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Sample records for cell-derived endothelial cells

  1. Endothelial cells derived from human embryonic stem cells

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    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  2. Pathogen sensing pathways in human embryonic stem cell derived-endothelial cells: role of NOD1 receptors.

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    Daniel M Reed

    Full Text Available Human embryonic stem cell-derived endothelial cells (hESC-EC, as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR Toll-like receptor (TLR-4 and nucleotide-binding oligomerisation domain-containing protein (NOD-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC. HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC, and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

  3. Stem Cells Derived from Tooth Periodontal Ligament Enhance Functional Angiogenesis by Endothelial Cells

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    Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J.; Tarle, Susan A.

    2014-01-01

    In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential

  4. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

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    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering.

  5. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

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    Katsunari Makino

    Full Text Available Endothelin (ET-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF-α and connective tissue growth factor (CTGF were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  6. Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

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    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D; Jordan, Maria C; Roos, Kenneth P; Yao, Yucheng; Boström, Kristina I

    2012-12-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

  7. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

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    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  8. Circulating apoptotic endothelial cell-derived microparticles are predicted metabolically unhealthy obesity

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    Alexander E. Berezin

    2017-01-01

    Full Text Available Introduction: Circulating apoptotic endothelial cell-derived micro particles (EMPs are a marker of endothelial dysfunction and cardiovascular (CV risk in type 2 diabetes mellitus patients. There is evidence regarding association between apoptotic EMP number and CV disease in obese individuals. The aim of the study to investigate whether increased number of circulating apoptotic EMPs may predict transformation of Met-HO into Met-UHO. Methods: The study was retrospectively evolved 89 patients with established abdominal obesity (47 patients with Met-UHO determined as MetS and 42 subjects with Met-HO from the large cohort of abdominal obesity patients (n=268. Thirty five healthy volunteers matched for age and sex were involved in the study as a control cohort. Obesity-related biomarker (adiponectin, leptin, vistafin and EMPs were measured at baseline. Flow cytometry was used to determine EMPs with immune phenotype CD31+/annexin V+ and CD144+/annexin V+. Results: There was not found a significant difference between numbers of EMPs labeled CD31+/ Annexin V+ in Met-UHO and Met-HO patients, while Met-UHO patients had a significantly increased level of circulating CD144+/ Annexin V+ compared with Met-HO individuals. Multivariate logistic regression analysis has revealed the HOMA-IR, number of CV risk factors, serum leptin and hs-CRP independently predicted numbers of circulating CD31+/ Annexin V+ and CD144+/ Annexin V+ EMPs in Met-UHO. In Met-HO patients HOMA-IR remained an independent predictor of increased numbers of circulating CD31+/ Annexin V+ and CD144+/ Annexin V+ EMPs. Conclusion: in the investigation we found that the increased number of CD31+/Annexin V+ and CD144+/ Annexin V+ EMPs added to the based predictive model (HOMA-IR may predict transformation of Met-HO into Met-UHO.

  9. Tumor and endothelial cell-derived microvesicles carry distinct CEACAMs and influence T-cell behavior.

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    Harrison T Muturi

    Full Text Available Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1000 nm in diameter, which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter, derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis.

  10. Limited gene expression variation in human embryonic stem cell and induced pluripotent stem cell-derived endothelial cells.

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    White, Mark P; Rufaihah, Abdul J; Liu, Lei; Ghebremariam, Yohannes T; Ivey, Kathryn N; Cooke, John P; Srivastava, Deepak

    2013-01-01

    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized, and defined growth factors were used to generate KDR(+) EC progenitors. Magnetic purification of a KDR(+) progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders.

  11. Isolation and transplantation of corneal endothelial cell-like cells derived from in-vitro-differentiated human embryonic stem cells.

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    Zhang, Kai; Pang, Kunpeng; Wu, Xinyi

    2014-06-15

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.

  12. Advances in pluripotent stem cell-derived endothelial cells: from biomaterials to organ regeneration.

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    Lui, Kathy O

    2014-01-01

    Human embryonic stem cells (ESCs), by virtue of their capability to self-renew and differentiate into a variety of cell types, represent the first type of pluripotent stem cells (PSCs) to be used in clinical transplantation during recent phase-I trials; however, it is still unclear whether hESC-derived tissues can self-organize and form part of the vascularized, functional organ following transplantation. Recently, endothelial cells (ECs) or angiogenic factors such as VEGFA have been demonstrated to support development and regeneration of multiple organ systems, including the heart, pancreas, liver, lung and bone marrow. Therefore, co-transplantation of ECs derived from the same parental PSCs that differentiate into cell types of interest; or overexpression of the inductive angiogenic factors responsible for organ regeneration might be beneficial to support function of hPSC-derived tissues. In this special issue, we discuss how protein kinases (Ng and colleagues); DNA methylation and histone modification (Tsui and colleagues) regulate cellular pluripotency and cell-fate specification of PSCs. In addition, we discuss how ECs and angiogenic factors could contribute to repair and regeneration of organs such as the heart (Yuan and colleagues), the cardiovascular system (Tse and colleagues) and the pancreas (Lui). We also discuss the role of mesenchymal stem cells or paracrine factors secreted by them in tissue repair (Li and colleagues). Lastly, we discuss how to generate self-organized and vascularized tissues derived from PSCs in a 2- or 3-dimensional format by fusing tissue bioengineering approaches with stem cell technology (Chen).

  13. Human embryonic stem cell-derived endothelial cells as cellular delivery vehicles for treatment of metastatic breast cancer.

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    Su, Weijun; Wang, Lina; Zhou, Manqian; Liu, Ze; Hu, Shijun; Tong, Lingling; Liu, Yanhua; Fan, Yan; Kong, Deling; Zheng, Yizhou; Han, Zhongchao; Wu, Joseph C; Xiang, Rong; Li, Zongjin

    2013-01-01

    Endothelial progenitor cells (EPCs) have shown tropism towards primary tumors or metastases and are thus potential vehicles for targeting tumor therapy. However, the source of adult EPCs is limited, which highlights the need for a consistent and renewable source of endothelial cells for clinical applications. Here, we investigated the potential of human embryonic stem cell-derived endothelial cells (hESC-ECs) as cellular delivery vehicles for therapy of metastatic breast cancer. In order to provide an initial assessment of the therapeutic potency of hESC-ECs, we treated human breast cancer MDA-MB-231 cells with hESC-EC conditioned medium (EC-CM) in vitro. The results showed that hESC-ECs could suppress the Wnt/β-catenin signaling pathway and thereby inhibit the proliferation and migration of MDA-MB-231 cells. To track and evaluate the possibility of hESC-EC-employed therapy, we employed the bioluminescence imaging (BLI) technology. To study the therapeutic potential of hESC-ECs, we established lung metastasis models by intravenous injection of MDA-MB-231 cells labeled with firefly luciferase (Fluc) and green fluorescent protein (GFP) to NOD/SCID mice. In mice with lung metastases, we injected hESC-ECs armed with herpes simplex virus truncated thymidine kinase (HSV-ttk) intravenously on days 11, 16, 21, and 26 after MDA-MB-231 cell injection. The NOD/SCID mice were subsequently treated with ganciclovir (GCV), and the growth status of tumor was monitored by Fluc imaging. We found that MDA-MB-231 tumors were significantly inhibited by intravenously injected hESC-ECs. The tumor-suppressive effects of the hESC-ECs, by inhibiting Wnt/β-catenin signaling pathway and inducing tumor cell death through bystander effect in human metastatic breast cancer model, provide previously unexplored therapeutic modalities for cancer treatment.

  14. Functions of Müller cell-derived vascular endothelial growthfactor in diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Müller cells are macroglia and play many essentialroles as supporting cells in the retina. To respond topathological changes in diabetic retinopathy (DR), amajor complication in the eye of diabetic patients,retinal Müller glia produce a high level of vascularendothelial growth factor (VEGF or VEGF-A). As VEGFis expressed by multiple retinal cell-types and Müllerglia comprise only a small portion of cells in the retina,it has been a great challenge to reveal the function ofVEGF or other globally expressed proteins produced byMüller cells. With the development of conditional genetargeting tools, it is now possible to dissect the functionof Müller cell-derived VEGF in vivo . By using conditionalgene targeting approach, we demonstrate that Müllerglia are a major source of retinal VEGF in diabetic miceand Müller cell-derived VEGF plays a significant role inthe alteration of protein expression and peroxynitration,which leads to retinal inflammation, neovascularization,vascular leakage, and vascular lesion, key pathologicalchanges in DR. Therefore, Müller glia are a potentialcellular target for the treatment of DR, a leading causeof blindness.

  15. Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells.

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    Kamprom, Witchayaporn; Kheolamai, Pakpoom; U-Pratya, Yaowalak; Supokawej, Aungkura; Wattanapanitch, Methichit; Laowtammathron, Chuti; Issaragrisil, Surapol

    2016-01-01

    Human mesenchymal stem cell (hMSC) is a potential source for cell therapy due to its property to promote tissue repair. Although, it has been known that hMSCs promote tissue repair via angiogenic cytokines, the interaction between hMSC-derived cytokines and the endothelial progenitor cells (EPCs), which play an important role in tissue neovascularization, is poorly characterized. We investigate the effect of cytokine released from different sources of hMSCs including bone marrow and gestational tissues on the EPC functions in vitro. The migration, extracellular matrix invasion and vessel formation of EPCs were studied in the presence or absence of cytokines released from various sources of hMSCs using transwell culture system. The migration of EPCs was highest when co-culture with secretory factors from placenta-derived hMSCs (PL-hMSCs) compared to those co-culture with other sources of hMSCs. For invasion and vessel formation, secretory factors from bone marrow-derived hMSCs (BM-hMSCs) could produce the maximal enhancement compared to other sources. We further identified the secreted cytokines and found that the migratory-enhancing cytokine from PL-hMSCs was PDGF-BB while the enhancing cytokine from BM-hMSCs on invasion was IGF-1. For vessel formation, the cytokines released from BM-hMSCs were IGF1 and SDF-1. In conclusion, hMSCs can release angiogenic cytokines which increase the migration, invasion and vessel forming capacity of EPCs. We can then use hMSCs as a source of angiogenic cytokines to induce neovascularization in injured/ischemic tissues.

  16. Biocompatibility of pure titanium modified by human endothelial cell-derived extracellular matrix

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    Xue Xiaoqing [Key Laboratory of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Wang Jin, E-mail: jinxxwang@263.net [Key Laboratory of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Zhu Ying; Tu Qiufen; Huang Nan [Key Laboratory of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China)

    2010-04-01

    Extracellular matrix (ECM) used to modify biomaterial surface is a promising method for improving cardiovascular material hemocompatibility. In the present work, human umbilical vein endothelial cells (HUVECs) are cultured and native ECM is obtained on pure titanium surface. Fourier infrared spectrum (FTIR) test proves the existence of amide I and amide II band on the modified titanium surface. X-ray photoelectron spectroscopy (XPS) further confirms the chemical composition and binding types of the ECM proteins on the titanium substrate. The results of light microscopy and atomic force microscopy (AFM) exhibit the morphology of HUVEC derived ECM. There are higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) are presented on ECM-covered titanium surface by immunofluorescence staining. The biological behavior of cultured HUVECs and adherent platelets on different samples are investigated by in vitro HUVECs culture and platelet adhesion. Cells exhibit better morphology and their proliferation ability greatly improve on the ECM-covered titanium. At the same time, the platelet adhesion and spreading are inhibited on ECM-covered titanium surface. These investigations demonstrate that ECM produced by HUVECs cannot only improve adhesion and proliferation ability of endothelial cell but also inhibit adhesion and activation of platelets. Thus, the approach described here may provide a basis for preparation of modified surface in cardiovascular implants application.

  17. In Vivo Vascularization of Endothelial Cells Derived from Bone Marrow Mesenchymal Stem Cells in SCID Mouse Model

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    Allameh Abdolamir

    2016-07-01

    Full Text Available Objective In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods We induced human mesenchymal stem cells (MSCs to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor (vWF, vascular endothelial growth factor (VEGF receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency (SCID mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry (DIHC staining. Results Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin (H&E staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site. Conclusion The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis.

  18. Limited Gene Expression Variation in Human Embryonic Stem Cell and Induced Pluripotent Stem Cell Derived Endothelial Cells

    OpenAIRE

    2013-01-01

    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based ...

  19. SIRT1 Overexpression Maintains Cell Phenotype and Function of Endothelial Cells Derived from Induced Pluripotent Stem Cells.

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    Jiang, Bin; Jen, Michele; Perrin, Louisiane; Wertheim, Jason A; Ameer, Guillermo A

    2015-12-01

    Endothelial cells (ECs) that are differentiated from induced pluripotent stem cells (iPSCs) can be used in establishing disease models for personalized drug discovery or developing patient-specific vascularized tissues or organoids. However, a number of technical challenges are often associated with iPSC-ECs in culture, including instability of the endothelial phenotype and limited cell proliferative capacity over time. Early senescence is believed to be the primary mechanism underlying these limitations. Sirtuin1 (SIRT1) is an NAD(+)-dependent deacetylase involved in the regulation of cell senescence, redox state, and inflammatory status. We hypothesize that overexpression of the SIRT1 gene in iPSC-ECs will maintain EC phenotype, function, and proliferative capacity by overcoming early cell senescence. SIRT1 gene was packaged into a lentiviral vector (LV-SIRT1) and transduced into iPSC-ECs at passage 4. Beginning with passage 5, iPSC-ECs exhibited a fibroblast-like morphology, whereas iPSC-ECs overexpressing SIRT1 maintained EC cobblestone morphology. SIRT1 overexpressing iPSC-ECs also exhibited a higher percentage of canonical markers of endothelia (LV-SIRT1 61.8% CD31(+) vs. LV-empty 31.7% CD31(+), P cell lifespan, overcoming critical hurdles associated with the use of iPSC-ECs in translational research.

  20. Suppression of Transforming Growth Factor-β Signaling Delays Cellular Senescence and Preserves the Function of Endothelial Cells Derived From Human Pluripotent Stem Cells.

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    Bai, Hao; Gao, Yongxing; Hoyle, Dixie L; Cheng, Tao; Wang, Zack Z

    2016-09-20

    : Transplantation of vascular cells derived from human pluripotent stem cells (hPSCs) offers an attractive noninvasive method for repairing the ischemic tissues and for preventing the progression of vascular diseases. Here, we found that in a serum-free condition, the proliferation rate of hPSC-derived endothelial cells is quickly decreased, accompanied with an increased cellular senescence, resulting in impaired gene expression of endothelial nitric oxide synthase (eNOS) and impaired vessel forming capability in vitro and in vivo. To overcome the limited expansion of hPSC-derived endothelial cells, we screened small molecules for specific signaling pathways and found that inhibition of transforming growth factor-β (TGF-β) signaling significantly retarded cellular senescence and increased a proliferative index of hPSC-derived endothelial cells. Inhibition of TGF-β signaling extended the life span of hPSC-derived endothelial and improved endothelial functions, including vascular network formation on Matrigel, acetylated low-density lipoprotein uptake, and eNOS expression. Exogenous transforming growth factor-β1 increased the gene expression of cyclin-dependent kinase inhibitors, p15(Ink4b), p16(Ink4a), and p21(CIP1), in endothelial cells. Conversely, inhibition of TGF-β reduced the gene expression of p15(Ink4b), p16(Ink4a), and p21(CIP1). Our findings demonstrate that the senescence of newly generated endothelial cells from hPSCs is mediated by TGF-β signaling, and manipulation of TGF-β signaling offers a potential target to prevent vascular aging.

  1. The hematopoietic chemokine CXCL12 promotes integration of human endothelial colony forming cell-derived cells into immature vessel networks.

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    Newey, Sarah E; Tsaknakis, Grigorios; Khoo, Cheen P; Athanassopoulos, Thanassi; Camicia, Rosalba; Zhang, Youyi; Grabowska, Rita; Harris, Adrian L; Roubelakis, Maria G; Watt, Suzanne M

    2014-11-15

    Proangiogenic factors, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF-2) prime endothelial cells to respond to "hematopoietic" chemokines and cytokines by inducing/upregulating expression of the respective chemokine/cytokine receptors. Coculture of human endothelial colony forming cell (ECFC)-derived cells with human stromal cells in the presence of VEGF and FGF-2 for 14 days resulted in upregulation of the "hematopoietic" chemokine CXCL12 and its CXCR4 receptor by day 3 of coculture. Chronic exposure to the CXCR4 antagonist AMD3100 in this vasculo/angiogenesis assay significantly reduced vascular tubule formation, an observation recapitulated by delayed AMD3100 addition. While AMD3100 did not affect ECFC-derived cell proliferation, it did demonstrate a dual action. First, over the later stages of the 14-day cocultures, AMD3100 delayed tubule organization into maturing vessel networks, resulting in enhanced endothelial cell retraction and loss of complexity as defined by live cell imaging. Second, at earlier stages of cocultures, we observed that AMD3100 significantly inhibited the integration of exogenous ECFC-derived cells into established, but immature, vascular networks. Comparative proteome profiler array analyses of ECFC-derived cells treated with AMD3100 identified changes in expression of potential candidate molecules involved in adhesion and/or migration. Blocking antibodies to CD31, but not CD146 or CD166, reduced the ECFC-derived cell integration into these extant vascular networks. Thus, CXCL12 plays a key role not only in endothelial cell sensing and guidance, but also in promoting the integration of ECFC-derived cells into developing vascular networks.

  2. Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells

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    Kim Yoon-Keun

    2009-11-01

    Full Text Available Abstract Background Various cancer cells, including those of colorectal cancer (CRC, release microvesicles (exosomes into surrounding tissues and peripheral circulation. These microvesicles can mediate communication between cells and affect various tumor-related processes in their target cells. Results We present potential roles of CRC cell-derived microvesicles in tumor progression via a global comparative microvesicular and cellular transcriptomic analysis of human SW480 CRC cells. We first identified 11,327 microvesicular mRNAs involved in tumorigenesis-related processes that reflect the physiology of donor CRC cells. We then found 241 mRNAs enriched in the microvesicles above donor cell levels, of which 27 were involved in cell cycle-related processes. Network analysis revealed that most of the cell cycle-related microvesicle-enriched mRNAs were associated with M-phase activities. The integration of two mRNA datasets showed that these M-phase-related mRNAs were differentially regulated across CRC patients, suggesting their potential roles in tumor progression. Finally, we experimentally verified the network-driven hypothesis by showing a significant increase in proliferation of endothelial cells treated with the microvesicles. Conclusion Our study demonstrates that CRC cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, suggesting that microvesicles of cancer cells can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. This information will help elucidate the pathophysiological functions of tumor-derived microvesicles, and aid in the development of cancer diagnostics, including colorectal cancer.

  3. Functional endothelial cells derived from embryonic stem cells labeled with HIV transactivator peptide-conjugated superparamagnetic nanoparticles

    Institute of Scientific and Technical Information of China (English)

    GAO Bin; FU Wei-guo; DONG Zhi-hui; FANG Zheng-dong; LIU Zhen-jie; SI Yi; ZHANG Xiang-man; WANG Yu-qi

    2011-01-01

    Background The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo,for example,magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles.Although ES cells have been labeled with SPIO particles,the potential adverse effects of the label have not been fully examined.The objective of this study was to determine whether SPIO labeling affects murine ES cell viability,proliferation,or ability to differentiate into functional endothelial cells (ECs).Methods Cross-linked iron oxide (CLIO,an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides,and murine ES cells were labeled with either CLiO-Tat,CLIO,or HIV-Tat.After labeling,ES cells were cultured for 4 days and FIk-1+ ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS).FIk-1+ cells were raplated on fibronectin-coated dishes,and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF).ES cell viability was determined using trypan blue exclusion,and the proportion of SPIO+ cells was evaluated using Prussian blue staining and transmission electron microscopy.After differentiation,the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction,flow cytometry,immunocytochemistry,Dil-labeled acetylated low-density lipoprotein (AcLDL) uptake,and Matrigel tube formation assay.Results CLIO-Tat was a highly effective label for ES cells,with >96% of cells incorporating the particles,and it did not alter the viability of the labeled cells.ECs derived from CLIO-Tat+ ES cells were very similar to murine aortic ECs in their morphology,expression of endothelial cell markers,ability to form vascular-like channels,and scavenging of AcLDL from the culture medium

  4. SIRT1 Overexpression Maintains Cell Phenotype and Function of Endothelial Cells Derived from Induced Pluripotent Stem Cells

    Science.gov (United States)

    Jiang, Bin; Jen, Michele; Perrin, Louisiane; Wertheim, Jason A.

    2015-01-01

    Endothelial cells (ECs) that are differentiated from induced pluripotent stem cells (iPSCs) can be used in establishing disease models for personalized drug discovery or developing patient-specific vascularized tissues or organoids. However, a number of technical challenges are often associated with iPSC-ECs in culture, including instability of the endothelial phenotype and limited cell proliferative capacity over time. Early senescence is believed to be the primary mechanism underlying these limitations. Sirtuin1 (SIRT1) is an NAD+-dependent deacetylase involved in the regulation of cell senescence, redox state, and inflammatory status. We hypothesize that overexpression of the SIRT1 gene in iPSC-ECs will maintain EC phenotype, function, and proliferative capacity by overcoming early cell senescence. SIRT1 gene was packaged into a lentiviral vector (LV-SIRT1) and transduced into iPSC-ECs at passage 4. Beginning with passage 5, iPSC-ECs exhibited a fibroblast-like morphology, whereas iPSC-ECs overexpressing SIRT1 maintained EC cobblestone morphology. SIRT1 overexpressing iPSC-ECs also exhibited a higher percentage of canonical markers of endothelia (LV-SIRT1 61.8% CD31+ vs. LV-empty 31.7% CD31+, P < 0.001; LV-SIRT1 46.3% CD144+ vs. LV-empty 20.5% CD144+, P < 0.02), with a higher nitric oxide synthesis, lower β-galactosidase production indicating decreased senescence (3.4% for LV-SIRT1 vs. 38.6% for LV-empty, P < 0.001), enhanced angiogenesis, increased deacetylation activity, and higher proliferation rate. SIRT1 overexpressing iPSC-ECs continued to proliferate through passage 9 with high purity of EC-like characteristics, while iPSC-ECs without SIRT1 overexpression became senescent after passage 5. Taken together, SIRT1 overexpression in iPSC-ECs maintains EC phenotype, improves EC function, and extends cell lifespan, overcoming critical hurdles associated with the use of iPSC-ECs in translational research. PMID:26413932

  5. Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor.

