronal Architecture and Gene Expression in Human Neural Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Jessica J. DeWitt

    2016-09-01

    Full Text Available We previously identified the long noncoding RNA (lncRNA MSNP1AS (moesin pseudogene 1, antisense as a functional element revealed by genome wide significant association with autism spectrum disorder (ASD. MSNP1AS expression was increased in the postmortem cerebral cortex of individuals with ASD and particularly in individuals with the ASD-associated genetic markers on chromosome 5p14.1. Here, we mimicked the overexpression of MSNP1AS observed in postmortem ASD cerebral cortex in human neural progenitor cell lines to determine the impact on neurite complexity and gene expression. ReNcell CX and SK-N-SH were transfected with an overexpression vector containing full-length MSNP1AS. Neuronal complexity was determined by the number and length of neuronal processes. Gene expression was determined by strand-specific RNA sequencing. MSNP1AS overexpression decreased neurite number and neurite length in both human neural progenitor cell lines. RNA sequencing revealed changes in gene expression in proteins involved in two biological processes: protein synthesis and chromatin remodeling. These data indicate that overexpression of the ASD-associated lncRNA MSNP1AS alters the number and length of neuronal processes. The mechanisms by which MSNP1AS overexpression impacts neuronal differentiation may involve protein synthesis and chromatin structure. These same biological processes are also implicated by rare mutations associated with ASD, suggesting convergent mechanisms.

  18. TGFβ inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy

    Institute of Scientific and Technical Information of China (English)

    Chengyan Wang; Liying Du; Yang Gao; Ming Yin; Mingxiao Ding; Hongkui Deng; Xuming Tang; Xiaomeng Sun; Zhenchuan Miao; Yaxin Lv; Yanlei Yang; Huidan Zhang; Pengbo Zhang; Yang Liu

    2012-01-01

    Embryonic hematopoiesis is a complex process.Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells.However,the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs)remains unknown.Here,on the basis of the emergence of CD43+ hematopoietic cells from hemogenic endothelial (HE) cells,we demonstrated that VEGF was essential and sufficient,and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43+ hematopoietic cells.Significantly,we identified TGFβ as a novel signal to regulate hematopoietic development,as the TGFβ inhibitor SB 431542 significantly promoted the transition from HE cells into CD43+ hematopoietic progenitor cells (HPCs) during hESC differentiation.By defining these critical signaling factors during hematopoietic differentiation,we can efficiently generate HPCs from hESCs.Our strategy could offer an in vitro model to study early human hematopoietic development.

  19. Efficient generation of human embryonic stem cell-derived cardiac progenitors based on tissue-specific enhanced green fluorescence protein expression.

    Science.gov (United States)

    Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I; Sarkadi, Balázs; Apáti, Ágota

    2015-01-01

    Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFP(high) rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFP(high) rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFP(high) rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications.

  20. Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors

    Institute of Scientific and Technical Information of China (English)

    Xiao-dong JU; Si-quan LOU; Wei-guo WANG; Jian-qiang PENG; Hua TIAN

    2004-01-01

    AIM: To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV). METHODS: Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10 % FBS or 5 % FBS and dexamethasone 1 μmol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC.After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization. RESULTS: Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiency compared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV. CONCLUSION: Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.

  1. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34+ hematopoietic stem cells and for chondrogenic differentiation

    Institute of Scientific and Technical Information of China (English)