    Science.gov (United States)

    Sivarapatna, Amogh; Ghaedi, Mahboobe; Le, Andrew V; Mendez, Julio J; Qyang, Yibing; Niklason, Laura E

    2015-01-01

    Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications.

  6. In vitro model of cerebral ischemia by using brain microvascular endothelial cells derived from human induced pluripotent stem cells.

    Science.gov (United States)

    Kokubu, Yasuhiro; Yamaguchi, Tomoko; Kawabata, Kenji

    2017-04-29

    Brain-derived microvascular endothelial cells (BMECs), which play a central role in blood brain barrier (BBB), can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported, they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy, drug screening, and pathological study. In the recent study, an induction method of BMECs from PSCs has been established, making it possible to more precisely study the in vitro human BBB function. Here, using induced pluripotent stem (iPS) cell-derived BMECs, we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function, and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly, TNF-α, which is known to be secreted from astrocytes and microglia in the cerebral ischemia, prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus, we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs.

  7. Hypoxia Affects the Structure of Breast Cancer Cell-Derived Matrix to Support Angiogenic Responses of Endothelial Cells.

    Science.gov (United States)

    Hielscher, Abigail; Qiu, Connie; Porterfield, Josh; Smith, Quinton; Gerecht, Sharon

    2013-01-01

    Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co-cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDA-MB-231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia-exposed co-cultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de-cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report that in vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for

  8. GM-CSF Exhibits Anti-Inflammatory Activity on Endothelial Cells Derived from Chronic Venous Disease Patients

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    Veronica Tisato

    2013-01-01

    Full Text Available Twenty patients affected by chronic venous disease (CVD in tertiary venous network and/or saphenous vein were analyzed before surgical ablation by echo-color-doppler for the hemodynamic parameters reflux time (RT and resistance index (RI, a negative and a positive prognostic factor, respectively. RT and RI were next correlated with relevant in vitro parameters of venous endothelial cells (VEC obtained from surgical specimens, such as cell migration in response to serum gradient, proliferation index, intercellular adhesion molecule (ICAM-1 and vascular cell adhesion molecule (VCAM-1 expression, as well as cytokines release. Of interest, ICAM-1 expression in patient-derived VEC cultures correlated positively with RT and negatively with RI. Moreover, RT showed a positive correlation with the baseline osteoprotegerin (OPG expression by VEC and an inverse correlation with VEC proliferation index. On the other hand, RI correlated positively with TNF-related apoptosis inducing ligand (TRAIL expression. Among the cytokines released by VEC, GM-CSF showed a positive correlation with VEC proliferation and TRAIL expression and a negative correlation with OPG, ICAM-1 and VCAM-1 expression. Since in vitro recombinant GM-CSF induced VEC proliferation and counteracted the induction of ICAM-1, VCAM-1 and OPG upon exposure to TNF-α, our data suggest an anti-inflammatory activity of GM-CSF on venous endothelial cells.

  9. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device.

    Science.gov (United States)

    van der Meer, Andries D; Orlova, Valeria V; ten Dijke, Peter; van den Berg, Albert; Mummery, Christine L

    2013-09-21

    Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here, we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells, human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h, the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell-cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels, inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity, highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary, we have

  10. Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial Nitric Oxide Synthase

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    ML Rossi

    2009-06-01

    Full Text Available Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs and impaired bioavailabilty of nitric oxide (NO are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5’ nuclease assays (TaqMan™ PCRs to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA and 15 with acute coronary syndromes (ACS without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001 in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype.

  11. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

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    Jonathan A Rose

    Full Text Available Pulmonary arterial hypertension (PAH is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

  12. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    OpenAIRE

    Katt, Moriah E.; Xu, Zinnia S.; Gerecht, Sharon; Searson, Peter C.

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial...

  13. Pattern of endothelial progenitor cells and apoptotic endothelial cell-derived microparticles in chronic heart failure patients with preserved and reduced left ventricular ejection fraction

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    Alexander E. Berezin

    2016-02-01

    Conclusion: We found that CD31+/annexin V+ EMPs to CD14+CD309+ cell ratio added to NT-proBNP, clinical data, and cardiovascular risk factors has exhibited the best discriminate value and higher reliability to predict HFpEF compared with NT-proBNP and clinical data/cardiovascular risk factors alone.

  14. Cell-derived microparticles and the lung

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    Dario Nieri

    2016-09-01

    Full Text Available Cell-derived microparticles are small (0.1–1 μm vesicles shed by most eukaryotic cells upon activation or during apoptosis. Microparticles carry on their surface, and enclose within their cytoplasm, molecules derived from the parental cell, including proteins, DNA, RNA, microRNA and phospholipids. Microparticles are now considered functional units that represent a disseminated storage pool of bioactive effectors and participate both in the maintenance of homeostasis and in the pathogenesis of diseases. The mechanisms involved in microparticle generation include intracellular calcium mobilisation, cytoskeleton rearrangement, kinase phosphorylation and activation of the nuclear factor-κB. The role of microparticles in blood coagulation and inflammation, including airway inflammation, is well established in in vitro and animal models. The role of microparticles in human pulmonary diseases, both as pathogenic determinants and biomarkers, is being actively investigated. Microparticles of endothelial origin, suggestive of apoptosis, have been demonstrated in the peripheral blood of patients with emphysema, lending support to the hypothesis that endothelial dysfunction and apoptosis are involved in the pathogenesis of the disease and represent a link with cardiovascular comorbidities. Microparticles also have potential roles in patients with asthma, diffuse parenchymal lung disease, thromboembolism, lung cancer and pulmonary arterial hypertension.

  15. Cellular Functions and Gene and Protein Expression Profiles in Endothelial Cells Derived from Moyamoya Disease-Specific iPS Cells

    Science.gov (United States)

    Hamauchi, Shuji; Shichinohe, Hideo; Uchino, Haruto; Yamaguchi, Shigeru; Nakayama, Naoki; Kazumata, Ken; Osanai, Toshiya; Abumiya, Takeo; Houkin, Kiyohiro; Era, Takumi

    2016-01-01

    Background and purpose Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal portions of the cerebral internal carotid artery. However, the functions and characteristics of the endothelial cells (ECs) in MMD are unknown. We analyzed these features using induced pluripotent stem cell (iPSC)-derived ECs. Methods iPSC lines were established from the peripheral blood of three patients with MMD carrying the variant RNF213 R4810K, and three healthy persons used as controls. After the endothelial differentiation of iPSCs, CD31+CD144+ cells were purified as ECs using a cell sorter. We analyzed their proliferation, angiogenesis, and responses to some angiogenic factors, namely VEGF, bFGF, TGF-β, and BMP4. The ECs were also analyzed using DNA microarray and proteomics to perform comprehensive gene and protein expression analysis. Results Angiogenesis was significantly impaired in MMD regardless of the presence of any angiogenic factor. On the contrary, endothelial proliferation was not significant between control- and MMD-derived cells. Regarding DNA microarray, pathway analysis illustrated that extracellular matrix (ECM) receptor-related genes, including integrin β3, were significantly downregulated in MMD. Proteomic analysis revealed that cytoskeleton-related proteins were downregulated and splicing regulation-related proteins were upregulated in MMD. Conclusions Downregulation of ECM receptor-related genes may be associated with impaired angiogenic activity in ECs derived from iPSCs from patients with MMD. Upregulation of splicing regulation-related proteins implied differences in splicing patterns between control and MMD ECs. PMID:27662211

  16. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

    Science.gov (United States)

    Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

  17. 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.

  18. Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

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    Wing-Hon Lai

    Full Text Available Functional endothelial-like cells (EC have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC, human embryonic stem cells (hESC-EC and human induced pluripotent stem cells (hiPSC-EC, and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC and BM-EC (P>0.05. While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05, the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05. Compared with medium, transplanting BM-EC (n = 6, HUVEC (n = 6, hESC-EC (n = 8 or hiPSC-EC (n = 8 significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as "off-the-shelf" format for the treatment

  19. Endothelial cells derived from the blood-brain barrier and islets of Langerhans differ in their response to the effects of bilirubin on oxidative stress under hyperglycemic conditions

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    Jaime eKapitulnik

    2012-07-01

    Full Text Available Unconjugated bilirubin (UCB is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS. High glucose levels (hyperglycemia generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB. In the current study we show that UCB (1-40 M induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langherans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM glucose levels. While UCB (0.1-40 M did not alter ROS production in cells exposed to normal glucose, relatively low ('physiological' UCB concentrations (0.1-5 M attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20-40 M increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic conditions.

  20. Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Expression of Proliferating Cell Nuclear Antigen and Vascular Endothelial Growth Factor in Primary Culture Cells Derived from Human Ovarian Mucinous Cystadenocarcino

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The effects of antisense oligodeoxynucleotide (antisense ODN) to follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone (FSH) on the expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma (OMC). The prlmary OMC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups (P<0.05 or P< 0.01), while decreased significantly in antisense ODN groups (P<0. 05 or P<0.01) and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly (P<0.01). It was suggested that FSH might promotethe development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH.

  1. Familial Follicular-Cell Derived Carcinoma

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    Eun Ju eSon

    2012-05-01

    Full Text Available Follicular cell-derived well-differentiated thyroid cancer, papillary (PTC and follicular thyroid carcinomas (FTC compose 95% of all thyroid malignancies. Familial follicular cell-derived well-differentiated thyroid cancers contribute to 5% of those cases. These familial follicular cell derived carcinomas or non-medullary thyroid carcinomas (NMTC divide into two clinical-pathological groups. One group, syndromic-associated, composed by predominately non-thyroidal tumors, is comprised of Pendred syndrome, Warner syndrome, Carney complex type 1, PTEN-hamartoma tumor syndrome (Cowden disease; PHTS, familial adenomatous polyposis (FAP/Gardner syndrome. Additionally other less established links correlated to the development of follicular cell-derived tumors have also included Ataxia-teleangiectasia syndrome, McCune Albright syndrome, and Peutz-Jeghers syndrome. The subsequent group encompasses syndromes typified by non-medullary thyroid carcinomas or NMTC, as well as, pure familial (f PTC with or without oxyphilia, fPTC with multinodular goiter and fPTC with papillary renal cell carcinoma. This heterogeneous group of diseases has not a established genotype-phenotype correlation as the well-known genetic events identified in the familial C-cell-derived tumors or medullary thyroid carcinomas (MTC. Clinicians should be have the knowledge to identify the likelihood of a patient presenting with thyroid cancer having an additional underlying familial syndrome stemming from characteristics through morphological findings that would alert the pathologist to have the patient undergo subsequent molecular genetics evaluations. This review will discuss the clinical and pathological findings of the patients with familial papillary thyroid carcinoma, such as familial adenomatous polyposis, Carney complex, Werner syndrome, and Pendred syndrome and the heterogeneous group of familial papillary thyroid carcinoma.

  2. Influence of basement membrane proteins and endothelial cell-derived factors on the morphology of human fetal-derived astrocytes in 2D.

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    Amanda F Levy

    Full Text Available Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB, however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF, laminin, basic fibroblast growth factor (bFGF, and leukemia inhibitory factor (LIF. Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models.

  3. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device

    NARCIS (Netherlands)

    Meer, van der Andries D.; Orlova, Valeria V.; Dijke, tem Peter; Berg, van den Albert; Mummery, Christine L.

    2013-01-01

    Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels

  4. Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4

    DEFF Research Database (Denmark)

    Jinquan, T; Jacobi, H H; Jing, C

    2000-01-01

    The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha) is the only known ligand for CXCR4. To date, the CXCR4 expression and function...... of SDF-1alpha in basophils are unknown....

  5. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  6. The role of endothelial cell derived toll-like receptor 4 acute lung injury%内皮源性toll样受体4在急性肺损伤中的作用

    Institute of Scientific and Technical Information of China (English)

    张进祥; 王慧; 仲照东; 李毅清; 梁慧芳; 吴河水; 田元; 蒋春舫; 郑启昌

    2008-01-01

    Objective To investigate the role of toll-like receptor 4 (TLR4) of endothelium or bone marrow derived cells in the acute lung injury (ALI) induced by lipopolyscccharide (LPS) in mice with reciprocal bone marrow transplantation. Method Chimeric mice were produced by reciprocal bone marrow transplantation between TLR4mut/mut and TLR4+/+ mice and divided into 4 groups: WT/WT (recipient/donor),WT/Mutant, Mutant/WT and Mutant/Mutant group. Six to eight weeks following transplantation, LPS was injected inot mice's tail vein in order to produce ALI model,and mice were sacrificed five hours later on.Samples of lung tissues were taken for the following analysis of wet/dry weight (W/D), lung permeabifity index (LPI), myeloperoxidase (MPO),levels of cytokines (TNF-α, IL-1β) and adhesion molecules (ICAM-1). Results Lung injury in the Mutant/Mutant mice was the mildest in the 4 groups. And lung injury in WT/Mutant mice was more serious than that in Mutant/WT mice. levels of MPO and ICAM-1 in WT/Mutant mice were much higher than those of Mutant/WT. In addition,the expression of ICAM-1 in WT/Mutant mice is comarable to that in WT/WT mice. Mutant/WT mice expressed higher levels of TNF-α and IL-1β than WT/Mutant mice. Conclusions Endothelial cell derived TLR4 plays ker-nel role in ALI induced by LPS via lung PMN recruitment,although bone marrow cells derived TLR4 are more im-portant for the release of cytokines.%目的 探索内皮源性或髓源性细胞表面toll样受体4(TLR4)介入急性肺损伤(ALI)的作用. 方法 采用TLR4基因突变(C3H/HeJ品系,TLR4mut/mut)及野生型(C3H/HeN,TLB4+/+)小鼠,通过骨髓移植建立"内皮细胞TLR4+/+髓系细胞TLR4mut/mut"(WT/Mutant:受体/供体)及Mutant/WT嵌合体小鼠,尾静脉注射LPS(5 mg·kg-1)复制ALI模型,5 h后测肺组织湿干重比(W/D),肺通透指数(LPI),肺组织髓过氧化物酶(MPO)水平、炎症因子及黏附分子水平. 结果 TLR4mut/mut小鼠肺损伤较TLR4+/+小鼠较轻,WT/Mutant组小鼠

  7. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  8. Stromal cell-derived factor-1/CXCR4 signaling modifies the capillary-like organization of human embryonic stem cell-derived endothelium in vitro.

    Science.gov (United States)

    Chen, Tong; Bai, Hao; Shao, Ying; Arzigian, Melanie; Janzen, Viktor; Attar, Eyal; Xie, Yi; Scadden, David T; Wang, Zack Z

    2007-02-01

    The molecular mechanisms that regulate human blood vessel formation during early development are largely unknown. Here we used human ESCs (hESCs) as an in vitro model to explore early human vasculogenesis. We demonstrated that stromal cell-derived factor-1 (SDF-1) and CXCR4 were expressed concurrently with hESC-derived embryonic endothelial differentiation. Human ESC-derived embryonic endothelial cells underwent dose-dependent chemotaxis to SDF-1, which enhanced vascular network formation in Matrigel. Blocking of CXCR4 signaling abolished capillary-like structures induced by SDF-1. Inhibition of the SDF-1/CXCR4 signaling pathway by AMD3100, a CXCR4 antagonist, disrupted the endothelial sprouting outgrowth from human embryoid bodies, suggesting that the SDF-1/CXCR4 axis plays a critical role in regulating initial vessel formation, and may function as a morphogen during human embryonic vascular development.

  9. Downregulation of Securin by the variant RNF213 R4810K (rs112735431, G>A) reduces angiogenic activity of induced pluripotent stem cell-derived vascular endothelial cells from moyamoya patients

    Energy Technology Data Exchange (ETDEWEB)

    Hitomi, Toshiaki [Department of Health and Environmental Sciences, Kyoto University, Kyoto (Japan); Habu, Toshiyuki [Radiation Biology Center, Kyoto University, Kyoto (Japan); Kobayashi, Hatasu; Okuda, Hiroko; Harada, Kouji H. [Department of Health and Environmental Sciences, Kyoto University, Kyoto (Japan); Osafune, Kenji [Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto (Japan); Taura, Daisuke; Sone, Masakatsu [Department of Medicine and Clinical Science, Kyoto University, Kyoto (Japan); Asaka, Isao; Ameku, Tomonaga; Watanabe, Akira; Kasahara, Tomoko; Sudo, Tomomi; Shiota, Fumihiko [Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto (Japan); Hashikata, Hirokuni; Takagi, Yasushi [Department of Neurosurgery, Kyoto University,Kyoto (Japan); Morito, Daisuke [Faculty of Life Sciences, Kyoto Sangyo University, Kyoto (Japan); Miyamoto, Susumu [Department of Neurosurgery, Kyoto University,Kyoto (Japan); Nakao, Kazuwa [Department of Medicine and Clinical Science, Kyoto University, Kyoto (Japan); Koizumi, Akio, E-mail: koizumi.akio.5v@kyoto-u.ac.jp [Department of Health and Environmental Sciences, Kyoto University, Kyoto (Japan)

    2013-08-16

    Highlights: •Angiogenic activities were reduced in iPSECs from MMD patients. •Many mitosis-regulated genes were downregulated in iPSECs from MMD patients. •RNF213 R4810K downregulated Securin and inhibited angiogenic activity. •Securin suppression by siRNA reduced angiogenic activities of iPSECs and HUVECs. -- Abstract: Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p < 0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p < 0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.

  10. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  11. Comprehensive proteomic characterization of stem cell-derived extracellular matrices.

    Science.gov (United States)

    Ragelle, Héloïse; Naba, Alexandra; Larson, Benjamin L; Zhou, Fangheng; Prijić, Miralem; Whittaker, Charles A; Del Rosario, Amanda; Langer, Robert; Hynes, Richard O; Anderson, Daniel G

    2017-06-01

    In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.

  12. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  13. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

  14. Enriched retinal ganglion cells derived from human embryonic stem cells

    Science.gov (United States)

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  15. 基质细胞衍生因子-1α和白细胞介素-1β诱导淋巴管内皮表型的作用%Effect of stromal cell derived factor-1αand interleukin-1βon inducing vascular endothelial cells expressing lymphatic phenotype

    Institute of Scientific and Technical Information of China (English)

    索宁; 王雪颖; 杨春林; 周辉; 李菲; 张宗璞; 万芳竹; 田铧

    2014-01-01

    目的:探讨基质细胞衍生因子-1α( SDF-1α)及白细胞介素-1β( IL-1β)诱导内皮细胞表达淋巴管表型的作用。方法 SDF-1α和IL-1β分别诱导内皮细胞株CRL-1730,用Real-time PCR、Western blotting 及免疫细胞化学等方法检测其内皮及淋巴管标志物,的表达情况。结果 SDF-1α诱导培养之后,CRL-1730细胞株的内皮细胞标志物血管性血友病因子(vWF)、血管内皮钙黏蛋白(VE-cadherin)、血管内皮生长因子受体(VEGFR)2随其浓度增高而表达降低,淋巴管标志物平足蛋白( podoplanin )、同源异形盒蛋白-1( Prox-1)和淋巴管内皮透明质酸受体-1(LYVE-1)随其浓度增高而表达增高。 IL-1β诱导之后,CRL-1730细胞株的vWF、VEGFR2和podoplanin、prox-1、LYVE-1的变化趋势同SDF-1α,而VE-cadherin的表达量基本不变。结论 SDF-1α和IL-1β都能够诱导血管内皮细胞表达淋巴管标志物。%Objective To investigate the effect of stromal cell-derived factor-1α( SDF-1α) and interleukin ( IL-1β) on inducing vascular endothelial cells to express lymphatic phenotype .Methods The CRL-1730 cell line was cultured and treated with SDF-1αor IL-1β.The expression of endothelial cell markers and lymphatic endothelial cell markers were investigated with Real-time PCR, Western blotting and immunocytochemistry .Results In CRL-1730 cell line, endothelial cell markers such as voln willebrand factor ( vWF ) , VE-cadherin , vascular endothelial growth factor receptor(VEGFR)2, were dose dependently down-regulated after SDF-1αstimulation, while lymphatic phenotypes such as Prox-1, podoplanin and lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1), were dose-dependently up-regulated after SDF-1αstimulation.The changes of vWF, VEGFR2 and podoplanin, Prox-1, LYVE-1 expression after IL-1βstimulation was similar to that after SDF-1αwhile expression of VE-cadherin changed slightly .Conclusion SDF-1αand IL-1

  16. Neural stem cell-derived exosomes mediate viral entry

    Directory of Open Access Journals (Sweden)

    Sims B

    2014-10-01

    Full Text Available Brian Sims,1,2,* Linlin Gu,3,* Alexandre Krendelchtchikov,3 Qiana L Matthews3,4 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental, and Integrative Biology, 3Division of Infectious Diseases, Department of Medicine, 4Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL, USA *These authors contributed equally to this work Background: Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells.Methods: To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5 for the proof-of-principle study. Results: Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4, which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry.Conclusion: Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines. Keywords: neural stem cell-derived exosomes, adenovirus type 5, TIM-4, viral entry, phospholipids

  17. 骨髓内皮细胞分泌的抑制CFU-F生长的因子%Bone Marrow Endothelial Cell-Derived Factors Inhibit the Growth of Marrow CFU-F

    Institute of Scientific and Technical Information of China (English)

    汪保和; 黄炜琦; 王绮如

    2000-01-01

    为探讨骨髓微环境中内皮细胞调节造血的可能机制,本实验研究了骨髓内皮细胞无血清条件培养液(BMEC-CM)及其不同分子量组分对骨髓成纤维祖细胞(CFU-F)生长的作用.在体外培养条件下,收集人和小鼠纯骨髓内皮细胞无血清条件培养液,应用连续超滤技术从其中制备出分子量>10 kD,3-10 kD和<3 kD三种组分,进行骨髓CFU-F集落形成实验,检测这些条件培养液及其超滤组分的作用.结果显示,人和小鼠骨髓内皮细胞无血清条件培养液和分子量<3 kD超滤组分都抑制CFU-F的生长,但是分子量>10kD和3-10 kD组分却不产生这种效应.两种条件培养液及其<3 kD组分不仅减少CFU-F集落形成数,同时也使集落变小,而且,<3 kD组分与CFU-F集落数之间有明显的负相关浓度依赖性关系.这些结果提示,骨髓内皮细胞至少能分泌一种分子量小于3 kD的对骨髓CFU-F生长有抑制作用的细胞因子.%The present study investigated the effects of the serum-free conditioned media of the bone marrow endothelial cells on CFU-F for potential mechanisms upon which hematopoiesis may be regulated by them within the bone marrow microenvironment. After obtaining the serum-free conditioned media of human and murine purified bone marrow endothelial cells (hBMEC-CM and mBMEC-CM) in vitro, MW>10 kD, 3 - 10 kD and <3 kD components were sifted out from these media by means of serial ultrafiltration. Assays of CFU-F were performed to test the effects of BMEC-CM and their ultrafiltrated components. The results showed that every one of hBMEC-CM, mBMEC-CM and their MW<3 kD components exerted a suppressive effect on the proliferation of corresponding CFU-F but MW>10 kDand 3 - 10 kD components. The BMEC-CM and MW <3 kD components decreased the number as well as the size of CFU-F. There were the markedly negative concentration-dependent relations between the concentrations of MW<3 kD component and the

  18. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    Science.gov (United States)

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.

  19. Pluripotent stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Schwartz, R E; Fleming, H E; Khetani, S R; Bhatia, S N

    2014-01-01

    Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are

  20. Early and Late Endothelial Progenitor Cells Derived from Rabbit Bone Marrow Isolated and Cultured by An Improved Method%改良法分离培养兔骨髓源性早晚期内皮祖细胞

    Institute of Scientific and Technical Information of China (English)

    王佐; 张凯; 王仁; 苏维; 李爽; 杨简; 姜志胜

    2011-01-01

    目的 探索简单有效分离培养兔骨髓源性内皮祖细胞的方法,并比较两种内皮祖细胞生物学性状.方法 4周龄左右的新西兰兔,于每侧胫骨取骨髓2mL,密度梯度离心后取单个核细胞接种于培养瓶,48h后将悬浮的细胞收集再次贴壁,血管内皮生长因子诱导其向内皮祖细胞分化.免疫细胞化学鉴定其表面标志物、免疫荧光功能学测定,对比前后两种贴壁细胞生长状况.结果 早期获取的单个核细胞,半小时后就开始贴壁,3天左右即可长出长梭形的细胞,胞体较大,有血岛样克隆形成,随后培养可形成管腔样结构,10天左右即可呈漩涡状融合整个培养瓶,但这种细胞传代能力差,为早期内皮祖细胞;第2次贴壁的晚期细胞于贴壁后呈椭圆形生长,贴壁后5-7天即可出现集落,片状生长,最后呈铺路石样融合,并可连续传至10代以上,为晚期内皮祖细胞.第2次贴壁的内皮祖细胞在分化过程中明显失去CD133+,而CD34+表达有所升高,大部分第1次贴壁内皮祖细胞可以吞噬乙酰化低密度脂蛋白和荆豆凝集素1,第2次贴壁内皮祖细胞功能学鉴定结果与第1次贴壁的结果类似.结论 改良后的密度梯度离心法结合差速贴壁法能有效分离培养兔骨髓源性内皮祖细胞,第2次贴壁的内皮祖细胞生长能力更强.%Aim To establish an available and convenient method to isolate and culture the rabbit bone marrow-derived endothelial progenitor cells (EPC) and compare the characteristics of the two different EPC. Methods Obtained 2 mL bone marrow from each shinbone of about 4 weeks old New Zealand rabbit, mononuclear cells ( MNC) were I-solated by Ficoll density gradient centrifugation method and planted in the first culture flask, after incubated for 48 h, collecting the suspended cells into the second flask, supplemented with vascular endothelial growth factor ( VEGF) in order to induce cells differentiation into EPC. To

  1. Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

    Directory of Open Access Journals (Sweden)

    Takahisa Furuta

    Full Text Available BACKGROUND: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase and -related cytokines (IL-4, -9, and -17 between patients with differing severity of Dengue fever and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF, Dengue hemorrhagic fever (DHF, and Dengue shock syndrome (DSS, as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. CONCLUSIONS: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

  2. Balancing Ethical Pros and Cons of Stem Cell Derived Gametes.

    Science.gov (United States)

    Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido

    2017-01-13

    In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

  3. Stromal cell-derived factor 1α (SDF-1α)

    DEFF Research Database (Denmark)

    Li, Dana; Bjørnager, Louise; Langkilde, Anne

    2016-01-01

    OBJECTIVES: Stromal cell-derived factor 1a (SDF-1α), is a chemokine and is able to home hematopoietic progenitor cells to injured areas of heart tissue for structural repair. Previous studies have found increased levels of SDF-1α in several cardiac diseases, but only few studies have investigated...... SDF-1α in patients with atrial fibrillation (AF). We aimed to test SDF-1α in a large cohort of patients with AF and its role as a prognostic marker. DESIGN: Between January 1st 2008 to December 1st 2012, 290 patients with ECG documented AF were enrolled from the in- and outpatient clinics...... at the Department of Cardiology, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark. Plasma levels of SDF-1α were measured using ELISA technique. Clinical data were registered and patient follow-up was conducted. RESULTS: Patients with permanent AF had significantly higher SDF-1α levels (2199.5 pg...

  4. Large-scale generation of cell-derived nanovesicles

    Science.gov (United States)

    Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim, S. C.; Gho, Y. S.; Park, J.

    2014-09-01

    Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

  5. Human embryonic stem cell derivation and directed differentiation.

    Science.gov (United States)

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  6. Dendritic Cell-Derived Exosomes Stimulate Stronger CD8+ CTL Responses and Antitumor Immunity than Tumor Cell-Derived Exosomes

    Institute of Scientific and Technical Information of China (English)

    Siguo Hao; Ou Bai; Jinying Yuan; Mabood Qureshi; Jim Xiang

    2006-01-01

    Exosomes (EXO) derived from dendritic cells (DC) and tumor cells have been used to stimulate antitumor immune responses in animal models and in clinical trials. However, there has been no side-by-side comparison of the stimulatory efficiency of the antitumor immune responses induced by these two commonly used EXO vaccines. In this study, we selected to study the phenotype characteristics of EXO derived from a transfected EG7 tumor cells expressing ovalbumin (OVA) and OVA-pulsed DC by flow cytometry. We compared the stimulatory effect in induction of OVA-specific immune responses between these two types of EXO. We found that OVA protein-pulsed DCovA-derived EXO (EXODC) can more efficiently stimulate naive OVA-specific CD8+ T cell proliferation and differentiation into cytotoxic T lymphocytes in vivo, and induce more efficient antitumor immunity than EG7 tumor cell-derived EXO (EXOEG7). In addition, we elucidated the important role of the host DC in EXO vaccines that the stimulatory effect of EXO is delivered to T cell responses by the host DC. Therefore, DC-derived EXO may represent a more effective EXO-based vaccine in induction of antitumor immunity.

  7. Mecanotransduction and Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    S.MULLER; JF.; STOLTZ2

    2005-01-01

    1 IntroductionAtherosclerosis preferentially occurs in areas of complex blood flow where there are disturbed flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective~([1]). Reports of others and our studies suggest a steady laminar flow decreases some molecules and genes expression of vascular endothelial cells (EC) that may promote atherosclerosis, as well as it can differentially regulate production of many vasoactive factors at the level of gene expression an...

  8. Contribution of cells derived from the area pellucida to extraembryonic mesodermal cell lineages in heterospecific quail chick blastodermal chimeras.

    Science.gov (United States)

    Karagenç, Levent; Sandikci, Mustafa

    2013-01-01

    The current study has two main objectives: first, to determine if cells derived from the area pellucida are able to populate extraembryonic membranes, and second, to determine if donor cells have the potential to differentiate to endothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes functioning as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting putative chimeras was examined using quail cell-specific antibody against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN(+) donor cells was examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin antibody. Evidence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of resulting heterospecific quail chick chimeras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages derived from the posterior mesoderm both in the yolk sac and allantois.

  9. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  10. Generating and characterizing the mechanical properties of cell-derived matrices using atomic force microscopy.

    Science.gov (United States)

    Tello, Marta; Spenlé, Caroline; Hemmerlé, Joseph; Mercier, Luc; Fabre, Roxane; Allio, Guillaume; Simon-Assmann, Patricia; Goetz, Jacky G

    2016-02-01

    Mechanical interaction between cells and their surrounding extracellular matrix (ECM) controls key processes such as proliferation, differentiation and motility. For many years, two-dimensional (2D) models were used to better understand the interactions between cells and their surrounding ECM. More recently, variation of the mechanical properties of tissues has been reported to play a major role in physiological and pathological scenarios such as cancer progression. The 3D architecture of the ECM finely tunes cellular behavior to perform physiologically relevant tasks. Technical limitations prevented scientists from obtaining accurate assessment of the mechanical properties of physiologically realistic matrices. There is therefore a need for combining the production of high-quality cell-derived 3D matrices (CDMs) and the characterization of their topographical and mechanical properties. Here, we describe methods that allow to accurately measure the young modulus of matrices produced by various cellular types. In the first part, we will describe and review several protocols for generating CDMs matrices from endothelial, epithelial, fibroblastic, muscle and mesenchymal stem cells. We will discuss tools allowing the characterization of the topographical details as well as of the protein content of such CDMs. In a second part, we will report the methodologies that can be used, based on atomic force microscopy, to accurately evaluate the stiffness properties of the CDMs through the quantification of their young modulus. Altogether, such methodologies allow characterizing the stiffness and topography of matrices deposited by the cells, which is key for the understanding of cellular behavior in physiological conditions.

  11. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.

    Science.gov (United States)

    Schwartz, Michael P; Hou, Zhonggang; Propson, Nicholas E; Zhang, Jue; Engstrom, Collin J; Santos Costa, Vitor; Jiang, Peng; Nguyen, Bao Kim; Bolin, Jennifer M; Daly, William; Wang, Yu; Stewart, Ron; Page, C David; Murphy, William L; Thomson, James A

    2015-10-01

    Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.

  12. Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Seta

    Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

  13. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    Science.gov (United States)

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  14. Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells

    OpenAIRE

    Islam, Mohammad S; Stemig, Melissa E.; Takahashi, Yutaka; Hui, Susanta K.

    2014-01-01

    Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harv...

  15. iPS-cell derived dendritic cells and macrophages for cancer therapy.

    Science.gov (United States)

    Senju, Satoru

    2016-08-01

    Antibody-based anti-cancer immunotherapy was recently recognized as one of the truly effective therapies for cancer patients. Antibodies against cell surface cancer antigens, such as CD20, and also those against immune-inhibitory molecules called "immune checkpoint blockers", such as CTLA4 or PD1, have emerged. Large-scale clinical trials have confirmed that, in some cases, antibody-based drugs are superior to conventional chemotherapeutic agents. These antibody-based drugs are now being manufactured employing a mass-production system by pharmaceutical companies. Anti-cancer therapy by immune cells, i.e. cell-based immunotherapy, is expected to be more effective than antibody therapy, because immune cells can recognize, infiltrate, and act in cancer tissues more directly than antibodies. In order to achieve cell-based anti-cancer immunotherapy, it is necessary to develop manufacturing systems for mass-production of immune cells. Our group has been studying immunotherapy with myeloid cells derived from ES cells or iPS cells. These pluripotent stem cells can be readily propagated under constant culture conditions, with expansion into a large quantity. We consider these stem cells to be the most suitable cellular source for mass-production of immune cells. This review introduces our studies on anti-cancer therapy with iPS cell-derived dendritic cells and iPS cell-derived macrophages.

  16. Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering.

    Science.gov (United States)

    Zhang, Weixiang; Zhu, Yun; Li, Jia; Guo, Quanyi; Peng, Jiang; Liu, Shichen; Yang, Jianhua; Wang, Yu

    2016-06-01

    The extracellular matrix (ECM) is a dynamic and intricate microenvironment with excellent biophysical, biomechanical, and biochemical properties, which can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. The ECM has attracted a great deal of attention with the rapid development of tissue engineering in the field of regenerative medicine. Tissue-derived ECM scaffolds (also referred to as decellularized tissues and whole organs) are considered a promising therapy for the repair of musculoskeletal defects, including those that are widely used in orthopedics, although there are a few shortcomings. Similar to tissue-derived ECM scaffolds, cell-derived ECM scaffolds also have highly advantageous biophysical and biochemical properties, in particular their ability to be produced in vitro from a number of different cell types. Furthermore, cell-derived ECM scaffolds more closely resemble native ECM microenvironments. The products of cell-derived ECM have a wide range of biomedical applications; these include reagents for cell culture substrates and biomaterials for scaffolds, hybrid scaffolds, and living cell sheet coculture systems. Although cell-derived ECM has only just begun to be investigated, it has great potential as a novel approach for cell-based tissue repair in orthopedic tissue engineering. This review summarizes and analyzes the various types of cell-derived ECM products applied in cartilage, bone, and nerve tissue engineering in vitro or in vivo and discusses future directions for investigation of cell-derived ECM.

  17. A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells.

    Science.gov (United States)

    Choudhary, Parul; Whiting, Paul John

    2016-09-02

    Cell replacement and regenerative therapy using embryonic stem cell-derived material holds promise for the treatment of several pathologies. However, the safety of this approach is of prime importance given the teratogenic potential of residual stem cells, if present in the differentiated cell product. Using the example of embryonic stem cell-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration, we present a novel strategy for ensuring the absence of stem cells in the RPE population. Based on an unbiased screening approach, we identify and validate the expression of CD59, a cell surface marker expressed on RPE but absent on stem cells. We further demonstrate that flow sorting on the basis of CD59 expression can effectively purify RPE and deplete stem cells, resulting in a population free from stem cell impurity. This purification helps to ensure removal of stem cells and hence increases the safety of cells that may be used for clinical transplantation. This strategy can potentially be applied to other pluripotent stem cell-derived material and help mitigate concerns of using such cells for therapy.

  18. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  19. Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes.

    Science.gov (United States)

    Kabeya, Tomoki; Matsumura, Wakana; Iwao, Takahiro; Hosokawa, Masakiyo; Matsunaga, Tamihide

    2017-04-22

    Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

  20. Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant

    Science.gov (United States)

    Mukhopadhyay, C. K.; Fox, P. L.

    1998-01-01

    Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

  1. Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture.

    Science.gov (United States)

    Zaniboni, Andrea; Bernardini, Chiara; Alessandri, Marco; Mangano, Chiara; Zannoni, Augusta; Bianchi, Francesca; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2014-02-15

    Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

  2. SDF-1对糖尿病外周血EPCs功能的影响及其与PI3K/AKT信号通路的关系%Effects of Stromal Cell-Derived-Factor-1 on Endothelial Progenitor Cells of Peripheral Blood and Their Relationship with PI3K/AKT Signal Transduction Pathway in Patients with Diabetes

    Institute of Scientific and Technical Information of China (English)

    黎金凤; 林安华; 邓颖; 霍亚南; 刘精东; 吴明斌; 王晨秀

    2014-01-01

    Objective To observe the effects of stromal cell-derived-factor-1(SDF-1) on the function of endotheli⁃al progenitor cells(EPCs)of peripheral blood in patients with diabetes, and to discuss the effects of PI3K/AKT signaling path⁃way on the role of SDF-1 in EPCs. Methods The peripheral blood samples (30 mL) were collected in 10 diabetes patients (DM group) and 10 healthy controls (HC group). (1) The 100μg/L SDF-1 was added in intervention group. EGM-2MV was added in non-intervention group. The Boyden chamber and in vitro angiogenesis kit were used to analyze the migration and in vitro angiogenesis of EPCs. (2) Cultured EPCs were divided into blank control group, 1μg/L SDF-1 group, 10μg/L SDF-1 group, 100μg/L SDF-1 group, pure AMD3100 group and 100μg/L SDF-1+AMD3100 group. AKT protein expression lev⁃els of endothelial progenitor cells were detected by Western blot assay in each group. Results (1) Without intervention with SDF-1, EPCs’migration and angiogenesis ability were lower in DM group than those in HC group. After intervention with SDF-1, the migration and angiogenesis ability were enhanced in two groups, but the increased level was higher in DM group than that of HC group. (2) Under the same concentration, AKT protein expression level was significantly lower in DM group than that in HC group (P<0.01). AKT protein expression levels were increased with the increased levels of SDF-1 in DM group and HC group (P<0.05). AKT protein expression was significantly lower in 100μg/L SDF-1+AMD3100 group than that of 100μg/L SDF-1 group (P<0.05). Conclusion SDF-1 can increase the chemotactic migration and angiogenesis ability of EPCs in peripheral blood, especially for patients with diabetes. The effects of SDF-1 on EPCs were related to the PI3K/AKT signaling pathway.%目的:观察基质细胞衍生因子-1(SDF-1)对糖尿病外周血内皮祖细胞(EPCs)功能的影响,探讨SDF-1对EPCs的影响是否与PI3K/AKT信号通路有关。方法采集

  3. Foetal stem cell derivation & characterization for osteogenic lineage

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    A Mangala Gowri

    2013-01-01

    Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

  4. Stem cell-derived hepatocytes and their use in toxicology.

    Science.gov (United States)

    Guguen-Guillouzo, Christiane; Corlu, Anne; Guillouzo, Andre

    2010-03-30

    Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.

  5. Biogenesis and function of T cell-derived exosomes

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    Miguel Angel Alonso

    2016-08-01

    Full Text Available Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins and nucleic acids confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes.

  6. Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Corinne A Lee-Kubli; Paul Lu

    2015-01-01

    The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell ther-apies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges.

  7. Stem cell-derived hepatocytes for functional liver replacement

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    Bruno eChrist

    2012-06-01

    Full Text Available Mesenchymal stem cells (MSC represent an alternate cell source to substitute for primary hepatocytes in hepatocyte transplantation because of their multiple differentiation potential and nearly unlimited availability. They may differentiate into hepatocyte-like cells in vitro and maintain specific hepatocyte functions also after transplantation into the regenerating livers of mice or rats both under injury and non-injury conditions. Depending on the underlying liver disease their mode of action is either to replace the diseased liver tissue or to support liver regeneration through their anti-inflammatory and anti-apoptotic as well as their pro-proliferative action.

  8. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Institute of Scientific and Technical Information of China (English)

    Li-Wei Zheng; Logan Linthicum; Pamela K DenBesten; Yan Zhang

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCI) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which ,vas also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  9. Molecular Characterization of Dendritic Cell-Derived Exosomes

    OpenAIRE

    Théry, Clotilde; Regnault, Armelle; Garin, Jérôme; Wolfers, Joseph; Zitvogel, Laurence; Ricciardi-Castagnoli, Paola; Raposo, Graça; Amigorena, Sebastian

    1999-01-01

    Exosomes are membrane vesicles secreted by hematopoietic cells upon fusion of late multivesicular endosomes with the plasma membrane. Dendritic cell (DC)-derived exosomes induce potent antitumor immune responses in mice, resulting in the regression of established tumors (Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Nat. Med. 4:594–600). To unravel the molecular basis of exosome-induced immune stimulation, w...

  10. Mast cell-derived histamine mediates cystitis pain.

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    Charles N Rudick

    Full Text Available BACKGROUND: Mast cells trigger inflammation that is associated with local pain, but the mechanisms mediating pain are unclear. Interstitial cystitis (IC is a bladder disease that causes debilitating pelvic pain of unknown origin and without consistent inflammation, but IC symptoms correlate with elevated bladder lamina propria mast cell counts. We hypothesized that mast cells mediate pelvic pain directly and examined pain behavior using a murine model that recapitulates key aspects of IC. METHODS AND FINDINGS: Infection of mice with pseudorabies virus (PRV induces a neurogenic cystitis associated with lamina propria mast cell accumulation dependent upon tumor necrosis factor alpha (TNF, TNF-mediated bladder barrier dysfunction, and pelvic pain behavior, but the molecular basis for pelvic pain is unknown. In this study, both PRV-induced pelvic pain and bladder pathophysiology were abrogated in mast cell-deficient mice but were restored by reconstitution with wild type bone marrow. Pelvic pain developed normally in TNF- and TNF receptor-deficient mice, while bladder pathophysiology was abrogated. Conversely, genetic or pharmacologic disruption of histamine receptor H1R or H2R attenuated pelvic pain without altering pathophysiology. CONCLUSIONS: These data demonstrate that mast cells promote cystitis pain and bladder pathophysiology through the separable actions of histamine and TNF, respectively. Therefore, pain is independent of pathology and inflammation, and histamine receptors represent direct therapeutic targets for pain in IC and other chronic pain conditions.

  11. Cell-derived matrix coatings for polymeric scaffolds.

    Science.gov (United States)

    Decaris, Martin L; Binder, Bernard Y; Soicher, Matthew A; Bhat, Archana; Leach, J Kent

    2012-10-01

    Cells in culture deposit a complex extracellular matrix that remains intact following decellularization and possesses the capacity to modulate cell phenotype. The direct application of such decellularized matrices (DMs) to 3D substrates is problematic, as transport issues influence the homogeneous deposition, decellularization, and modification of DM surface coatings. In an attempt to address this shortcoming, we hypothesized that DMs deposited by human mesenchymal stem cells (MSCs) could be transferred to the surface of polymeric scaffolds while maintaining their capacity to direct cell fate. The ability of the transferred DM (tDM)-coated scaffolds to enhance the osteogenic differentiation of undifferentiated and osteogenically induced MSCs under osteogenic conditions in vitro was confirmed. tDM-coated scaffolds increased MSC expression of osteogenic marker genes (BGLAP, IBSP) and intracellular alkaline phosphatase production. In addition, undifferentiated MSCs deposited significantly more calcium when seeded onto tDM-coated scaffolds compared with control scaffolds. MSC-seeded tDM-coated scaffolds subcutaneously implanted in nude rats displayed significantly higher blood vessel density after 2 weeks compared with cells on uncoated scaffolds, but we did not observe significant differences in mineral deposition after 8 weeks. These data demonstrate that DM-coatings produced in 2D culture can be successfully transferred to 3D substrates and retain their capacity to modulate cell phenotype.

  12. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure.

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    Biju Parekkadan

    Full Text Available Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.