    JIN-FUWANG; LI-JUANWANG; YI-FANWU; YINGXIANG; CHUN-GANGXIE; BING-BINGJIA; JENNYHARRINGTON; IANK.MCNIECE

    2005-01-01

    Background and Objectives. Human mesenchymal stem/progenitor cells (MSPC) ar pluripotent, being the precursors for marrow stroma, bone, cartilage, muscle and connective tissues. Although the presence of hematopoietic stem/progenitor cells (HSPC) in umbilical cord blood (UCB) is well known, that of MSPC has been not fully evaluated. Design and Methods. In this study, we examined the immunophenotype, the supporting function in relation to exvivo expansion of hematopoietic stem progenitor cells and the chondrogenic differentiation of cultured cells with characteristics of MSPC from UCB. When UCB nucleated cells were isolated and 107 cells cultured in IMDM with 20% fetal bovine serum, the mean number of adherent fibroblastlike colonies was 3.5±0.7/106 monuclear cells. Results. UCB-derived MSPC could be expanded for at least 15 passages. In their undifferentiated state, UCB-derived MSPC were CD 13+, CD29+, CD90+, CD105+, CD166+, SH2+,SH3+, SH4+, CD45-, CD34-, and CD14-; they produced stem cell factor, interleukin 6 and tumor necrosis factor α.UCB-derived MSPC cultured in chondrogenic media differentiated into chondrogenic cells. UCB-derived MSPC supported the proliferation and differentiation of CD34+ cells from UCB in vitro. Interpretation and Conclusions. UCB-derived MSPC have the potential to support ex vivo expansion of HSPC and chondrogenic differentiation. UCB should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells and may provide a unique source of fetal cells for cellular and gene therapy.

  2. Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

    Science.gov (United States)

    Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

    2017-11-01

    Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis.

    Science.gov (United States)

    Sun, Hongliang; Tsai, Ying; Nowak, Irena; Liesveld, Jane; Chen, Yuhchyau

    2012-09-01

    Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.

  4. 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

    AMD3100 inhibits the interaction between SDF-1 and CXCR4, and rapidly mobilizes hematopoietic progenitors for clinical transplantation. However, the repopulating function of human cells mobilized with AMD3100 has not been characterized in comparison to cells mobilized with granulocyte-colony stim......AMD3100 inhibits the interaction between SDF-1 and CXCR4, and rapidly mobilizes hematopoietic progenitors for clinical transplantation. However, the repopulating function of human cells mobilized with AMD3100 has not been characterized in comparison to cells mobilized with granulocyte...

  5. Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

    Science.gov (United States)

    Yin, Ping; Ono, Masanori; Moravek, Molly B; Coon, John S; Navarro, Antonia; Monsivais, Diana; Dyson, Matthew T; Druschitz, Stacy A; Malpani, Saurabh S; Serna, Vanida A; Qiang, Wenan; Chakravarti, Debabrata; Kim, J Julie; Bulun, Serdar E

    2015-04-01

    Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

  6. Collagen-Coated Polytetrafluoroethane Membrane Inserts Enhances Chondrogenic Differentiation of Human Cord Blood Multi-Lineage Progenitor Cells

    DEFF Research Database (Denmark)

    Munir, Samir; Søballe, Kjeld; Ulrich-Vinther, Michael

    Background: Articular chondrocytes and bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the favoured cells for cartilage tissue engineering. Umbilical cord blood has proven an alternative source of MSCs and moreover they may be more potent chondroprogenitor cells than bonemarrow...... MSCs. Purpose / Aim of Study: Multilineage progenitor cells (MLPCs) are clonal cord blood-derived MSCs and may therefore provide a cell source with more reproducible outcomes compared to heterogeneous primary MSC cultures. Materials and Methods: We evaluated the chondrogenic potency of MLPCs...

  7. Molecular cross talk between Notch1, Shh and Akt pathways during erythroid differentiation of K562 and HEL cell lines.

    Science.gov (United States)

    Roy, Anita; Haldar, Srijan; Basak, Nandini Pal; Banerjee, Subrata

    2014-01-01

    Erythropoiesis is a tightly regulated process dependent on extrinsic signals conveyed by the bone marrow niche. The signalling pathways thus activated or repressed do not act in isolation; rather an intricate cross talk among these pathways ensues homoeostasis within the erythroid compartment. In this study, we describe the effects of two such signalling pathways namely the Notch1 and the Shh pathway on erythropoiesis in immortalised K562 and HEL cell lines as well as the cross talk that ensues between them. We show that while activation of the Notch1 pathway inhibits differentiation of erythroid lineage cell lines as well as in in-vitro primary erythroid cultures from the human CD34(+) cells; Shh pathway favours erythroid differentiation. Further, the Notch1 pathway activates the Akt pathway and constitutively active Akt partially mimics the effect of Notch1 activation on erythropoiesis. Moreover, the Notch1, Akt and Shh pathways were found to cross talk with each other. In this process, activation of Notch1 was found to down regulate the Shh pathway independent of Akt activation. Significantly, Notch1 not only down regulated the Shh pathway, but also inhibited recombinant Shh mediated erythropoiesis. Our study thus reveals an intricate crosstalk among the Notch1, Shh and Akt pathways wherein Notch1 emerges as a key regulator of erythropoiesis.