  13. In-vitro stem cell derived red blood cells for transfusion: are we there yet?

    Science.gov (United States)

    Kim, Hyun Ok

    2014-03-01

    To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices.

  14. Characterization of mesenchymal stem cells derived from equine adipose tissue

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    A.M. Carvalho

    2013-08-01

    Full Text Available Stem cell therapy has shown promising results in tendinitis and osteoarthritis in equine medicine. The purpose of this work was to characterize the adipose-derived mesenchymal stem cells (AdMSCs in horses through (1 the assessment of the capacity of progenitor cells to perform adipogenic, osteogenic and chondrogenic differentiation; and (2 flow cytometry analysis using the stemness related markers: CD44, CD90, CD105 and MHC Class II. Five mixed-breed horses, aged 2-4 years-old were used to collect adipose tissue from the base of the tail. After isolation and culture of AdMSCs, immunophenotypic characterization was performed through flow cytometry. There was a high expression of CD44, CD90 and CD105, and no expression of MHC Class II markers. The tri-lineage differentiation was confirmed by specific staining: adipogenic (Oil Red O, osteogenic (Alizarin Red, and chondrogenic (Alcian Blue. The equine AdMSCs are a promising type of adult progenitor cell for tissue engineering in veterinary medicine.

  15. Identification of Stem Leydig Cells Derived from Pig Testicular Interstitium

    Science.gov (United States)

    Yu, Shuai; Zhang, Pengfei; Dong, Wuzi; Zeng, Wenxian

    2017-01-01

    Stem Leydig cells (SLCs), located in the testicular interstitial compartment in the mammalian testes, are capable of differentiating to testosterone-synthesizing Leydig cells (LCs), thus providing a new strategy for treating testosterone deficiency. However, no previous reports have identified and cultured SLCs derived from the pig. The aim of the current study was to isolate, identify, and culture SLCs from pigs. Haematoxylin and eosin staining and immunochemical analysis showed that SLCs were present and that PDGFRα was mainly expressed in the pig testicular interstitium, indicating that PDGFRα was a marker for SLCs in the neonatal pig. In addition, reverse transcription-PCR results showed that SLC markers were expressed in primary isolated LCs, indicating that they were putative SLCs. The putative SLCs were subsequently cultured with a testicular fluid of piglets (pTF) medium. Clones formed after 7 days and the cells expressed PDGFRα. However, no clones grew in the absence of pTF, but the cells expressed CYP17A1, indicating that pTF could sustain the features of porcine SLCs. To summarize, we isolated porcine SLCs and identified their basic characteristics. Taken together, these results may help lay the foundation for research in the clinical application of porcine SLCs.

  16. Chronotropic Biosensing Via Stem-Cell Derived Myocyte Aggregates

    Science.gov (United States)

    2007-11-02

    the example of Figure 2, the myocyte aggregates showed a high degree of ab - solute tracking; for the 6 sustained-activity aggregates, the cor- relation...chronotropy. Indeed, excitable-cell biosensor systems could lead to the development of long-term, physiologically- tuned, functionally integrated bioprocessing ...H. Ko- dama, J. Pan, M. Sano, T. Takahashi, S. Hori, H. Abe , J. Hata, A. Umezawa, and S. Ogawa, “Cardiomyocytes can be generated from marrow stromal

  17. Biogenesis and function of T cell-derived exosomes

    OpenAIRE

    Miguel Angel Alonso; Leandro N. Ventimiglia

    2016-01-01

    Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins, and nucleic acids) confers on them the capacity to modulate the activity of receptor cells, even if these ce...

  18. Impairment of mesenchymal stem cells derived from oral leukoplakia.

    Science.gov (United States)

    Zhang, Zhihui; Song, Jiangyuan; Han, Ying; Mu, Dongdong; Su, Sha; Ji, Xiaoli; Liu, Hongwei

    2015-01-01

    Oral leukoplakia is one of the common precancerous lesions in oral mucosa. To compare the biological characteristics and regenerative capacities of mesenchymal stem cells (MSCs) from oral leukoplakia (epithelial hyperplasia and dysplasia) and normal oral mucosa, MSCs were isolated by enzyme digestion. Then these cells were identified by the expression of MSC related markers, STRO-1, CD105 and CD90, with the absent for the hematopoietic stem cell marker CD34 by flow cytometric detection. The self-renewal ability of MSCs from oral leukoplakia was enhanced, while the multipotent differentiation was descended, compared with MSCs from normal oral mucosa. Fibrin gel was used as a carrier for MSCs transplanted into immunocompromised mice to detect their regenerative capacity. The regenerative capacities of MSCs from oral leukoplakia became impaired partly. Collagen IV (Col IV) and matrix metalloproteinases-9 (MMP-9) were selected to analyze the potential mechanism for the functional changes of MSCs from oral leukoplakia by immunochemical and western blot analysis. The expression of Col IV was decreased and that of MMP-9 was increased by MSCs with the progression of oral leukoplakia, especially in MSCs from epithelial dysplasia. The imbalance between regenerative and metabolic self-regulatory functions of MSCs from oral leukoplakia may be related to the progression of this premalignant disorder.

  19. Mesenchymal Stem Cells Derived from Dental Pulp: A Review

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    Edgar Ledesma-Martínez

    2016-01-01

    Full Text Available The mesenchymal stem cells of dental pulp (DPSCs were isolated and characterized for the first time more than a decade ago as highly clonogenic cells that were able to generate densely calcified colonies. Now, DPSCs are considered to have potential as stem cell source for orthopedic and oral maxillofacial reconstruction, and it has been suggested that they may have applications beyond the scope of the stomatognathic system. To date, most studies have shown that, regardless of their origin in third molars, incisors, or exfoliated deciduous teeth, DPSCs can generate mineralized tissue, an extracellular matrix and structures type dentin, periodontal ligament, and dental pulp, as well as other structures. Different groups worldwide have designed and evaluated new efficient protocols for the isolation, expansion, and maintenance of clinically safe human DPSCs in sufficient numbers for various therapeutics protocols and have discussed the most appropriate route of administration, the possible contraindications to their clinical use, and the parameters to be considered for monitoring their clinical efficacy and proper biological source. At present, DPSC-based therapy is promising but because most of the available evidence was obtained using nonhuman xenotransplants, it is not a mature technology.

  20. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

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    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  1. Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

    Science.gov (United States)

    Marton, Annamaria; Vizler, Csaba; Kusz, Erzsebet; Temesfoi, Viktoria; Szathmary, Zsuzsa; Nagy, Krisztina; Szegletes, Zsolt; Varo, Gyorgy; Siklos, Laszlo; Katona, Robert L; Tubak, Vilmos; Howard, O M Zack; Duda, Erno; Minarovits, Janos; Nagy, Katalin; Buzas, Krisztina

    2012-01-01

    To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

  2. Classification of follicular cell-derived thyroid cancer by global RNA profiling

    DEFF Research Database (Denmark)

    Rossing, Maria

    2013-01-01

    The incidence of thyroid cancer is increasing worldwide and thyroid nodules are a frequent clinical finding. Diagnosing follicular cell-derived cancers is, however, challenging both histopathologically and especially cytopathologically. The advent of high-throughput molecular technologies has...... profiling of follicular cell-derived thyroid cancers....... prompted many researchers to explore the transcriptome and, in recent years, also the miRNome in order to generate new molecular classifiers capable of classifying thyroid tumours more accurately than by conventional cytopathological and histopathological methods. This has led to a number of molecular...

  3. Calcium Imaging in Pluripotent Stem Cell-Derived Cardiac Myocytes.

    Science.gov (United States)

    Walter, Anna; Šarić, Tomo; Hescheler, Jürgen; Papadopoulos, Symeon

    2016-01-01

    The possibility to generate cardiomyocytes (CMs) from disease-specific induced pluripotent stem cells (iPSCs) is a powerful tool for the investigation of various cardiac diseases in vitro. The pathological course of various cardiac conditions, causatively heterogeneous, often converges into disturbed cellular Ca(2+) cycling. The gigantic Ca(2+) channel of the intracellular Ca(2+) store of CMs, the ryanodine receptor type 2 (RyR2), controls Ca(2+) release and therefore plays a crucial role in Ca(2+) cycling of CMs. In the present protocol we describe ways to measure and analyze global as well as local cellular Ca(2+) release events in CMs derived from a patient carrying a CPVT-causing RyR2 mutation.

  4. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  5. PDX1-engineered embryonic stem cell-derived insulin producing cells regulate hyperglycemia in diabetic mice

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    Raikwar Sudhanshu P

    2012-10-01

    Full Text Available Abstract Background Type 1 diabetes can be treated by the transplantation of cadaveric whole pancreata or isolated pancreatic islets. However, this form of treatment is hampered by the chronic shortage of cadaveric donors. Embryonic stem (ES cell-derived insulin producing cells (IPCs offer a potentially novel source of unlimited cells for transplantation to treat type 1 and possibly type 2 diabetes. However, thus far, the lack of a reliable protocol for efficient differentiation of ES cells into IPCs has hindered the clinical exploitation of these cells. Methods To efficiently generate IPCs using ES cells, we have developed a double transgenic ES cell line R1Pdx1AcGFP/RIP-Luc that constitutively expresses pancreatic β-cell-specific transcription factor pancreatic and duodenal homeobox gene 1 (Pdx1 as well as rat insulin promoter (RIP driven luciferase reporter. We have established several protocols for the reproducible differentiation of ES cells into IPCs. The differentiation of ES cells into IPCs was monitored by immunostaining as well as real-time quantitative RT-PCR for pancreatic β-cell-specific markers. Pancreatic β-cell specific RIP became transcriptionally active following the differentiation of ES cells into IPCs and induced the expression of the luciferase reporter. Glucose stimulated insulin secretion by the ES cell-derived IPCs was measured by ELISA. Further, we have investigated the therapeutic efficacy of ES cell-derived IPCs to correct hyperglycemia in syngeneic streptozotocin (STZ-treated diabetic mice. The long term fate of the transplanted IPCs co-expressing luciferase in syngeneic STZ-induced diabetic mice was monitored by real time noninvasive in vivo bioluminescence imaging (BLI. Results We have recently demonstrated that spontaneous in vivo differentiation of R1Pdx1AcGFP/RIP-Luc ES cell-derived pancreatic endoderm-like cells (PELCs into IPCs corrects hyperglycemia in diabetic mice. Here, we investigated whether R1Pdx1Ac

  6. Mesenchymal stem cell derived hematopoietic cells are permissive to HIV-1 infection

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    Mondal Debasis

    2011-01-01

    Full Text Available Abstract Background Tissue resident mesenchymal stem cells (MSCs are multipotent, self-renewing cells known for their differentiation potential into cells of mesenchymal lineage. The ability of single cell clones isolated from adipose tissue resident MSCs (ASCs to differentiate into cells of hematopoietic lineage has been previously demonstrated. In the present study, we investigated if the hematopoietic differentiated (HD cells derived from ASCs could productively be infected with HIV-1. Results HD cells were generated by differentiating clonally expanded cultures of adherent subsets of ASCs (CD90+, CD105+, CD45-, and CD34-. Transcriptome analysis revealed that HD cells acquire a number of elements that increase their susceptibility for HIV-1 infection, including HIV-1 receptor/co-receptor and other key cellular cofactors. HIV-1 infected HD cells (HD-HIV showed elevated p24 protein and gag and tat gene expression, implying a high and productive infection. HD-HIV cells showed decreased CD4, but significant increase in the expression of CCR5, CXCR4, Nef-associated factor HCK, and Vpu-associated factor BTRC. HIV-1 restricting factors like APOBEC3F and TRIM5 also showed up regulation. HIV-1 infection increased apoptosis and cell cycle regulatory genes in HD cells. Although undifferentiated ASCs failed to show productive infection, HIV-1 exposure increased the expression of several hematopoietic lineage associated genes such as c-Kit, MMD2, and IL-10. Conclusions Considering the presence of profuse amounts of ASCs in different tissues, these findings suggest the possible role that could be played by HD cells derived from ASCs in HIV-1 infection. The undifferentiated ASCs were non-permissive to HIV-1 infection; however, HIV-1 exposure increased the expression of some hematopoietic lineage related genes. The findings relate the importance of ASCs in HIV-1 research and facilitate the understanding of the disease process and management strategies.

  7. Suppression of Th1-mediated autoimmunity by embryonic stem cell-derived dendritic cells.

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    Tokunori Ikeda

    Full Text Available We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs using two models of autoimmune disease, namely non-obese diabetic (NOD mice and experimental autoimmune encephalomyelitis (EAE. Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.

  8. SDF-1α及其受体CXCR4与HIF-1α、VEGF在脑动静脉畸形中的表达%Expressions of stromal-cell derived factor-1α and its receptor CXCR4, hypoxia inducible factor-1α and vascular endothelial growth factor in brain arteriovenous malformation

    Institute of Scientific and Technical Information of China (English)

    王凌雁; 郭少雷; 齐铁伟; 梁丰; 黄正松

    2014-01-01

    Objective To investigate the expressions ofstromal-cell derived factor-1α (SDF-1α)and its receptor CXCR4 in brain arteriovenous malformation (AVM) and to explore the relationships of SDF-1α with hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF).Methods Surgical specimens from 48 patients accepted brain AVM resection,collected in our hospital from January 2012 to December 2013,were studied for expressions ofSDF-1α,CXCR4,VEGF and HIF-1α by immunohistochemical staining.The relationships of SDF-1α with VEGF and HIF-1α were analyzed and influences of embolism,hemorrhage and Spetzer-Martin classification in SDF-1α expression were assessed.Results SDF-1α and CXCR4 expressed in 100% and 83.3% AVM specimens,respectively.The positive staining for SDF-1α was observed in the cytoplasm of vascular endothelium within the nidus and smooth muscle cells of vascular wall.CXCR4 expressed in vascular endothelium and perivascular cells located in the space between the abnormal vessels.SDF-1α expression was significantly associated with VEGF and HIF-1α (r=0.537 and 0.437,respectively,P<0.05).SDF-1α showed more intense expression in embolized patients than that in non-embolized patients (P< 0.05),while no significant difference was noted between patients with and without hemorrhage and between patients of different Spetzer-Martin classifications (P>0.05).Conclusion SDF-1α and its receptor CXCR4 highly express in brain AVM; preoperative embolization might induce expression of SDF-1α.%目的 观察脑动静脉畸形(AVM)病灶内间质细胞衍生因子-1α(SDF-1α)及其受体CXCR4的表达情况,以及SDF-1α与低氧诱导因子-1α(HIF-1α)和血管内皮生长因子(VEGF)表达的关系. 方法 选择中山大学附属第一医院神经外科自2012年1月至2013年12月经手术切除并经病理组织学证实的脑AVM标本共48例,应用免疫组化染色方法检测SDF-1α、CXCR4、HIF-1α和VEGF的表达情况,

  9. Two Polymorphisms in the Epithelial Cell-Derived Neutrophil-Activating Peptide (ENA-78 Gene

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    Mahsa M. Amoli

    2005-01-01

    Full Text Available Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78 has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing.

  10. Muse Cells, a New Type of Pluripotent Stem Cell Derived from Human Fibroblasts.

    Science.gov (United States)

    Liu, Qi; Zhang, Ru-zhi; Li, Di; Cheng, Sai; Yang, Yu-hua; Tian, Ting; Pan, Xiao-ru

    2016-04-01

    A new type of mesenchymal stem cells (MSCs) that expresses stage-specific embryonic antigen 3 (SSEA-3) and the mesenchymal cell marker CD105 are known as multilineage-differentiating stress-enduring (Muse) cells. Studies have shown that stem cells in suspension cultures are more likely to generate embryoid body-like stem cell spheres and maintain an undifferentiated phenotype and pluripotency. We separated Muse cells derived from human dermal fibroblasts by long-term trypsin incubation (LTT) through suspension cultures in methylcellulose. The Muse cells obtained expressed several pluripotency markers, including Nanog, Oct4, Sox2, and SSEA-3, and could differentiate in vitro into cells of the three germ layers, such as hepatocytes (endodermal), neural cells (ectodermal) and adipocytes, and osteocytes (mesodermal cells). These cells showed a low level of DNA methylation and a high nucleo-cytoplasmic ratio. Our study provides an innovative and exciting platform for exploring the potential cell-based therapy of various human diseases using Muse cells as well as their great possibility for regenerative medicine.

  11. Embryonic stem cell-derived microvesicles induce gene expression changes in Muller cells of the retina.

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    Diana Katsman

    Full Text Available Cell-derived microvesicles (MVs, recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  12. 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

  13. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

    Science.gov (United States)

    Rezania, Alireza; Bruin, Jennifer E; Arora, Payal; Rubin, Allison; Batushansky, Irina; Asadi, Ali; O'Dwyer, Shannon; Quiskamp, Nina; Mojibian, Majid; Albrecht, Tobias; Yang, Yu Hsuan Carol; Johnson, James D; Kieffer, Timothy J

    2014-11-01

    Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

  14. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  15. Microencapsulation technology by nature: Cell derived extracellular vesicles with therapeutic potential.

    Science.gov (United States)

    Kittel, A; Falus, A; Buzás, E

    2013-06-01

    Cell derived extracellular vesicles are submicron structures surrounded by phospholipid bilayer and released by both prokaryotic and eukaryotic cells. The sizes of these vesicles roughly fall into the size ranges of microbes, and they represent efficient delivery platforms targeting complex molecular information to professional antigen presenting cells. Critical roles of these naturally formulated units of information have been described in many physiological and pathological processes. Extracellular vesicles are not only potential biomarkers and possible pathogenic factors in numerous diseases, but they are also considered as emerging therapeutic targets and therapeutic vehicles. Strikingly, current drug delivery systems, designed to convey therapeutic proteins and peptides (such as liposomes), show many similarities to extracellular vesicles. Here we review some aspects of therapeutic implementation of natural, cell-derived extracellular vesicles in human diseases. Exploration of molecular and functional details of extracellular vesicle release and action may provide important lessons for the design of future drug delivery systems.

  16. Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

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    Siebler Mario

    2009-08-01

    Full Text Available Abstract Background The present work was performed to investigate the ability of two different embryonic stem (ES cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs, progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. Results While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2, only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2–4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated

  17. Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening.

    Science.gov (United States)

    Greenhough, Sebastian; Medine, Claire N; Hay, David C

    2010-12-30

    Despite considerable progress in modelling human liver toxicity, the requirement still exists for efficient, predictive and cost effective in vitro models to reduce attrition during drug development. Thousands of compounds fail in this process, with hepatotoxicity being one of the significant causes of failure. The cost of clinical studies is substantial, therefore it is essential that toxicological screening is performed early on in the drug development process. Human hepatocytes represent the gold standard model for evaluating drug toxicity, but are a limited resource. Current alternative models are based on immortalised cell lines and animal tissue, but these are limited by poor function, exhibit species variability and show instability in culture. Pluripotent stem cells are an attractive alternative as they are capable of self-renewal and differentiation to all three germ layers, and thereby represent a potentially inexhaustible source of somatic cells. The differentiation of human embryonic stem cells and induced pluripotent stem cells to functional hepatocyte like cells has recently been reported. Further development of this technology could lead to the scalable production of hepatocyte like cells for liver toxicity screening and clinical therapies. Additionally, induced pluripotent stem cell derived hepatocyte like cells may permit in vitro modelling of gene polymorphisms and genetic diseases.

  18. Stem cell derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model

    Science.gov (United States)

    Donegan, Jennifer J.; Tyson, Jennifer A.; Branch, Sarah Y.; Beckstead, Michael J.; Anderson, Stewart A.; Lodge, Daniel J.

    2016-01-01

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol (MAM) rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity, and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia. PMID:27480492

  19. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Hiromasa [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Oki, Yoshinao [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Bono, Hidemasa [Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Kano, Koichiro, E-mail: kkano@brs.nihon-u.ac.jp [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan)

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  20. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes.

    Science.gov (United States)

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  1. Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

    Science.gov (United States)

    Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

    2012-01-01

    Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

  2. Effect of endothelial progenitor cells in neovascularization and their application in tumor therapy

    Institute of Scientific and Technical Information of China (English)

    DONG Fang; HA Xiao-qin

    2010-01-01

    Objective To review the effect of endothelial progenitor cells in neovascularization as well as their application to the therapy of tumors.Data sources The data used in this review were mainly from PubMed for relevant English language articles published from 1997 to 2009. The search term was "endothelial progenitor cells".Study selection Articles regarding the role of endothelial progenitor cells in neovascularization and their application to the therapy of tumors were selected.Results Endothelial progenitor cells isolated from bone marrow, umbilical cord blood and peripheral blood can proliferate, mobilize and differentiate into mature endothelial cells. Experiments suggest endothelial progenitor cells take part in forming the tumor vascular through a variety of mechanisms related to vascular endothelial growth factor, matrix metalloproteinases, chemokine stromal cell-derived factor 1 and its receptor C-X-C receptor-4, erythropoietin, Notchsignal pathway and so on. Evidence demonstrates that the number and function change of endothelial progenitor cells in peripheral blood can be used as a biomarker of the response of cancer patients to anti-tumor therapy and predict the prognosis and recurrence. In addition, irradiation temporarily increased endothelial cells number and decreased the endothelial progenitor cell counts in animal models. Meanwhile, in preclinical experiments, therapeutic gene-modified endothelial progenitor cells have been approved to attenuate tumor growth and offer a novel strategy for cell therapy and gene therapy of cancer.Conclusions Endothelial progenitor cells play a particular role in neovascularization and have attractively potential prognostic and therapeutic applications to malignant tumors. However, a series of problems, such as the definitive biomarkers of endothelial progenitor cells, their interrelationship with radiotherapy and their application in cell therapy and gene therapy of tumors, need further investigation.

  3. The perivascular phenotype and behaviors of dedifferentiated cells derived from human mature adipocytes.

    Science.gov (United States)

    Song, Ning; Kou, Liang; Lu, Xiao-Wen; Sugawara, Atsunori; Shimizu, Yutaka; Wu, Min-Ke; Du, Li; Wang, Hang; Sato, Soh; Shen, Jie-Fei

    2015-02-13

    Derived from mature adipocytes, dedifferentiated fat (DFAT) cells represent a special group of multipotent cells. However, their phenotype and cellular nature remain unclear. Our study found that human DFAT cells adopted perivascular characteristics and behaviors. Flow cytometry and immunofluorescent staining revealed that human DFAT cells positively expressed markers highly related to perivascular cell lineages, such as CD140b, NG2 and desmin, but were negative for common endothelial markers, including CD31, CD34, and CD309. Furthermore, DFAT cells displayed vascular network formation ability in Matrigel, and they noticeably promoted and stabilized the vessel structures formed by human umbilical vascular endothelial cells (HUVECs) in vitro. These results provide novel evidence on the pericyte nature of human DFAT cells, further supporting the recent model for the perivascular origin of adult stem cells, in which tissue-specific progenitor cells in mesenchymal tissues associate with blood vessels, exhibiting perivascular characteristics and functions.

  4. Differences in Cell Activation by Chlamydophila pneumoniae and Chlamydia trachomatis Infection in Human Endothelial Cells

    Science.gov (United States)

    Krüll, M.; Kramp, J.; Petrov, T.; Klucken, A. C.; Hocke, A. C.; Walter, C.; Schmeck, B.; Seybold, J.; Maass, M.; Ludwig, S.; Kuipers, Jens G.; Suttorp, N.; Hippenstiel, S.

    2004-01-01

    Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis. PMID:15501794

  5. Tropism mechanism of stem cells targeting injured brain tissues by stromal cell-derived factor-1

    Institute of Scientific and Technical Information of China (English)

    ZHANG Sai; LIU Xiao-zhi; LIU Zhen-lin; SHANG Chong-zhi; HU Qun-liang

    2009-01-01

    Objective: To explore the role and function of stromal cell-derived factor- 1 (SDF- 1) in stem cells migrating into injured brain area.Methods: Rat-derived nerve stem cells (NSCs) were isolated and cultured routinely. Transwell system was used to observe the migration ability of NSCs into injured nerve cells. Immunocytochemistry was used to explore the expression of chemotactic factor receptor-4 (CXCR-4) in NSCs. In vivo, we applied immunofluorescence technique to observe the migration of NSCs into injured brain area. Immunofluorescence technique and Western blotting were used to test expression level of SDF- 1. After AMD3100 (a special chemical blocker) blocking CXCR-4, the migration ability of NSCs was tested in vivo and in vitro, respectively.Results: NSCs displayed specific tropism for injured nerve cells or traumatic brain area in vivo and in vitro. The expression level of SDF-1 in traumatic brain area increased remarkably and the expression level of CXCR-4 in the NSCs increased simultaneously. After AMD3100 blocking the expression of CXCR-4, the migration ability of NSCs decreased significantly both in vivo and in vitro.Conclusions: SDF-1 may play a key role in stem cells migrating into injured brain area through specially combining with CXCR-4.

  6. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    Science.gov (United States)

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

  7. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

    Directory of Open Access Journals (Sweden)

    Takuya Matsumoto

    2016-03-01

    Full Text Available Modeling of neurological diseases using induced pluripotent stem cells (iPSCs derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs. We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases.

  8. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    OpenAIRE

    LIANG, YIN-KU; Ping, Wei; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with t...

  9. Chemotherapy and anti-angiogenic drugs affect composition and coagulant phenotype of cell-derived vesicles in cancer patients

    NARCIS (Netherlands)

    Kleinjan, A.; Verhoeff, J.; Berckmans, R.; Kunst, P.; Van Doormaal, F.; Di Nisio, M.; Richel, D.; Kamphuisen, P.W.; Büller, H.R.; Nieuwland, R.

    2013-01-01

    Background: The relationship between chemotherapy and circulating microparticles in patients with cancer is complex. First, release of cancer cell-derived microparticles may contribute to resistance of cancer cells to chemotherapy. Second, chemotherapy and angiogenesis inhibiting agents promote a pr

  10. Function of cancer cell-derived extracellular matrix in tumor progression

    Institute of Scientific and Technical Information of China (English)

    Gao-Feng Xiong; Ren Xu

    2016-01-01

    Extracellular matrix (ECM) is an essential component of the tumor microenvironment. Cancer development and progression are associated with increased ECM deposition and crosslink. The chemical and physical signals elicited from ECM are necessary for cancer cell proliferation and invasion. It is well recognized that stromal cells are a major source of ECM proteins. However, recent studies showed that cancer cells are also an active and important component in ECM remodeling. Cancer cells deposit a signiifcant amount of collagen, ifbronectin, and tenascin C (TNC). Recent studies demonstrate that these cancer cell-derived ECM proteins enhance cancer cell survival and promote cancer cell colonization at distant sites. ECM-related enzymes and chaperone proteins, such as prolyl-4-hydroxylase, lysyl-hydroxylase, lysyl oxidase, and heat shock protein 47, are also highly expressed in cancer cells. Inhibition of these enzymes signiifcantly reduces cancer growth, invasion, and metastasis. These factors suggest that the cancer cell-derived ECM is crucial for cancer progression and metastasis. Therefore, targeting these ECM proteins and ECM-related enzymes is a potential strategy for cancer treatment.

  11. Characterization of Adherent Nonhematopoietic Cells Derived from Human Umbilical Cord Blood

    Institute of Scientific and Technical Information of China (English)

    安小惠; 蔡国平

    2003-01-01

    To confirm and characterize the adherent fibroblast-like progenitors in human umbilical cord blood, we isolated mononuclear cells from human umbilical cord blood by Ficoll-Hypaque.Two main morphologically different kinds of cells were formed by culturing the cells in collagen-coated 24-well plastic dishes and flasks.One type was the adherent fibroblast-like cells, while the other was loosely adherent clonally expanded round cells.Our experiments demonstrate that the adherent fibroblast-like cells possess multilineage potential, including the ability to differentiate into endothelial-like cells and to express the mesenchymal cell marker.

  12. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    Science.gov (United States)

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  13. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jan Pavel Kucera

    2015-09-01

    Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  14. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  15. Producing fully ES cell-derived mice from eight-cell stage embryo injections.

    Science.gov (United States)

    DeChiara, Thomas M; Poueymirou, William T; Auerbach, Wojtek; Frendewey, David; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT). To skip the chimera stage, we developed the VelociMouse method, in which injection of genetically modified ES cells into eight-cell embryos followed by maturation to the blastocyst stage and transfer to a surrogate mother produces F0 generation mice that are fully derived from the injected ES cells and exhibit a 100% GLT efficiency. The method is simple and flexible. Both male and female ES cells can be introduced into the eight-cell embryo by any method of injection or aggregation and using all ES cell and host embryo combinations from inbred, hybrid, and outbred genetic backgrounds. The VelociMouse method provides several unique opportunities for shortening project timelines and reducing mouse husbandry costs. First, as VelociMice exhibit 100% GLT, there is no need to test cross chimeras to establish GLT. Second, because the VelociMouse method permits efficient production of ES cell-derived mice from female ES cells, XO ES cell subclones, identified by screening for spontaneous loss of the Y chromosome, can be used to generate F0 females that can be bred with isogenic F0 males derived from the original targeted ES cell clone to obtain homozygous mutant mice in the F1 generation. Third, as VelociMice are genetically identical to the ES cells from which they were derived, the VelociMouse method opens up myriad possibilities for creating mice with

  16. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    Science.gov (United States)

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

  17. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

    Science.gov (United States)

    Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

    2017-02-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

  18. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice

    Science.gov (United States)

    Boylan, Brendan T.; Moreira, Fernando R.; Carlson, Tim W.

    2017-01-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses. PMID:28187142

  19. Exosomes Secreted from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Prevent Osteonecrosis of the Femoral Head by Promoting Angiogenesis

    Science.gov (United States)

    Liu, Xiaolin; Li, Qing; Niu, Xin; Hu, Bin; Chen, Shengbao; Song, Wenqi; Ding, Jian; Zhang, Changqing; Wang, Yang

    2017-01-01

    Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues, but whether they could promote angiogenesis in ONFH has not been reported, and little is known regarding the underlying mechanism. Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining, micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation, migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt. Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss, and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation, migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover, the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells. Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on

  20. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Directory of Open Access Journals (Sweden)

    Laura Camacho

    Full Text Available Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM versus non-brain metastatic (non-BM cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210 and two down-regulated miRNAs (miR-19a and miR-29c in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  1. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Science.gov (United States)

    Camacho, Laura; Guerrero, Paola; Marchetti, Dario

    2013-01-01

    Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM) versus non-brain metastatic (non-BM) cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM) variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210) and two down-regulated miRNAs (miR-19a and miR-29c) in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  2. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.

    Science.gov (United States)

    Lippmann, Ethan S; Azarin, Samira M; Kay, Jennifer E; Nessler, Randy A; Wilson, Hannah K; Al-Ahmad, Abraham; Palecek, Sean P; Shusta, Eric V

    2012-08-01

    The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.

  3. A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

    Directory of Open Access Journals (Sweden)

    Stephen W Waldo

    Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

  4. A Novel Minimally-Invasive Method to Sample Human Endothelial Cells for Molecular Profiling

    Science.gov (United States)

    Waldo, Stephen W.; Brenner, Daniel A.; McCabe, James M.; Dela Cruz, Mark; Long, Brian; Narla, Venkata A.; Park, Joseph; Kulkarni, Ameya; Sinclair, Elizabeth; Chan, Stephen Y.; Schick, Suzaynn F.; Malik, Namita; Ganz, Peter; Hsue, Priscilla Y.

    2015-01-01

    Objective The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity. Methods Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34 / CD105 / CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b / CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR). Results A median of 4,212 (IQR: 2161 – 6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001). Conclusion This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets. PMID:25679506

  5. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    Science.gov (United States)

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

  6. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    Science.gov (United States)

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-07

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  7. Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

    Science.gov (United States)

    Cusella-De Angelis, Maria Gabriella; Laino, Gregorio; Piattelli, Adriano; Pacifici, Maurizio; De Rosa, Alfredo; Papaccio, Gianpaolo

    2007-01-01

    Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and

  8. Myeloid and T Cell-Derived TNF Protects against Central Nervous System Tuberculosis

    Science.gov (United States)

    Hsu, Nai-Jen; Francisco, Ngiambudulu M.; Keeton, Roanne; Allie, Nasiema; Quesniaux, Valérie F. J.; Ryffel, Bernhard; Jacobs, Muazzam

    2017-01-01

    Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF−/−) to assess the contribution of myeloid and T cell-derived TNF in immune responses during CNS-TB. These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF−/− mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection. PMID:28280495

  9. Genetic strategies to investigate neuronal circuit properties using stem cell-derived neurons

    Directory of Open Access Journals (Sweden)

    Isabella eGarcia

    2012-12-01

    Full Text Available The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC and induced pluripotent stem cell (iPSC technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs towards investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches.

  10. Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

    Science.gov (United States)

    Muppidi, Jagan R; Schmitz, Roland; Green, Jesse A; Xiao, Wenming; Larsen, Adrien B; Braun, Sterling E; An, Jinping; Xu, Ying; Rosenwald, Andreas; Ott, German; Gascoyne, Randy D; Rimsza, Lisa M; Campo, Elias; Jaffe, Elaine S; Delabie, Jan; Smeland, Erlend B; Braziel, Rita M; Tubbs, Raymond R; Cook, J R; Weisenburger, Dennis D; Chan, Wing C; Vaidehi, Nagarajan; Staudt, Louis M; Cyster, Jason G

    2014-12-11

    Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

  11. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jason P. Weick

    2016-01-01

    Full Text Available Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.

  12. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

    Directory of Open Access Journals (Sweden)

    Hiroki eSakai

    2013-09-01

    Full Text Available AbstractIn Bombyx mori, polar body nuclei are observed until 9h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe. The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/ +pe of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/ +pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells. Analyses of serosal cells pigmentation indicated that approximately 30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26 % of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25. Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

  13. Stimulation of human embryonic stem cell-derived cardiomyocytes on thin-film microelectrodes.

    Science.gov (United States)

    Viitanen, Jouko; Heimala, Päivi; Hokkanen, Ari; Iljin, Kristiina; Kerkelä, Erja; Kolari, Kai; Kattelus, Hannu

    2011-05-01

    We describe successful long-term stimulation of human embryonic stem cell-derived cardiomyocyte clusters on thin-film microelectrode structures in vitro. Interdigitated electrode structures were constructed using plain titanium on glass as the electrode material. Titanium rapidly oxidizes in atmospheric conditions to produce an insulating TiO(χ) layer with high relative permittivity. Capacitive coupling to the incubation medium and to the cells adherent to the electrodes was still efficient, and the dielectric layer prevented electrolysis, allowing a wider window of possible stimulation amplitudes to be used, relative to conducting surfaces. A common hypothesis suggests that to achieve proper differentiation of electroactive cells from the stem cells electrical stimuli are also needed. Spontaneously beating cardiomyocyte clusters were seeded on the glass-electrode surfaces, and we successfully altered and resynchronized a clearly different beat interval. The new pace was reliably maintained for extended periods of several tens of minutes.

  14. Microvascular endothelial cells of the corpus luteum

    Directory of Open Access Journals (Sweden)

    Spanel-Borowski Katherina

    2003-11-01

    Full Text Available Abstract The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of

  15. Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations.

    Science.gov (United States)

    Farra, N; Zhang, W-B; Pasceri, P; Eubanks, J H; Salter, M W; Ellis, J

    2012-12-01

    Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Here, we describe the first characterization and neuronal differentiation of induced pluripotent stem (iPS) cells derived from Mecp2-deficient mice. Fully reprogrammed wild-type (WT) and heterozygous female iPS cells express endogenous pluripotency markers, reactivate the X-chromosome and differentiate into the three germ layers. We directed iPS cells to produce glutamatergic neurons, which generated action potentials and formed functional excitatory synapses. iPS cell-derived neurons from heterozygous Mecp2(308) mice showed defects in the generation of evoked action potentials and glutamatergic synaptic transmission, as previously reported in brain slices. Further, we examined electrophysiology features not yet studied with the RTT iPS cell system and discovered that MeCP2-deficient neurons fired fewer action potentials, and displayed decreased action potential amplitude, diminished peak inward currents and higher input resistance relative to WT iPS-derived neurons. Deficiencies in action potential firing and inward currents suggest that disturbed Na(+) channel function may contribute to the dysfunctional RTT neuronal network. These phenotypes were additionally confirmed in neurons derived from independent WT and hemizygous mutant iPS cell lines, indicating that these reproducible deficits are attributable to MeCP2 deficiency. Taken together, these results demonstrate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in primary neurons, and these identified phenotypes further illustrate the requirement of MeCP2 in neuronal development and/or in the maintenance of normal function. By validating the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, we identify deficiencies that can be targeted for in vitro translational screens.

  16. Effect of purine alkaloids on the proliferation of lettuce cells derived from protoplasts.

    Science.gov (United States)

    Sasamoto, Hamako; Fujii, Yoshiharu; Ashihara, Hiroshi

    2015-05-01

    To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

  17. NF-κB Regulates B-Cell-Derived Nerve Growth Factor Expression

    Institute of Scientific and Technical Information of China (English)

    Klaus Heese; Noriko Inoue; Tohru Sawada

    2006-01-01

    In the mammalian brain, four neurotrophins have been identified: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). NGF exerts an important role in the development and functions of the central and peripheral nervous system. However, it has recently been documented that several types of immune cells, such as mast cells, lymphocytes, basophils and eosinophils, produce,store and release NGF. Accumulating preclinical and clinical data indicate that dysfunctions of NGF and the other neurotrophins may contribute to impaired immune responses and concentration of NGF frequently correlates with disease severity. Thus, the aim of this study was to elucidate the potential signaling mechanisms of cytokineneurotrophins interactions contributing to increased NGF levels. Our data show that the transcription factorNF-κB plays a pivotal role in regulating B-cell-derived NGF expression.

  18. A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

    Science.gov (United States)

    Wiley, Luke A; Beebe, David C; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-05-12

    This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

  19. clickECM: Development of a cell-derived extracellular matrix with azide functionalities.

    Science.gov (United States)

    Ruff, S M; Keller, S; Wieland, D E; Wittmann, V; Tovar, G E M; Bach, M; Kluger, P J

    2016-12-10

    In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM.

  20. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    Science.gov (United States)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  1. Homing of circulating blood endothelial progenitor cells after myocardial infarction is mediated by Akt-SDF-1-signal pathway

    Institute of Scientific and Technical Information of China (English)

    赵岚

    2013-01-01

    Objective To investigate the expressions of protein kinase B(Akt) and stromal cell-derived factor-1(SDF-1) and their relations with circulating blood endothelial progenitor cell homing after myocardial infarction(MI). Methods MI was induced in the

  2. A cloned toy poodle produced from somatic cells derived from an aged female dog.

    Science.gov (United States)

    Jang, G; Hong, S G; Oh, H J; Kim, M K; Park, J E; Kim, H J; Kim, D Y; Lee, B C

    2008-03-15

    To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.

  3. Cross-Talk between CLL Cells and Bone Marrow Endothelial Cells: Role of Signal Transducer and Activator of Transcription-3

    Science.gov (United States)

    Badoux, Xavier; Bueso-Ramos, Carlos; Harris, David; Li, Ping; Liu, Zhiming; Burger, Jan; O’Brien, Susan; Ferrajoli, Alessandra; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Summary Chronic lymphocytic leukemia (CLL) bone marrow is characterized by increased angiogenesis. However, the molecular mediators of neovascularization and the biological significance of increased endothelial cell proliferation in CLL require further investigation. Because signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL we studied the role of STAT3 in modulating vascular endothelial growth factor (VEGF) expression and the effect of vascular endothelial cells on CLL cells. Using chromatin immunoprecipitation (ChIP) we found that anti-STAT3 antibodies immunoprecipitated DNA of STAT3, VEGF and other STAT3-regulated genes. In addition, STAT3-short interfering RNA significantly reduced mRNA levels of VEGF in CLL cells suggesting that STAT3 induces VEGF expression in CLL. Remarkably, bone marrow CLL cells expressed high levels of VEGF and high VEGF levels were detected in the plasma of patients with untreated CLL and correlated with white blood cell count. CLL bone marrow biopsies revealed increased microvascular density and attachment of CLL cells to endothelial cells. Co-culture of CLL and human umbilical vein endothelial cells (HUVEC) cells showed a similar attachment. Furthermore, co-culture studies with HUVEC showed that HUVEC protected CLL cells from spontaneous apoptosis by direct cell-to-cell contact as assessed by flow cytometry using Annexin V. Our data suggest that constitutively activated STAT3 induces VEGF production by CLL cells and CLL cells derive a survival advantage from endothelial cells via cell-to cell contact. PMID:21733558

  4. Cryopreservation of Human Pluripotent Stem Cell-derived Cardiomyocytes: Strategies, Challenges, and Future Directions

    Science.gov (United States)

    Preininger, Marcela K.; Singh, Monalisa; Xu, Chunhui

    2017-01-01

    In recent years, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a vital cell source for in vitro modeling of genetic cardiovascular disorders, drug screening, and in vivo cardiac regeneration research. Looking forward, the ability to efficiently cryopreserve hPSC-CMs without compromising their normal biochemical and physiologic functions will dramatically facilitate their various biomedical applications. Although working protocols for freezing, storing, and thawing hPSC-CMs have been established, the question remains as to whether they are optimal. In this chapter, we discuss our current understanding of cryopreservation appertaining to hPSC-CMs, and proffer key questions regarding the mechanical, contractile, and regenerative properties of cryopreserved hPSC-CMs. PMID:27837559

  5. Electrophysiological properties and calcium handling of embryonic stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jae Boum Youm

    2016-03-01

    Full Text Available Embryonic stem cell-derived cardiomyocytes (ESC-CMs hold great interest in many fields of research including clinical applications such as stem cell and gene therapy for cardiac repair or regeneration. ESC-CMs are also used as a platform tool for pharmacological tests or for investigations of cardiac remodeling. ESC-CMs have many different aspects of morphology, electrophysiology, calcium handling, and bioenergetics compared with adult cardiomyocytes. They are immature in morphology, similar to sinus nodal-like in the electrophysiology, higher contribution of trans-sarcolemmal Ca2+ influx to Ca2+ handling, and higher dependence on anaerobic glycolysis. Here, I review a detailed electrophysiology and Ca2+ handling features of ESC-CMs during differentiation into adult cardiomyocytes to gain insights into how all the developmental changes are related to each other to display cardinal features of developing cardiomyocytes.

  6. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    Directory of Open Access Journals (Sweden)

    Daniel R. Bayzigitov

    2016-01-01

    Full Text Available Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

  7. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    Science.gov (United States)

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

  8. Generation of Neural Crest-Like Cells From Human Periodontal Ligament Cell-Derived Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Tomokiyo, Atsushi; Hynes, Kim; Ng, Jia; Menicanin, Danijela; Camp, Esther; Arthur, Agnes; Gronthos, Stan; Mark Bartold, Peter

    2017-02-01

    Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC- and FF iPSC-derived NCLC, which highly expressed HNK1. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly, the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC. J. Cell. Physiol. 232: 402-416, 2017. © 2016 Wiley Periodicals, Inc.

  9. Target-cell-derived tRNA-like primers for reverse transcription support retroviral infection at low efficiency

    DEFF Research Database (Denmark)

    Schmitz, Alexander; Lund, Anders H; Hansen, Anette C;

    2002-01-01

    by cases of correction of single mismatches between Akv-MLV vectors and complementary tRNA primers toward the primer sequence in the integrated vector. Thus, target-cell-derived tRNA-like primers are able to initiate first-strand cDNA synthesis and plus-strand transfer leading to a complete provirus......Reverse transcription of a retroviral genome takes place in the cytoplasm of an infected cell by a process primed by a producer-cell-derived tRNA annealed to an 18-nucleotide primer-binding site (PBS). By an assay involving primer complementation of PBS-mutated vectors we analyzed whether tRNA...... primers derived from the target cell can sustain reverse transcription during murine leukemia virus (MLV) infection. Transduction efficiencies were 4-5 orders of magnitude below those of comparable producer-cell complementations. However, successful usage of a target-cell-derived tRNA primer was proven...