  8. Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the NASA rotating wall vessel bioreactor

    Science.gov (United States)

    Sytkowski, A. J.; Davis, K. L.

    2001-01-01

    Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.

  9. Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

    Institute of Scientific and Technical Information of China (English)

    YiZhanga; ChangdongLi; XiaoxiaJiang; ShuangxiZhang; YingWu; BingLiu; PeihsienTang; NingMao

    2005-01-01

    Objective. Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ ceils from UCB in vitro.Materials and Methods. Human placenta MPC was isolated and characterized by morphologic,immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculturewith placeutal MPC. Suitable aliquots of cells were used to monitor cell production, elonogenie activity, and tong-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay.Results. In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin,and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and α-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes.In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of eytokines, was significantly enhanced: CD34+ cells by 14.89±2.32 fold; colonyforming cell (CFC) by 36.73±5.79 told; and LTC-IC by 7.43±2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli.Conclusion. These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.

  10. Intravenous administration of human umbilical cord blood-derived AC133+ endothelial progenitor cells in rat stroke model reduces infarct volume: magnetic resonance imaging and histological findings.

    Science.gov (United States)

    Iskander, Asm; Knight, Robert A; Zhang, Zheng Gang; Ewing, James R; Shankar, Adarsh; Varma, Nadimpalli Ravi S; Bagher-Ebadian, Hassan; Ali, Meser M; Arbab, Ali S; Janic, Branislava

    2013-09-01

    Endothelial progenitor cells (EPCs) hold enormous therapeutic potential for ischemic vascular diseases. Previous studies have indicated that stem/progenitor cells derived from human umbilical cord blood (hUCB) improve functional recovery in stroke models. Here, we examined the effect of hUCB AC133+ EPCs on stroke development and resolution in a middle cerebral artery occlusion (MCAo) rat model. Since the success of cell therapies strongly depends on the ability to monitor in vivo the migration of transplanted cells, we also assessed the capacity of magnetic resonance imaging (MRI) to track in vivo the magnetically labeled cells that were administered. Animals were subjected to transient MCAo and 24 hours later injected intravenously with 10(7) hUCB AC133+ EPCs. MRI performed at days 1, 7, and 14 after the insult showed accumulation of transplanted cells in stroke-affected hemispheres and revealed that stroke volume decreased at a significantly higher rate in cell-treated animals. Immunohistochemistry analysis of brain tissues localized the administered cells in the stroke-affected hemispheres only and indicated that these cells may have significantly affected the magnitude of endogenous proliferation, angiogenesis, and neurogenesis. We conclude that transplanted cells selectively migrated to the ischemic brain parenchyma, where they exerted a therapeutic effect on the extent of tissue damage, regeneration, and time course of stroke resolution.

  11. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors.

    Science.gov (United States)

    Takayama, Naoya; Nishikii, Hidekazu; Usui, Joichi; Tsukui, Hiroko; Sawaguchi, Akira; Hiroyama, Takashi; Eto, Koji; Nakauchi, Hiromitsu

    2008-06-01

    Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

  12. A Novel Molecular and Functional Stemness Signature Assessing Human Cord Blood-Derived Endothelial Progenitor Cell Immaturity.