  10. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

    Directory of Open Access Journals (Sweden)

    Rebecca Josowitz

    Full Text Available The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN. When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

  11. Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

    Science.gov (United States)

    Rajamohan, Divya; Kalra, Spandan; Duc Hoang, Minh; George, Vinoj; Staniforth, Andrew; Russell, Hugh; Yang, Xuebin

    2016-01-01

    Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose–response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation. PMID:26906236

  12. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    Science.gov (United States)

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  13. Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche

    Science.gov (United States)

    Luarte, Alejandro; Bátiz, Luis Federico; Wyneken, Ursula; Lafourcade, Carlos

    2016-01-01

    Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future. PMID:27195011

  14. Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche

    Directory of Open Access Journals (Sweden)

    Alejandro Luarte

    2016-01-01

    Full Text Available Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future.

  15. Myocardial regeneration strategies using human embryonic stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Capi, Oren; Gepstein, Lior

    2006-11-28

    Regenerative medicine is a new biomedicine discipline that takes advantage of the recent advancements in the fields of stem cell biology, molecular biology, and tissue engineering to derive tissue substitutes, in an attempt to replace or modify the function of diseased organs. The heart represents an attractive candidate for these emerging technologies since adult cardiac tissue has limited regenerative capacity. Consequentially, myocardial cell replacement therapy has emerged as a novel therapeutic paradigm for restoration of the myocardial electromechanical function. This innovative strategy has been significantly hampered, however, by the paucity of cell sources for human cardiomyocytes. The recent establishment of the human embryonic stem cell (hESC) lines may provide a possible solution for this cell-sourcing problem. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. This review will describe the hESC system, their differentiation into cardiomyocytes, and the structural and functional characterization of these cardiac lineage derivatives. The potential applications of this unique differentiating system in several research areas will be discussed with special emphasis on the steps required to fully harness their unique potential in the emerging field of cardiovascular regenerative medicine.

  16. PROPOSED CARDIAC STEM CELLS DERIVED FROM “CARDIOSPHERES” LACK CARDIOMYOGENIC POTENTIAL

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline

       Recent studies have reported that clinical relevant numbers of cardiac stem cells (CSCs) with cardiomyogenic potential can be obtained from small heart tissue biopsies, by an intrinsic ability of CSCs to form beating cardiospheres (CSs) during ex vivo culture. Such data have provided optimism...... that injuried heart tissue may be repaired by stem cell therapy using autologous CS derived cells, and pre-clinical studies have already been described in literature.    Herein, we established CSs from neonatal rats, and by immunofluorescence, qRT-PCR, and microscopic examination we demonstrated...... to form CSs by themselves. Phenotypically, CS cells largely resembled fibroblasts, and they lacked cardiomyogenic as well as endothelial differentiation potential.    Our data imply that at least the murine cardiosphere model seems unsuitable for enrichment of cardiac stem cells with cardiomyogenic...

  17. Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo.

    Science.gov (United States)

    Sritanaudomchai, Hathaitip; Pavasuthipaisit, Kanok; Kitiyanant, Yindee; Kupradinun, Piengchai; Mitalipov, Shoukhrat; Kusamran, Thanit

    2007-10-01

    Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, alpha-skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and alpha-fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes.

  18. Stromal Cell-Derived Growth Factor-1 Alpha-Elastin Like Peptide Fusion Protein Promotes Cell Migration and Revascularization of Experimental Wounds in Diabetic Mice

    Science.gov (United States)

    Yeboah, Agnes; Maguire, Tim; Schloss, Rene; Berthiaume, Francois; Yarmush, Martin L.

    2017-01-01

    Objective: In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 × 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro. Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro. The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro. SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo, SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds. PMID:28116224

  19. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model?

    Science.gov (United States)

    Knollmann, Björn C

    2013-03-15

    This article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize antiarrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders, such as long QT syndrome, Brugada Syndrome, or Catecholaminergic Polymorphic Ventricular Tachycardia.

  20. Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease

    Science.gov (United States)

    Suzuki, Etsu; Fujita, Daishi; Takahashi, Masao; Oba, Shigeyoshi; Nishimatsu, Hiroaki

    2016-01-01

    Mesenchymal stem cells (MSCs) have been used to treat patients suffering from acute myocardial infarction (AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microRNAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease (CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD. PMID:27679686

  1. Characterization of inflammatory markers and transcriptome profiles of differentially activated embryonic stem cell-derived microglia.

    Science.gov (United States)

    Beins, Eva; Ulas, Thomas; Ternes, Svenja; Neumann, Harald; Schultze, Joachim L; Zimmer, Andreas

    2016-06-01

    Microglia, the immune cells of the CNS, are highly adaptive cells that can acquire different pro- and anti-inflammatory activation states with distinct functions in CNS homeostasis and pathologies. To study microglial function in vitro, primary microglia or immortalized cell lines are commonly used. An alternative to these cells are embryonic stem cell-derived microglia (ESdM). ESdM have previously been shown to be very similar to primary microglia in terms of expression profiles and surface molecules. In this study, ESdM and primary microglia were treated with different inflammatory stimulants to analyze their ability to adopt different activation states. Using quantitative real-time PCR, comparative transcriptomics, ELISA, and flow cytometry, we found that different activation states can be induced in ESdM, which are similar to those found in primary microglia. These states are characterized by specific sets of inflammatory marker molecules and differential transcriptome signatures. Our results show that ESdM are a valuable alternative cell model to study microglial functions and neuroinflammatory mechanisms.

  2. Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation.

    Science.gov (United States)

    Higashino, Kosaku; Viggeswarapu, Manjula; Bargouti, Maggie; Liu, Hui; Titus, Louisa; Boden, Scott D

    2011-02-01

    The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.

  3. Endothelial progenitor cells in cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    Poay; Sian; Sabrina; Lee; Kian; Keong; Poh

    2014-01-01

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells(EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vas-culogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk fac-tors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardio-vascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evalu-ate the challenges facing EPC research and how these may be overcome.

  4. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  5. Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development:do all roads lead to Mitf?

    Institute of Scientific and Technical Information of China (English)

    Ling Hou; William J Pavan

    2008-01-01

    Human neurocristopathies include a number of syndromes,tumors,and dysmorphologies of neural crest (NC) stem cell derivatives.In recent years,many white spotting genes have been associated with hypopigmentary disorders and deafness in neurocristopathies resulting from NC stem cell-derived melanocyte deficiency during development.These include PAX3,SOX10,MITF,SNAI2,EDNRB,EDN3,KIT,and KITL.Recent studies have revealed surprising new insights into a central role of MITF in the complex network of interacting genes in melanocyte development.In this perspective,we provide an overview of some of the current findings and explore complex functional roles of these genes during NC stem cell-derived melanocyte development.

  6. Modulating Innate Immunity Improves Hepatitis C Virus Infection and Replication in Stem Cell-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Xiaoling Zhou

    2014-07-01

    Full Text Available In this study, human embryonic stem cell-derived hepatocytes (hESC-Heps were investigated for their ability to support hepatitis C virus (HCV infection and replication. hESC-Heps were capable of supporting the full viral life cycle, including the release of infectious virions. Although supportive, hESC-Hep viral infection levels were not as great as those observed in Huh7 cells. We reasoned that innate immune responses in hESC-Heps may lead to the low level of infection and replication. Upon further investigation, we identified a strong type III interferon response in hESC-Heps that was triggered by HCV. Interestingly, specific inhibition of the JAK/STAT signaling pathway led to an increase in HCV infection and replication in hESC-Heps. Of note, the interferon response was not evident in Huh7 cells. In summary, we have established a robust cell-based system that allows the in-depth study of virus-host interactions in vitro.

  7. Embryonic stem cell-derived M2-like macrophages delay cutaneous wound healing.

    Science.gov (United States)

    Dreymueller, Daniela; Denecke, Bernd; Ludwig, Andreas; Jahnen-Dechent, Willi

    2013-01-01

    In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar-free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2-like macrophages. Embryonic/fetal macrophages are M2-like, and this may promote scar-free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell-derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow-derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2-like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll-like receptors, and reduced bacterial phagocytosis. Despite this anti-inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell-free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds.

  8. Importance of being Nernst: Synaptic activity andfunctional relevance in stem cell-derived neurons

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Functional synaptogenesis and network emergence aresignature endpoints of neurogenesis. These behaviorsprovide higher-order confirmation that biochemicaland cellular processes necessary for neurotransmitterrelease, post-synaptic detection and network propagation of neuronal activity have been properly expressed andcoordinated among cells. The development of synapticneurotransmission can therefore be considered a definingproperty of neurons. Although dissociated primaryneuron cultures readily form functioning synapsesand network behaviors in vitro , continuously culturedneurogenic cell lines have historically failed to meet thesecriteria. Therefore, in vitro -derived neuron models thatdevelop synaptic transmission are critically needed for awide array of studies, including molecular neuroscience,developmental neurogenesis, disease research andneurotoxicology. Over the last decade, neurons derivedfrom various stem cell lines have shown varying ability todevelop into functionally mature neurons. In this review,we will discuss the neurogenic potential of various stemcells populations, addressing strengths and weaknessesof each, with particular attention to the emergenceof functional behaviors. We will propose methods tofunctionally characterize new stem cell-derived neuron(SCN) platforms to improve their reliability as physiologicalrelevant models. Finally, we will review howsynaptically active SCNs can be applied to accelerateresearch in a variety of areas. Ultimately, emphasizingthe critical importance of synaptic activity and networkresponses as a marker of neuronal maturation is anticipatedto result in in vitro findings that better translateto efficacious clinical treatments.

  9. Human embryonic stem cell-derived mesenchymal stem cells as cellular delivery vehicles for prodrug gene therapy of glioblastoma.

    Science.gov (United States)

    Bak, Xiao Ying; Lam, Dang Hoang; Yang, Jingye; Ye, Kai; Wei, Esther Lee Xing; Lim, Sai Kiang; Wang, Shu

    2011-11-01

    Mesenchymal stem cells (MSCs) possess tumor-tropic properties and consequently have been used to deliver therapeutic agents for cancer treatment. Their potential in cancer therapy highlights the need for a consistent and renewable source for the production of uniform human MSCs suitable for clinical applications. In this study, we seek to investigate whether human embryonic stem cells can be used as a cell source to fulfill this goal. We generated MSC-like cells from two human embryonic stem cell lines, HuES9 and H1, and observed that MSC-like cells derived from human embryonic stem cells were able to migrate into human glioma intracranial xenografts after being injected into the cerebral hemisphere contralateral to the tumor inoculation site. We engineered these cells with baculoviral and lentiviral vectors, respectively, for transient and stable expression of the herpes simplex virus thymidine kinase gene. In tumor-bearing mice the engineered MSC-like cells were capable of inhibiting tumor growth and prolonging survival in the presence of ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere. Our findings suggest that human embryonic stem cell-derived MSCs may be a viable and attractive alternative for large-scale derivation of targeting vehicles for cancer therapy.

  10. Stromal cell-derived factor-1α promotes angiogenesis in the peri-infarct region in adults with cerebral infarction

    Institute of Scientific and Technical Information of China (English)

    凌莉

    2014-01-01

    Objective To investigate the possible effects of exogenous stromal cell-derived factor-1α(SDF-1α)on cell proliferation and angiogenesis in the ipsilateral thalamic ventroposterior nucleus(VPN)in adult rats with focal cortical infarction.Methods Thirty-six hypertensive rats with focal cortical infarction were divided randomly into the SDF-1αgroup,vehicle

  11. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    NARCIS (Netherlands)

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J.

    2015-01-01

    The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) th

  12. Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras.

    Science.gov (United States)

    Karagenç, Levent; Sandikci, Mustafa

    2010-01-01

    The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-mum intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm.

  13. Heparanase regulates secretion, composition, and function of tumor cell-derived exosomes.

    Science.gov (United States)

    Thompson, Camilla A; Purushothaman, Anurag; Ramani, Vishnu C; Vlodavsky, Israel; Sanderson, Ralph D

    2013-04-05

    Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.

  14. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, J.D., E-mail: jennifer.cohen@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Babiarz, J.E., E-mail: joshua.babiarz@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Abrams, R.M., E-mail: rory.abrams@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Guo, L., E-mail: liang.guo@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Kameoka, S., E-mail: sei.kameoka@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Chiao, E., E-mail: eric.chiao@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Taunton, J., E-mail: taunton@cmp.ucsf.edu [Howard Hughes Medical Institute, Cellular and Molecular Pharmacology, University California San Francisco, San Francisco, CA 94158 (United States); Kolaja, K.L., E-mail: kyle.kolaja@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States)

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  15. Towards Personalized Regenerative Cell Therapy: Mesenchymal Stem Cells Derived from Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Lin, Lin; Bolund, Lars; Luo, Yonglun

    2016-01-01

    Mesenchymal stem cells (MSCs) are adult stem cells with the capacity of self-renewal and multilineage differentiation, and can be isolated from several adult tissues. However, isolating MSCs from adult tissues for cell therapy is hampered by the invasive procedure, the rarity of the cells and their attenuated proliferation capacity when cultivated and expanded in vitro. Human MSCs derived from induced pluripotent stem cells (iPSC-MSCs) have now evolved as a promising alternative cell source for MSCs and regenerative medicine. Several groups, including ours, have reported successful derivation of functional iPSC-MSCs and applied these cells in MSC-based therapeutic testing. Still, the current experience and understanding of iPSC-MSCs with respect to production methods, safety and efficacy are primitive. In this review, we highlight the methodological progress in iPSC-MSC research, describing the importance of choosing the right sources of iPSCs, iPSC reprogramming methods, iPSC culture systems, embryoid body intermediates, pathway inhibitors, basal medium, serum, growth factors and culture surface coating. We also highlight some progress in the application of iPSC-MSCs in direct cell therapy, tissue engineering and gene therapy.

  16. A Non-invasive Platform for Functional Characterization of Stem-Cell-Derived Cardiomyocytes with Applications in Cardiotoxicity Testing

    Directory of Open Access Journals (Sweden)

    Mahnaz Maddah

    2015-04-01

    Full Text Available We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes.

  17. Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHEN XU; MIN XIONG SHEN; DONG ZHU MA; LI YING WANG; XI LIANG ZHA

    2003-01-01

    Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 × l06 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

  18. Process Extension from Embryonic Stem Cell-Derived Motor Neurons through Synthetic Extracellular Matrix Mimics

    Science.gov (United States)

    McKinnon, Daniel Devaud

    This thesis focuses on studying the extension of motor axons through synthetic poly(ethylene glycol) PEG hydrogels that have been modified with biochemical functionalities to render them more biologically relevant. Specifically, the research strategy is to encapsulate embryonic stem cell-derived motor neurons (ESMNs) in synthetic PEG hydrogels crosslinked through three different chemistries providing three mechanisms for dynamically tuning material properties. First, a covalently crosslinked, enzymatically degradable hydrogel is developed and exploited to study the biophysical dynamics of axon extension and matrix remodeling. It is demonstrated that dispersed motor neurons require a battery of adhesive peptides and growth factors to maintain viability and extend axons while those in contact with supportive neuroglial cells do not. Additionally, cell-degradable crosslinker peptides and a soft modulus mimicking that of the spinal cord are requirements for axon extension. However, because local degradation of the hydrogel results in a cellular environment significantly different than that of the bulk, enzymatically degradable peptide crosslinkers were replaced with reversible covalent hydrazone bonds to study the effect of hydrogel modulus on axon extension. This material is characterized in detail and used to measure forces involved in axon extension. Finally, a hydrogel with photocleavable linkers incorporated into the network structure is exploited to explore motor axon response to physical channels. This system is used to direct the growth of motor axons towards co-cultured myotubes, resulting in the formation of an in vitro neural circuit.

  19. Excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Christopher eKane

    2015-09-01

    Full Text Available Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation-contraction coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With calcium handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology.Here we discuss the implications of differences in excitation-contraction coupling when considering the potential applications of iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.

  20. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    Science.gov (United States)

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  1. Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

    Science.gov (United States)

    Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro

    2013-12-01

    Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies

  2. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    Science.gov (United States)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  3. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM deficient blastocysts.

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    Duancheng Wen

    Full Text Available Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs by injection of diploid (2n ESCs into tetraploid (4n blastocysts (ESC-derived mice. This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS cells. However, the underlying mechanism(s of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM. We designate these as type a (presence of ICM at blastocyst stage or type b (absence of ICM. ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  4. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts.

    Science.gov (United States)

    Wen, Duancheng; Saiz, Nestor; Rosenwaks, Zev; Hadjantonakis, Anna-Katerina; Rafii, Shahin

    2014-01-01

    Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  5. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    Science.gov (United States)

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  6. Good Cell Culture Practice for stem cells and stem-cell-derived models.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas

    2017-01-01

    The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

  7. Application of a Persistent Heparin Treatment Inhibits the Malignant Potential of Oral Squamous Carcinoma Cells Induced by Tumor Cell-Derived Exosomes.

    Science.gov (United States)

    Sento, Shinya; Sasabe, Eri; Yamamoto, Tetsuya

    2016-01-01

    Exosomes are 30-100 nm-sized membranous vesicles, secreted from a variety of cell types into their surrounding extracellular space. Various exosome components including lipids, proteins, and nucleic acids are transferred to recipient cells and affect their function and activity. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. However, the effect of exosomes released from oral squamous cell carcinoma (OSCC) into the tumor microenvironment remains unclear. In the present study, we isolated exosomes from OSCC cells and investigated the influence of OSCC cell-derived exosomes on the tumor cell behavior associated with tumor development. We demonstrated that OSCC cell-derived exosomes were taken up by OSCC cells themselves and significantly promoted proliferation, migration, and invasion through the activation of the PI3K/Akt, MAPK/ERK, and JNK-1/2 pathways in vitro. These effects of OSCC cell-derived exosomes were obviously attenuated by treatment with PI3K, ERK-1/2, and JNK-1/2 pharmacological inhibitors. Furthermore, the growth rate of tumor xenografts implanted into nude mice was promoted by treatment with OSCC cell-derived exosomes. The uptake of exosomes by OSCC cells and subsequent tumor progression was abrogated in the presence of heparin. Taken together, these data suggest that OSCC cell-derived exosomes might be a novel therapeutic target and the use of heparin to inhibit the uptake of OSCC-derived exosomes by OSCC cells may be useful for treatment.

  8. Stromal cell-derived factor-1/CXCL12 contributes to MMTV-Wnt1 tumor growth involving Gr1+CD11b+ cells.

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    Bob Y Liu

    Full Text Available BACKGROUND: Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12 but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1(+ myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1(+ cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1(+ cells. CONCLUSIONS/SIGNIFICANCE: We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1(+ myeloid cells to facilitate growth and/or angiogenesis.

  9. Degradation of amyloid beta by human induced pluripotent stem cell-derived macrophages expressing Neprilysin-2

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    Koutaro Takamatsu

    2014-11-01

    Full Text Available The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS cell-derived macrophage-like cells for Alzheimer's disease (AD. In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2, which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2 in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.

  10. The in Vitro Assessment of Biochemical Factors in Hepatocyte like Cells Derived from Umbilical Cord Blood Stem Cells

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    A KHoramroodi

    2009-10-01

    Full Text Available Introduction & Objective: Umbilical cord blood (UCB is a source of Hematopoietic Stem Cells (HSC and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders. Mesenchymal stem cell-derived from umbilical cord blood (UCB have been differentiated to some kind of cells, such as osteobblast, adipoblast and chondroblast in Vitro. This study examined the differentiation of Umbilical Cord Blood (UCB derived stem cells to functional hepatocytes. Materials & Methods: The present study was an experimental study which was carried out in the Payam-e-Noor University of Tehran in cooperation with Hamedan University of Medical Sciences in 2008. Umbilical cord blood (UCB was obtained from Fatemieh hospital (Hamadan, Iran. Stem cells were isolated from the cord blood by combining density gradient centrifugation with plastic adherence. When the isolated cells reached 80% confluence, they differentiated to hepatocyte like cells. The medium which was used was consists of DMEM and 10% Fetal Bovine Serum (FBS supplemented with 20 ng/mL Hepatocyte Growth Factor (HGF, 10 ng/mL basic Fibroblast Growth Factor (bFGF and 20 ng/mL Oncostatin M (OSM.The medium was changed every 3 days and stored for Albumin (ALB, Alpha Fetoprotein (AFP, Alkaline Phosphatase (ALP, and urea assay. Finally PAS stain was done to study Glycogen storage in the differentiated cell. Results: Measurement of biochemical factors in different days showed that concentration of albumin (ALB, alpha fetoprotein (AFP, alkaline phosphatase (ALP, and Urea gradually increased. Also, PAS staining showed the storage of glycogen in these cells. Conclusion: Stem cell-derived from human umbilical cord blood (HUCB is a new source of cell types for cell transplantation therapy of hepatic diseases and under certain conditions these cells can differentiate into liver cells.

  11. CHCHD2 is down-regulated in neuronal cells differentiated from iPS cells derived from patients with lissencephaly.

    Science.gov (United States)

    Shimojima, Keiko; Okumura, Akihisa; Hayashi, Masaharu; Kondo, Takayuki; Inoue, Haruhisa; Yamamoto, Toshiyuki

    2015-10-01

    The human cerebral cortex is peculiar for a six-layered cellular-sheet structure with convolution, which is a consequence of neuronal migration. Dysfunctions of the pathways contributing to this mechanism typically lead to lissencephaly manifesting smooth brain surfaces. To investigate the unknown mechanism underlying neuronal migration disorders, we generated induced pluripotent stem (iPS) cells from two patients with lissencephaly. Whole gene expression study for iPS cells derived from a patient with a LIS1 deletion showed reduced expression of the coiled-coil-helix-coiled-coil-helix domain containing 2 gene (CHCHD2), which was also confirmed in iPS cells derived from a patient with a TUBA1A mutation. CHCHD2 expression was detected in neuronal cells differentiated from normal iPS cells in a time-dependent manner, as well as in the brain of a fetus at 26-28 week gestational age, suggesting development-dependent expression. Migrating neuronal cells showed CHCHD2 expression, suggesting its functional relevance to neuronal migration.

  12. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  13. Induced pluripotent stem cell-derived neuron as a human model for testing environmentally induced developmental neurotoxicity

    Science.gov (United States)

    Induced pluripotent stem cell-derived neurons as a human model for testing environmentally induced developmental neurotoxicity Ingrid L. Druwe1, Timothy J. Shafer2, Kathleen Wallace2, Pablo Valdivia3 ,and William R. Mundy2. 1University of North Carolina, Curriculum in Toxicology...

  14. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

    Science.gov (United States)

    Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

    2016-01-01

    Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

  15. Induced pluripotent stem cell derived macrophages as a cellular system to study salmonella and other pathogens.

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    Christine Hale

    Full Text Available A number of pathogens, including several human-restricted organisms, persist and replicate within macrophages (Mφs as a key step in pathogenesis. The mechanisms underpinning such host-restricted intracellular adaptations are poorly understood, in part, due to a lack of appropriate model systems. Here we explore the potential of human induced pluripotent stem cell derived macrophages (iPSDMs to study such pathogen interactions. We show iPSDMs express a panel of established Mφ-specific markers, produce cytokines, and polarise into classical and alternative activation states in response to IFN-γ and IL-4 stimulation, respectively. iPSDMs also efficiently phagocytosed inactivated bacterial particles as well as live Salmonella Typhi and S. Typhimurium and were able to kill these pathogens. We conclude that iPSDMs can support productive Salmonella infection and propose this as a flexible system to study host/pathogen interactions. Furthermore, iPSDMs can provide a flexible and practical cellular platform for assessing host responses in multiple genetic backgrounds.

  16. "Kill" the messenger: Targeting of cell-derived microparticles in lupus nephritis.

    Science.gov (United States)

    Nielsen, Christoffer T; Rasmussen, Niclas S; Heegaard, Niels H H; Jacobsen, Søren

    2016-07-01

    Immune complex (IC) deposition in the glomerular basement membrane (GBM) is a key early pathogenic event in lupus nephritis (LN). The clarification of the mechanisms behind IC deposition will enable targeted therapy in the future. Circulating cell-derived microparticles (MPs) have been proposed as major sources of extracellular autoantigens and ICs and triggers of autoimmunity in LN. The overabundance of galectin-3-binding protein (G3BP) along with immunoglobulins and a few other proteins specifically distinguish circulating MPs in patients with systemic lupus erythematosus (SLE), and this is most pronounced in patients with active LN. G3BP co-localizes with deposited ICs in renal biopsies from LN patients supporting a significant presence of MPs in the IC deposits. G3BP binds strongly to glomerular basement membrane proteins and integrins. Accordingly, MP surface proteins, especially G3BP, may be essential for the deposition of ICs in kidneys and thus for the ensuing formation of MP-derived electron dense structures in the GBM, and immune activation in LN. This review focuses on the notion of targeting surface molecules on MPs as an entirely novel treatment strategy in LN. By targeting MPs, a double hit may be achieved by attenuating both the autoantigenic fueling of immune complexes and the triggering of the adaptive immune system. Thereby, early pathogenic events may be blocked in contrast to current treatment strategies that primarily target and modulate later events in the cellular and humoral immune response.

  17. Mast cell-derived heparin proteoglycans as a model for a local antithrombotic.

    Science.gov (United States)

    Lassila, Riitta; Jouppila, Annukka

    2014-11-01

    Mast cell-derived heparin proteoglycans (HEP-PG) reside in vascular tissue and serve as a local antithrombotic. Heparin, as used clinically, is isolated from its protein backbone from porcine and bovine gut mucosa. The isolated heparin is an anticoagulant; however, when bound to a protein carrier the heparin conjugates will become antiplatelet agents by inhibiting collagen-induced platelet aggregation and procoagulant activity, as distinct from soluble heparin. HEP-PG, whether soluble or immobilized to a surface, inhibit platelet deposition on the collagen surface in flowing blood. Mimics of HEP-PG, can be tailored to molecules carrying both antiplatelet and anticoagulant (APAC) properties. These molecules can target the vascular injury site and take residence there. Inhibition of thrombus growth using these APACs under these conditions has been demonstrated in several animal models. Although efficacious for antiplatelet and anticoagulant effects, the bleeding time is shorter with APACs than with unfractionated heparin, suggestive of beneficial efficacy/safety ratio. These strategies may be utilized in drug development, where many vascular injury-related problems can be tackled locally.

  18. A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

    Science.gov (United States)

    Schmetzer, Oliver; Valentin, Patricia; Smorodchenko, Anna; Domenis, Rossana; Gri, Giorgia; Siebenhaar, Frank; Metz, Martin; Maurer, Marcus

    2014-11-01

    The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs.

  19. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

    Science.gov (United States)

    Song, Kai; Song, Yong; Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-Lin; Liu, Ke; Shang, Zheng-Jun

    2014-10-15

    Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression.

  20. Discovery and characterization of novel microRNAs during endothelial differentiation of human embryonic stem cells.

    Science.gov (United States)

    Yoo, Jung Ki; Kim, Jumi; Choi, Seong-Jun; Noh, Hye Min; Kwon, Young Do; Yoo, Hanna; Yi, Hyo Seon; Chung, Hyung Min; Kim, Jin Kyeoung

    2012-07-20

    MicroRNAs (miRNAs) are small RNAs that participate in the regulation of genes associated with the differentiation and proliferation. In this study, 5 novel miRNAs were identified from human mesenchymal stem cells and characterized using various analyses. To investigate the potential functions associated with the regulation of cell differentiation, the differences in miRNA expression were examined in undifferentiated and differentiated human embryonic stem (ES) cells using reverse transcription (RT)-PCR analysis. Specifically, 3 miRNAs exhibited decreased expression levels in human umbilical vein endothelial cells (HUVECs) and endothelial cells derived from human ES cells. Putative target genes related to differentiation or maturation of endothelial cells were predicted by seed sequences of 2 novel miRNAs and analyzed for their expression via miRNA-mediated regulation using a luciferase assay. In HUVECs, CDH5 gene expression was directly repressed by hsa-miR-6086. Similarly, hsa-miR-6087 significantly downregulated endoglin expression. Therefore, the roles of these 2 miRNAs may be to directly suppress their target genes, popularly known as endothelial cell markers. Taken together, our results demonstrate that several novel miRNAs perform critical roles in human endothelial cell development.

  1. Effect of intracranial transplantation of CD34+ cells derived from human umbilical cord blood in rats with cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    LIU Hai-ying; ZHANG Qing-jun; LI Hong-jun; HAN Zhong-chao

    2006-01-01

    @@ As a source of transplantable stem cells, the CD34+ subpopulation in human umbilical cord blood (HUCB) has been used extensively to treat some hematopoietic system diseases. However,whether CD34+ cells hold the therapeutic potential to cerebral ischemia is unknown. The purpose of this study was to observe the recovery of neural function after transplantation of CD34+ cells derived from HUCB into ischemic cerebral tissue in rats.

  2. Novel application of stem cell-derived factors for periodontal regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Inukai, Takeharu, E-mail: t-inukai@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan); Katagiri, Wataru, E-mail: w-kat@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan); Yoshimi, Ryoko, E-mail: lianzi@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan); Osugi, Masashi, E-mail: masashi@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan); Kawai, Takamasa, E-mail: takamasa@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan); Hibi, Hideharu, E-mail: hibihi@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan); Ueda, Minoru, E-mail: mueda@med.nagoya-u.ac.jp [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine (Japan)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer Mesenchymal stem cells (MSCs) secrete a variety of cytokines. Black-Right-Pointing-Pointer Cytokines were detected in conditioned medium from cultured MSCs (MSC-CM). Black-Right-Pointing-Pointer MSC-CM enhanced activation of dog MSCs and periodontal ligament cells. Black-Right-Pointing-Pointer MSC-CM significantly promoted alveolar bone and cementum regeneration. Black-Right-Pointing-Pointer Multiple cytokines contained in MSC-CM promote periodontal regeneration. -- Abstract: The effect of conditioned medium from cultured mesenchymal stem cells (MSC-CM) on periodontal regeneration was evaluated. In vitro, MSC-CM stimulated migration and proliferation of dog MSCs (dMSCs) and dog periodontal ligament cells (dPDLCs). Cytokines such as insulin-like growth factor, vascular endothelial growth factor, transforming growth factor-{beta}1, and hepatocyte growth factor were detected in MSC-CM. In vivo, one-wall critical-size, intrabony periodontal defects were surgically created in the mandible of dogs. Dogs with these defects were divided into three groups that received MSC-CM, PBS, or no implants. Absorbable atelo-collagen sponges (TERUPLUG Registered-Sign ) were used as a scaffold material. Based on radiographic and histological observation 4 weeks after transplantation, the defect sites in the MSC-CM group displayed significantly greater alveolar bone and cementum regeneration than the other groups. These findings suggest that MSC-CM enhanced periodontal regeneration due to multiple cytokines contained in MSC-CM.

  3. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  4. Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

    Directory of Open Access Journals (Sweden)

    Nicolas Christoforou

    Full Text Available Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

  5. Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury.

    Science.gov (United States)

    Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J; Wright, Megan C; Lepore, Angelo C

    2015-09-01

    Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor

  6. Exposure to phthalates affects calcium handling and intercellular connectivity of human stem cell-derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Nikki Gillum Posnack

    Full Text Available The pervasive nature of plastics has raised concerns about the impact of continuous exposure to plastic additives on human health. Of particular concern is the use of phthalates in the production of flexible polyvinyl chloride (PVC products. Di-2-ethylhexyl-phthalate (DEHP is a commonly used phthalate ester plasticizer that imparts flexibility and elasticity to PVC products. Recent epidemiological studies have reported correlations between urinary phthalate concentrations and cardiovascular disease, including an increased risk of high blood pressure and coronary risk. Yet, there is little direct evidence linking phthalate exposure to adverse effects in human cells, including cardiomyocytes.The effect of DEHP on calcium handling was examined using monolayers of gCAMP3 human embryonic stem cell-derived cardiomyocytes, which contain an endogenous calcium sensor. Cardiomyocytes were exposed to DEHP (5 - 50 μg/mL, and calcium transients were recorded using a Zeiss confocal imaging system. DEHP exposure (24 - 72 hr had a negative chronotropic and inotropic effect on cardiomyocytes, increased the minimum threshold voltage required for external pacing, and modified connexin-43 expression. Application of Wy-14,643 (100 μM, an agonist for the peroxisome proliferator-activated receptor alpha, did not replicate DEHP's effects on calcium transient morphology or spontaneous beating rate.Phthalates can affect the normal physiology of human cardiomyocytes, including DEHP elicited perturbations in cardiac calcium handling and intercellular connectivity. Our findings call for additional studies to clarify the extent by which phthalate exposure can alter cardiac function, particularly in vulnerable patient populations who are at risk for high phthalate exposure.

  7. Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

    Directory of Open Access Journals (Sweden)

    Koichi Murata

    Full Text Available Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/- mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/- mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/- mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/- mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/- mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/- mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/- mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/- mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

  8. Pharmacoelectrophysiology of viral-free induced pluripotent stem cell-derived human cardiomyocytes.

    Science.gov (United States)

    Mehta, Ashish; Chung, YingYing; Sequiera, Glen Lester; Wong, Philip; Liew, Reginald; Shim, Winston

    2013-02-01

    Development of pharmaceutical agents for cardiac indication demands elaborate safety screening in which assessing repolarization of cardiac cells remains a critical path in risk evaluations. An efficient platform for evaluating cardiac repolarization in vitro significantly facilitates drug developmental programs. In a proof of principle study, we examined the effect of antiarrhythmogenic drugs (Vaughan Williams class I-IV) and noncardiac active drugs (terfenadine and cisapride) on the repolarization profile of viral-free human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Extracellular field potential (FP) recording using microelectrode arrays demonstrated significant delayed repolarization as prolonged corrected FP durations (cFPDs) by class I (quinidine and flecainide), class III (sotalol and amiodarone), and class IV (verapamil), whereas class II drugs (propranolol and nadolol) had no effects. Consistent with their sodium channel-blocking ability, class I drugs also significantly reduced FPmin and conduction velocity. Although lidocaine (class IB) had no effects on cFPDs, verapamil shortened cFPD and FPmin by 25 and 50%, respectively. Furthermore, verapamil reduced beating frequencies drastically. Importantly, the examined drugs exhibited dose-response curve on prolongation of cFPDs at an effective range that correlated significantly with therapeutic plasma concentrations achieved clinically. Consistent with clinical outcomes, drug-induced arrhythmia of tachycardia and bigeminy-like waveforms by quinidine, flecainide, and sotalol was demonstrated at supraphysiological concentrations. Furthermore, off-target effects of terfenadine and cisapride on cFPD and Na( + ) channel blockage were similarly revealed. These results suggest that hiPSC-CMs may be useful for safety evaluation of cardioactive and noncardiac acting drugs for personalized medicine.

  9. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

    Directory of Open Access Journals (Sweden)

    Jan Tønnesen

    Full Text Available Intrastriatal grafts of stem cell-derived dopamine (DA neurons induce behavioral recovery in animal models of Parkinson's disease (PD, but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2 and halorhodopsin (NpHR, respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  10. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

    Science.gov (United States)

    Tønnesen, Jan; Parish, Clare L; Sørensen, Andreas T; Andersson, Angelica; Lundberg, Cecilia; Deisseroth, Karl; Arenas, Ernest; Lindvall, Olle; Kokaia, Merab

    2011-03-04

    Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  11. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  12. Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

    Science.gov (United States)

    Avaliani, Natalia; Sørensen, Andreas Toft; Ledri, Marco; Bengzon, Johan; Koch, Philipp; Brüstle, Oliver; Deisseroth, Karl; Andersson, My; Kokaia, Merab

    2014-12-01

    Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation

  13. Embryonic stem cell-derived microvesicles induce gene expression changes in Müller cells of the retina.

    Science.gov (United States)

    Katsman, Diana; Stackpole, Emma J; Domin, Daniel R; Farber, Debora B

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  14. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    OpenAIRE

    Jan David Kijlstra; Dongjian Hu; Nikhil Mittal; Eduardo Kausel; Peter van der Meer; Arman Garakani; Ibrahim J. Domian

    2015-01-01

    Summary The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can b...

  15. Hepatocyte growth factor protects endothelial cells against gamma ray irradiation-induced damage

    Institute of Scientific and Technical Information of China (English)

    Shun-ying HU; Hai-feng DUAN; Qing-fang LI; Yue-feng YANG; Jin-long CHEN; Li-sheng WANG; Hua WANG

    2009-01-01

    Aim:To investigate the effect of HGF on proliferation, apoptosis and migratory ability of human vascular endothelial cells against gamma ray irradiation.Methods: ECV304 cells derived from adult human umbilical vein endothelial cells (HUVEC) were irradiated with a single gamma ray dose of 20 Gy. Immunocytochemistry and Western blot analysis were used to detect c-Met protein expression and HGF/c-Met signal pathway. In the HGF-treated groups, ECV304 cells were incubated with HGF (20 or 40 ng/mL) 3 h prior to irradiation. At 48 h post-irradiation, the proliferation of ECV304 cells was measured by MTT assay, the apoptosis was assessed by flow cytometry, and the migratory ability of ECV304 cells was measured by transwell chamber assay.Results: c-Met protein is expressed in ECV304 cells and can be activated by HGF. Gamma ray irradiation inhibits proliferation and migration of ECV304 cells in a dose-dependent manner. HGF significantly promoted the proliferation of ECV304 cells, and flow cytom-etry revealed that HGF can inhibit apoptosis of ECV304 cells. Transwell chamber assay also showed that HGF increases migration activity of endothelial cells.Conclusion: HGF may afford protection to vascular endothelial cells against gamma ray irradiation-induced damage.

  16. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes.

    Science.gov (United States)

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  17. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    Directory of Open Access Journals (Sweden)

    Kyung Hoon Lee

    2016-01-01

    Full Text Available Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4, which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  18. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, ptissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  19. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  20. Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling

    Directory of Open Access Journals (Sweden)

    Kathleen G. Neiva

    2009-06-01

    Full Text Available It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3, Akt, and extracellular signal-regulated kinase (ERK in a panel of head and neck squamous cell carcinoma (HNSCC cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A. Gene expression analysis demonstrated that interleukin-6 (IL- 6, interleukin-8 (CXCL8, and epidermal growth factor (EGF are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

  1. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, pfunction (shortening fraction 9.2 [4.9] vs 17.2 [4.2]%, n=8 per group, pmyocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  2. Adult stromal cells derived from human adipose tissue provoke pancreatic cancer cell death both in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Beatrice Cousin

    Full Text Available BACKGROUND: Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC on pancreatic tumor cell proliferation. PRINCIPAL FINDINGS: Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate. ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth. CONCLUSION: These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.

  3. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, pmyocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  4. Pluripotent muse cells derived from human adipose tissue: a new perspective on regenerative medicine and cell therapy.

    Science.gov (United States)

    Simerman, Ariel A; Dumesic, Daniel A; Chazenbalk, Gregorio D

    2014-01-01

    In 2010, Multilineage Differentiating Stress Enduring (Muse) cells were introduced to the scientific community, offering potential resolution to the issue of teratoma formation that plagues both embryonic stem (ES) and induced pluripotent (iPS) stem cells. Isolated from human bone marrow, dermal fibroblasts, adipose tissue and commercially available adipose stem cells (ASCs) under severe cellular stress conditions, Muse cells self-renew in a controlled manner and do not form teratomas when injected into immune-deficient mice. Furthermore, Muse cells express classic pluripotency markers and differentiate into cells from the three embryonic germ layers both spontaneously and under media-specific induction. When transplanted in vivo, Muse cells contribute to tissue generation and repair. This review delves into the aspects of Muse cells that set them apart from ES, iPS, and various reported adult pluripotent stem cell lines, with specific emphasis on Muse cells derived from adipose tissue (Muse-AT), and their potential to revolutionize the field of regenerative medicine and stem cell therapy.

  5. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  6. Isolation and analysis of SSEA-4 positive cells derived from fetal marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LIU Daqing; PEI Xuetao; YANG Yinxiang; GAO Yanhong; YUAN Hongfeng; QIN Lipeng; WANG Yunfang; NAN Xue; SHI Shuangshuang; YUE Wen

    2006-01-01

    A big issue in stem cell research is to derive prospective totipotential stem cells. In this study, fMSC-SSEA-4 cells expressing SSEA-4 antigen were isolated from fetal marrow masenchymal stem cells (fMSCs) using immunomagnetic bead sorting technique. The totipotent cells were identified and their biological characteristics were further studied. The expression of Oct-4 and SSEA-4, carcino- genicity, and the ability to differentiation of fMSC- SSEA-4 cells were evaluated to verify the totipotent potential. fMSC-SSEA-4 cells were isolated successfully from fMSCs (2.5% among fMSCs), while no obvious differences were seen in morphology, growth curve, cell cycle and immunophenotype, Oct-4 and SSEA-4 expression between fMSC-SSEA-4 cells and fMSCs. fMSC-SSEA-4 cells showed normal diploid chromosome karyotype and no carcinoma was induced after inoculation into nude mice. fMSC- SSEA-4 cells could be induced to fat cells, osteogenic cells and neuron-like cells in vitro with different induced factors. The results indicated that there may be a few totipotent cells among the fMSCs and it may offer the experimental basis for the further study and application of fMSCs.

  7. Cancer stem cell-like cells derived from malignant peripheral nerve sheath tumors.

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    Melanie Spyra

    Full Text Available This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST. Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5 × 10(5. These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors.

  8. Autocrine regulation of pulmonary inflammation by effector T-cell derived IL-10 during infection with respiratory syncytial virus.

    Directory of Open Access Journals (Sweden)

    Jie Sun

    2011-08-01

    Full Text Available Respiratory syncytial virus (RSV infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that certain polymorphisms in the gene encoding the regulatory cytokine IL-10 are associated with the development of severe bronchiolitis in RSV infected infants. Here, we examined the role of IL-10 in a murine model of primary RSV infection and found that high levels of IL-10 are produced in the respiratory tract by anti-viral effector T cells at the onset of the adaptive immune response. We demonstrated that the function of the effector T cell -derived IL-10 in vivo is to limit the excess pulmonary inflammation and thereby to maintain critical lung function. We further identify a novel mechanism by which effector T cell-derived IL-10 controls excess inflammation by feedback inhibition through engagement of the IL-10 receptor on the antiviral effector T cells. Our findings suggest a potentially critical role of effector T cell-derived IL-10 in controlling disease severity in clinical RSV infection.

  9. The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts.

    Science.gov (United States)

    Tzukerman, Maty; Rosenberg, Tzur; Reiter, Irena; Ben-Eliezer, Shoshana; Denkberg, Galit; Coleman, Raymond; Reiter, Yoram; Skorecki, Karl

    2006-04-01

    The awareness of the important role that the surrounding tissue microenvironment and stromal response play in the process of tumorigenesis has grown as a result of in vivo models of tumor xenograft growth in immunocompromised mice. In the current study, we used human embryonic stem cells in order to study the interactions of tumor cells with the surrounding microenvironment of differentiated human cell tissues and structures. Several cancer cell types stably expressing an H2A-green fluorescence protein fusion protein, which allowed tracking of tumor cells, were injected into mature teratomas and developed into tumors. The salient findings were: (a) the observation of growth of tumor cells with high proliferative capacity within the differentiated microenvironment of the teratoma, (b) the identification of invasion by tumor cells into surrounding differentiated teratoma structures, and (c) the identification of blood vessels of human teratoma origin, growing adjacent to and within the cancer cell-derived tumor. Mouse embryonic stem cell-derived teratomas also supported cancer cell growth, but provided a less suitable model for human tumorigenesis studies. Anticancer immunotherapy treatment directed against A431 epidermoid carcinoma cell-related epitopes induced the complete regression of A431-derived tumor xenografts following direct i.m. injection in immunocompromised mice, as opposed to corresponding tumors growing within a human embryonic stem cell-derived microenvironment, wherein remnant foci of viable tumor cells were detected and resulted in tumor recurrence. We propose using this novel experimental model as a preclinical platform for investigating and manipulating the stromal response in tumor cell growth as an additional tool in cancer research.