    Directory of Open Access Journals (Sweden)

    Oriane Guillevic

    Full Text Available Endothelial Colony Forming Cells (ECFCs, a distinct population of Endothelial Progenitor Cells (EPCs progeny, display phenotypic and functional characteristics of endothelial cells while retaining features of stem/progenitor cells. Cord blood-derived ECFCs (CB-ECFCs have a high clonogenic and proliferative potentials and they can acquire different endothelial phenotypes, this requiring some plasticity. These properties provide angiogenic and vascular repair capabilities to CB-ECFCs for ischemic cell therapies. However, the degree of immaturity retained by EPCs is still confused and poorly defined. Consequently, to better characterize CB-ECFC stemness, we quantified their clonogenic potential and demonstrated that they were reprogrammed into induced pluripotent stem cells (iPSCs more efficiently and rapidly than adult endothelial cells. Moreover, we analyzed the transcriptional profile of a broad gene panel known to be related to stem cells. We showed that, unlike mature endothelial cells, CB-ECFCs expressed genes involved in the maintenance of embryonic stem cell properties such as DNMT3B, GDF3 or SOX2. Thus, these results provide further evidence and tools to appreciate EPC-derived cell stemness. Moreover this novel stem cell transcriptional signature of ECFCs could help better characterizing and ranging EPCs according to their immaturity profile.

  13. Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice

    National Research Council Canada - National Science Library

    Lin, Kou-Ray; Yang-Yen, Hsin-Fang; Lien, Huang-Wei; Liao, Wei-Hao; Huang, Chang-Jen; Lin, Liang-In; Li, Chung-Leung; Yen, Jeffrey Jong-Young

    2016-01-01

    ...) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis...

  14. Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK).

    Science.gov (United States)

    Kleszczyński, Konrad; Zillikens, Detlef; Fischer, Tobias W

    2016-09-01

    Melatonin is an ubiquitous molecule with a variety of functions including potent antioxidative properties. Due to its lipophilic character, it easily crosses cellular and intracellular membranes and reaches all subcellular organelles. Because of its ability to scavenge free radicals, melatonin protects against oxidative stress, for example, induced by ultraviolet radiation (UVR). Here, we investigated, in a dose-dependent (0, 10, 25, and 50 mJ/cm(2) ) and time-dependent (0, 4, 24, 48 hr post-UVR) manner, whether melatonin prevents the UVR-mediated alterations in ATP synthesis and the generation of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEK). Additionally, we evaluated the molecular mechanism of action of melatonin with regard to activation of phase-2 antioxidative enzymes via nuclear erythroid 2-related factor (Nrf2). We found that (i) melatonin counteracted UVR-induced alterations in the ATP synthesis and reduced free radical formation; (ii) melatonin induced the translocation of Nrf2 transcription factor from the cytosol into the nucleus resulting in, (iii) melatonin enhanced gene expression of phase-2 antioxidative enzymes including γ-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1), and NADPH: quinone dehydrogenase-1 (NQO1) representing an elevated antioxidative response of keratinocytes. These results suggest that melatonin not only directly scavenges ROS, but also significantly induces the activation of phase-2 antioxidative enzymes via the Nrf2 pathway uncovering a new action mechanism that supports the ability of keratinocytes to protect themselves from UVR-mediated oxidative stress.

  15. Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E, CFU-E, and CFU-GM): species-related susceptibility.

    Science.gov (United States)

    Roda, E; Coccini, T; Acerbi, D; Castoldi, A F; Manzo, L

    2010-02-01

    Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001-50microg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3-3mg/kgb.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data. We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC(50) values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC(50): 2.45+/-1.08 and 1.82+/-0.8microM for humans, and 11.08+/-2.92 and 1.81+/-0.20microM for mice, respectively), than the mature CFU-E (AFB1 IC(50): 12.58+/-5.4 and 40.27+/-6.05microM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.

  16. Enhancing and suppressing effects of recombinant murine macrophage inflammatory proteins on colony formation in vitro by bone marrow myeloid progenitor cells.

    Science.gov (United States)

    Broxmeyer, H E; Sherry, B; Lu, L; Cooper, S; Oh, K O; Tekamp-Olson, P; Kwon, B S; Cerami, A

    1990-09-15

    Purified recombinant (r) macrophage inflammatory proteins (MIPs) 1 alpha, 1 beta, and 2 were assessed for effects on murine (mu) and human (hu) marrow colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Recombinant MIP-1 alpha, -1 beta, and -2 enhanced muCFU-GM colonies above that stimulated with 10