  10. Interaction between antigen presenting cells and autoreactive T cells derived from BXSB mice with murine lupus

    Institute of Scientific and Technical Information of China (English)

    Peng Yang; Bo Li; Ping Lv; Yan Zhang; XiaoMing Gao

    2007-01-01

    Systemic lupus erythematosus (SLE) is a typical autoimmune disease involving multiple systems and organs. Ample evidence suggests that autoreactive T cells play a pivotal role in the development of this autoimmune disorder. This study was undertaken to investigate the mechanisms of interaction between antigen presenting cells (APCs) and an autoreactive T cell (ATL1) clone obtained from lupus-prone BXSB mice. ATL1 cells, either before or after γ-ray irradiation, were able to activate naive B cells, as determined by B cell proliferation assays. Macrophages from BXSB mice were able to stimulate the proliferation of resting ATL1 cells at a responder/stimulator (R/S) ratio of 1/2.5. Dendritic cells (DCs) were much more powerful stimulators for ATL1 cells on a per cell basis. The T cell stimulating ability of macrophages and B cells, but not DCs, was sensitive toγ-ray irradiation. Monoclonal antibodies against mouse MHC-Ⅱand CD4 were able to block DC-mediated stimulation of ATL1 proliferation, indicating cognate recognition between ATL1 and APCs. Our data suggest that positive feedback loops involving macrophages, B cells and autoreactive T cells may play a pivotal role in keeping the momentum of autoimmune responses leading to autoimmune diseases.

  11. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    Science.gov (United States)

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  12. Pluripotent stem cell-derived neural stem cells: From basic research to applications

    Institute of Scientific and Technical Information of China (English)

    Masahiro; Otsu; Takashi; Nakayama; Nobuo; Inoue

    2014-01-01

    Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.

  13. Liver stem cell-derived β-cell surrogates for treatment of type 1 diabetes☆

    Science.gov (United States)

    Yang, Li-Jun

    2012-01-01

    Consistent with the common embryonic origin of liver and pancreas as well the similar glucose-sensing systems in hepatocytes and pancreatic β-cells, it should not be surprising that liver stem cells/hepatocytes can transdifferentiate into insulin-producing cells under high-glucose culture conditions or by genetic reprogramming. Persistent expression of the pancreatic duodenal homeobox-1 (Pdx1) transcription factor or its super-active form Pdx1-VP16 fusion protein in hepatic cells reprograms these cells into pancreatic β-cell precursors. In vitro culture at elevated glucose concentrations or in vivo exposure to a hyperglycemia are required for further differentiation and maturation of liver-derived pancreatic β-cell precursor into functional insulin-producing pancreatic β-like cells. Under appropriate conditions, multiple pancreatic transcription factors can work in concert to reprogram liver stem/adult liver cells into functional insulin-producing cells. If such autologous liver-derived insulin-producing cells can be made to escape the type 1 diabetes-associated autoimmunity, they may serve as a valuable cell source for future cell replacement therapy without the need for life-long immunosuppression. PMID:16890895

  14. Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain.

    Science.gov (United States)

    Labuz, Dominika; Berger, Stephan; Mousa, Shaaban A; Zöllner, Christian; Rittner, Heike L; Shaqura, Mohammed A; Segovia-Silvestre, Toni; Przewlocka, Barbara; Stein, Christoph; Machelska, Halina

    2006-04-19

    Endomorphins (EMs) are endogenous selective mu-opioid receptor agonists. Their role in inflammatory pain has not been fully elucidated. Here we examine peripheral antinociception elicited by exogenously applied EM-1 and EM-2 and the contribution of EM-containing leukocytes to stress- and corticotropin-releasing factor (CRF)-induced antinociception. To this end, we applied behavioral (paw pressure) testing, radioligand binding, immunohistochemistry, and flow cytometry in rats with unilateral hindpaw inflammation induced with Freund's adjuvant. EMs injected directly into both hindpaws produced antinociception exclusively in inflamed paws. This was blocked by locally applied mu-receptor-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) but not kappa-receptor-selective (nor-binaltorphimine) antagonists. Delta-receptor antagonists (naltrindole and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu) did not influence EM-1-induced but dose-dependently decreased EM-2-induced antinociception. Antibodies against beta-endorphin, methionine-enkephalin, or leucine-enkephalin did not significantly change EM-2-induced antinociception. Both EMs displaced binding of [3H]-[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to mu-receptors in dorsal root ganglia (DRG). Using [3H]-naltrindole or [(125)I]-[D-Pen2,5]-enkephalin, no detectable delta-binding was found in DRG of inflamed hindlimbs. Numerous beta-endorphin-containing and fewer EM-1- and EM-2-containing leukocytes were detected in subcutaneous tissue of inflamed paws. Leukocyte-depleting serum decreased the number of immigrating opioid-containing immune cells and attenuated swim stress- and CRF-induced antinociception in inflamed paws. Both forms of antinociception were strongly attenuated by anti-beta-endorphin and to a lesser degree by anti-EM-1 and anti-EM-2 antibodies injected into inflamed paws. Together, exogenously applied and immune cell-derived EMs alleviate prolonged inflammatory pain through selective activation of peripheral opioid receptors

  15. 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.

  16. Endothelial cell promotion of early liver and pancreas development.

    Science.gov (United States)

    Freedman, Deborah A; Kashima, Yasushige; Zaret, Kenneth S

    2007-01-01

    Different steps of embryonic pancreas and liver development require inductive signals from endothelial cells. During liver development, interactions between newly specified hepatic endoderm cells and nascent endothelial cells are crucial for the endoderm's subsequent growth and morphogenesis into a liver bud. Reconstitution of endothelial cell stimulation of hepatic cell growth with embryonic tissue explants demonstrated that endothelial signalling occurs independent of the blood supply. During pancreas development, midgut endoderm interactions with aortic endothelial cells induce Ptf1a, a crucial pancreatic determinant. Endothelial cells also have a later effect on pancreas development, by promoting survival of the dorsal mesenchyme, which in turn produces factors supporting pancreatic endoderm. A major goal of our laboratory is to determine the endothelial-derived molecules involved in these inductive events. Our data show that cultured endothelial cells induce Ptf1a in dorsal endoderm explants lacking an endogenous vasculature. We are purifying endothelial cell line product(s) responsible for this effect. We are also identifying endothelial-responsive regulatory elements in genes such as Ptf1a by genetic mapping and chromatin-based assays. These latter approaches will allow us to track endothelial-responsive signal pathways from DNA targets within progenitor cells. The diversity of organogenic steps dependent upon endothelial cell signalling suggests that cross-regulation of tissue development with its vasculature is a general phenomenon.

  17. Epigenetic rejuvenation of mesenchymal stromal cells derived from induced pluripotent stem cells

    NARCIS (Netherlands)

    Frobel, Joana; Hemeda, Hatim; Lenz, Michael; Abagnale, Giulio; Joussen, Sylvia; Denecke, Bernd; Sarić, Tomo; Zenke, Martin; Wagner, Wolfgang

    2014-01-01

    Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground stat

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kai [Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Shandong Province (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Song, Yong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Department of Stomatology, Liu Zhou People' s Hospital, Guangxi (China); Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-lin [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Liu, Ke, E-mail: liuke.1999@aliyun.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China)

    2014-10-15

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo.

  20. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions.

    Science.gov (United States)

    Klimchenko, Olena; Di Stefano, Antonio; Geoerger, Birgit; Hamidi, Sofiane; Opolon, Paule; Robert, Thomas; Routhier, Mélanie; El-Benna, Jamel; Delezoide, Anne-Lise; Boukour, Siham; Lescure, Bernadette; Solary, Eric; Vainchenker, William; Norol, Françoise

    2011-03-17

    The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

  1. Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

    Science.gov (United States)

    Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y Eugene; Ma, Peter X

    2015-12-01

    Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue.

  2. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure.

    Science.gov (United States)

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G J; Mummery, Christine L; Casini, Simona

    2015-11-27

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.

  3. Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds

    Directory of Open Access Journals (Sweden)

    Chaudhry G. R.

    2004-01-01

    Full Text Available An approach for 3D bone tissue generation from embryonic stem (ES cells was investigated. The ES cells were induced to differentiate into osteogenic precursors, capable of proliferating and subsequently differentiating into bone-forming cells. The differentiated cells and the seeded scaffolds were characterized using von Kossa and Alizarin Red staining, electron microscopy, and RT-PCR analysis. The results demonstrated that ES-derived bone-forming cells attached to and colonized the biocompatible and biodegradable scaffolds. Furthermore, these cells produced bone nodules when grown for 3–4 weeks in mineralization medium containing ascorbic acid and beta-glycerophosphate both in tissue culture plates and in scaffolds. The differentiated cells also expressed osteospecific markers when grown both in the culture plates and in 3D scaffolds. Osteogenic cells expressed alkaline phosphatase, osteocalcin, and osteopontin, but not an ES cell-specific marker, oct-4. These findings suggest that ES cell can be used for in vitro tissue engineering and cultivation of graftable skeletal structures.

  4. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential.

    Science.gov (United States)

    Ishii, Tetsuya

    2014-10-13

    Recently, fertile spermatozoa and oocytes were generated from mouse induced pluripotent (iPS) cells using a combined in vitro and in vivo induction system. With regard to germ cell induction from human iPS cells, progress has been made particularly in the male germline, demonstrating in vitro generation of haploid, round spermatids. Although iPS-derived germ cells are expected to be developed to yield a form of assisted reproductive technology (ART) that can address unmet reproductive needs, genetic and/or epigenetic instabilities abound in iPS cell generation and germ cell induction. In addition, there is still room to improve the induction protocol in the female germline. However, rapid advances in stem cell research are likely to make such obstacles surmountable, potentially translating induced germ cells into the clinical setting in the immediate future. This review examines the current status of the induction of germ cells from human iPS cells and discusses the clinical potential, as well as future directions.

  5. Stromal Cell-Derived Factor-1 Promotes Cell Migration, Tumor Growth of Colorectal Metastasis

    Directory of Open Access Journals (Sweden)

    Otto Kollmar

    2007-10-01

    Full Text Available In a mouse model of established extrahepatic colorectal metastasis, we analyzed whether stromal cellderived factor (SDF 1 stimulates tumor cell migration in vitro, angiogenesis, tumor growth in vivo. METHODS: Using chemotaxis chambers, CT26.WT colorectal tumor cell migration was studied under stimulation with different concentrations of SDF-1. To evaluate angiogenesis, tumor growth in vivo, green fluorescent protein-transfected CT26.WT cells were implanted in dorsal skinfold chambers of syngeneic BALB/c mice. After 5 days, tumors were locally exposed to SDF-1. Cell proliferation, tumor microvascularization, growth were studied during a further 9-day period using intravital fluorescence microscopy, histology, immunohistochemistry. Tumors exposed to PBS only served as controls. RESULTS:In vitro, > 30% of unstimulated CT26.WT cells showed expression of the SDF-1 receptor CXCR4. On chemotaxis assay, SDF-1 provoked a dose-dependent increase in cell migration. In vivo, SDF-1 accelerated neovascularization, induced a significant increase in tumor growth. Capillaries of SDF-1-treated tumors showed significant dilation. Of interest, SDF-1 treatment was associated with a significantly increased expression of proliferating cell nuclear antigen, a downregulation of cleaved caspase-3. CONCLUSION: Our study indicates that the CXC chemokine SDF-1 promotes tumor cell migration in vitro, tumor growth of established extrahepatic metastasis in vivo due to angiogenesis-dependent induction of tumor cell proliferation, inhibition of apoptotic cell death.

  6. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential

    Directory of Open Access Journals (Sweden)

    Tetsuya Ishii

    2014-10-01

    Full Text Available Recently, fertile spermatozoa and oocytes were generated from mouse induced pluripotent (iPS cells using a combined in vitro and in vivo induction system. With regard to germ cell induction from human iPS cells, progress has been made particularly in the male germline, demonstrating in vitro generation of haploid, round spermatids. Although iPS-derived germ cells are expected to be developed to yield a form of assisted reproductive technology (ART that can address unmet reproductive needs, genetic and/or epigenetic instabilities abound in iPS cell generation and germ cell induction. In addition, there is still room to improve the induction protocol in the female germline. However, rapid advances in stem cell research are likely to make such obstacles surmountable, potentially translating induced germ cells into the clinical setting in the immediate future. This review examines the current status of the induction of germ cells from human iPS cells and discusses the clinical potential, as well as future directions.

  7. Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Seong-Ho Koh

    Full Text Available Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9 has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs. However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs, and endothelial progenitor cells (EPCs. Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM, and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.

  8. Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro.

    Science.gov (United States)

    Zhou, Quan; Wang, Mei; Yuan, Yan; Wang, Xuepeng; Fu, Rui; Wan, Haifeng; Xie, Mingming; Liu, Mingxi; Guo, Xuejiang; Zheng, Ying; Feng, Guihai; Shi, Qinghua; Zhao, Xiao-Yang; Sha, Jiahao; Zhou, Qi

    2016-03-03

    In vitro generation of functional gametes is a promising approach for treating infertility, although faithful replication of meiosis has proven to be a substantial obstacle to deriving haploid gamete cells in culture. Here we report complete in vitro meiosis from embryonic stem cell (ESC)-derived primordial germ cells (PGCLCs). Co-culture of PGCLCs with neonatal testicular somatic cells and sequential exposure to morphogens and sex hormones reproduced key hallmarks of meiosis, including erasure of genetic imprinting, chromosomal synapsis and recombination, and correct nuclear DNA and chromosomal content in the resulting haploid cells. Intracytoplasmic injection of the resulting spermatid-like cells into oocytes produced viable and fertile offspring, showing that this robust stepwise approach can functionally recapitulate male gametogenesis in vitro. These findings provide a platform for investigating meiotic mechanisms and the potential generation of human haploid spermatids in vitro.

  9. Smooth-muscle-like cells derived from human embryonic stem cells support and augment cord-like structures in vitro.

    Science.gov (United States)

    Vo, Elaine; Hanjaya-Putra, Donny; Zha, Yuanting; Kusuma, Sravanti; Gerecht, Sharon

    2010-06-01

    Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers--including alpha-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain--to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

  10. Methods for derivation of multipotent neural crest cells derived from human pluripotent stem cells

    Science.gov (United States)

    Avery, John; Dalton, Stephen

    2016-01-01

    Summary Multipotent, neural crest cells (NCCs) produce a wide-range of cell types during embryonic development. This includes melanocytes, peripheral neurons, smooth muscle cells, osteocytes, chondrocytes and adipocytes. The protocol described here allows for highly-efficient differentiation of human pluripotent stem cells to a neural crest fate within 15 days. This is accomplished under feeder-free conditions, using chemically defined medium supplemented with two small molecule inhibitors that block glycogen synthase kinase 3 (GSK3) and bone morphogenic protein (BMP) signaling. This technology is well-suited as a platform to understand in greater detail the pathogenesis of human disease associated with impaired neural crest development/migration. PMID:25986498

  11. Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro

    Institute of Scientific and Technical Information of China (English)

    Sheng FANG; Yu-dong QIU; Liang MAO; Xiao-lei SHI; De-cai YU; Yi-tao DING

    2007-01-01

    Aim: Embryonic stem (ES) cells are being widely investigated as a promising source of hepatocytes with their proliferative, renewable, and pluripotent capacities. However, controlled and scalable ES cell differentiation culture into functional hepatocytes is challenging. In this study, we examined the differentiat- ing potential of embryoid-body cells derived from ES cells into hepatocytes in alginate microbeads containing exogenous growth factors in vitro. Methods: Embryoid bodies were formed from ES cells by suspension methods. Embryoid bodies cultured for 5 d were treated with trypsin-EDTA. The disaggregated cells were encapsulated in alginate microbeads and stimulated with exogenous growth factors to induce hepatic differentiation. In the course of cell differentiation, cell morphology and viability were observed, and the expression patterns of some genes of the hepatocyte were confirmed by RT-PCR. An immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK18). Hepatocyte functional assays were confirmed by the secretion of ALB and urea. Results: We showed that embryoid-body cells could maintain cell viability in alginate microbeads in vitro. We also found that directed differentiated cells expressed several hepatocyte genes including α-fetoprotein (AFP), ALB, Cyp7al, CK18, transthyretin (TTR) and tyrosine aminotransferase (TAT) and produced ALB and urea in alginate microbeads. The directed differentiated cells expressed ALB and CK18 proteins on d 14. However, embryoid-body cells could not form hepatocytes without exogenous growth factors in alginate microbeads. Conclusion: The differentiation of embryoid-body cells into hepatocytes con- taining exogenous growth factors in alginate microbeads gives rise to functional hepatocytes and may develop scalable stem cell differentiation strategies for bioartificial livers and hepatocyte transplantation.

  12. Cell polarity and neurogenesis in embryonic stem cell-derived neural rosettes.

    Science.gov (United States)

    Banda, Erin; McKinsey, Anna; Germain, Noelle; Carter, James; Anderson, Nickesha Camille; Grabel, Laura

    2015-04-15

    Embryonic stem cells (ESCs) undergoing neural differentiation form radial arrays of neural stem cells, termed neural rosettes. These structures manifest many of the properties associated with embryonic and adult neurogenesis, including cell polarization, interkinetic nuclear migration (INM), and a gradient of neuronal differentiation. We now identify novel rosette structural features that serve to localize key regulators of neurogenesis. Cells within neural rosettes have specialized basal as well as apical surfaces, based on localization of the extracellular matrix receptor β1 integrin. Apical processes of cells in mature rosettes terminate at the lumen, where adherens junctions are apparent. Primary cilia are randomly distributed in immature rosettes and tightly associated with the neural stem cell's apical domain as rosettes mature. Components of two signaling pathways known to regulate neurogenesis in vivo and in rosettes, Hedgehog and Notch, are apically localized, with the Hedgehog effector Smoothened (Smo) associated with primary cilia and the Notch pathway γ-secretase subunit Presenilin 2 associated with the adherens junction. Increased neuron production upon treatment with the Notch inhibitor DAPT suggests a major role for Notch signaling in maintaining the neural stem cell state, as previously described. A less robust outcome was observed with manipulation of Hedgehog levels, though consistent with a role in neural stem cell survival or proliferation. Inhibition of both pathways resulted in an additive effect. These data support a model by which cells extending a process to the rosette lumen maintain neural stem cell identity whereas release from this association, either through asymmetric cell division or apical abscission, promotes neuronal differentiation.

  13. Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

    Science.gov (United States)

    Mikhailova, Alexandra; Ilmarinen, Tanja; Ratnayake, Anjula; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli; Rafat, Mehrdad

    2016-05-01

    Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for

  14. Prospect of Human Pluripotent Stem Cell-Derived Neural Crest Stem Cells in Clinical Application

    Science.gov (United States)

    Zhu, Qian; Lu, Qiqi; Gao, Rong

    2016-01-01

    Neural crest stem cells (NCSCs) represent a transient and multipotent cell population that contributes to numerous anatomical structures such as peripheral nervous system, teeth, and cornea. NCSC maldevelopment is related to various human diseases including pigmentation abnormalities, disorders affecting autonomic nervous system, and malformations of teeth, eyes, and hearts. As human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can serve as an unlimited cell source to generate NCSCs, hESC/hiPSC-derived NCSCs can be a valuable tool to study the underlying mechanisms of NCSC-associated diseases, which paves the way for future therapies for these abnormalities. In addition, hESC/hiPSC-derived NCSCs with the capability of differentiating to various cell types are highly promising for clinical organ repair and regeneration. In this review, we first discuss NCSC generation methods from human pluripotent stem cells and differentiation mechanism of NCSCs. Then we focus on the clinical application potential of hESC/hiPSC-derived NCSCs on peripheral nerve injuries, corneal blindness, tooth regeneration, pathological melanogenesis, Hirschsprung disease, and cardiac repair and regeneration. PMID:28090209

  15. Dendritic cell-derived IL-15 controls the induction of CD8 T cell immune responses.

    Science.gov (United States)

    Rückert, René; Brandt, Katja; Bulanova, Elena; Mirghomizadeh, Farhad; Paus, Ralf; Bulfone-Paus, Silvia

    2003-12-01

    The development and the differentiation of CD8(+) T cells are dependent on IL-15. Here, we have studied the source and mechanism of how IL-15 modulates CD8(+) T cell-mediated Th1 immune responses by employing two delayed-type hypersensitivity (DTH) models. IL-15-deficient (IL-15(-/-)) mice or mice treated with soluble IL-15Ralpha as an IL-15 antagonist showed significantly reduced CD8(+) T cell-dependent DTH responses, while activation of CD4(+) T cell and B cell functions remained unaffected. Injection of antigen-labeled dendritic cells (DC) from IL-15(+/+), IL-15(-/-) or IL-15Ralpha(-/-) mice revealed that DC-derived IL-15 is an absolute requirement for the initiation of DTH response. The re-establishment of the interaction of IL-15 with the IL-15Ralpha by incubating IL-15(-/-) DC with IL-15 completely restored the capacity to prime T cells for DTH induction in vivo. Moreover, IL-15 also enhanced secretion of pro-inflammatory cytokines by DC and triggered in vitro CD8(+) T cell proliferation and IL-2 release. Taken together, the data suggest that an autocrine IL-15/IL-15Ralpha signaling loop in DC is essential for inducing CD8(+)-dependent Th1 immune responses in mice. Therefore, targeted manipulation of this loop promises to be an effective, novel strategy for therapeutic modulation of clinically relevant DTH reactions.

  16. Prospect of Human Pluripotent Stem Cell-Derived Neural Crest Stem Cells in Clinical Application

    Directory of Open Access Journals (Sweden)

    Qian Zhu

    2016-01-01

    Full Text Available Neural crest stem cells (NCSCs represent a transient and multipotent cell population that contributes to numerous anatomical structures such as peripheral nervous system, teeth, and cornea. NCSC maldevelopment is related to various human diseases including pigmentation abnormalities, disorders affecting autonomic nervous system, and malformations of teeth, eyes, and hearts. As human pluripotent stem cells including human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs can serve as an unlimited cell source to generate NCSCs, hESC/hiPSC-derived NCSCs can be a valuable tool to study the underlying mechanisms of NCSC-associated diseases, which paves the way for future therapies for these abnormalities. In addition, hESC/hiPSC-derived NCSCs with the capability of differentiating to various cell types are highly promising for clinical organ repair and regeneration. In this review, we first discuss NCSC generation methods from human pluripotent stem cells and differentiation mechanism of NCSCs. Then we focus on the clinical application potential of hESC/hiPSC-derived NCSCs on peripheral nerve injuries, corneal blindness, tooth regeneration, pathological melanogenesis, Hirschsprung disease, and cardiac repair and regeneration.

  17. Techniques of Human Embryonic Stem Cell and Induced Pluripotent Stem Cell Derivation.

    Science.gov (United States)

    Lewandowski, Jarosław; Kurpisz, Maciej

    2016-10-01

    Developing procedures for the derivation of human pluripotent stem cells (PSCs) gave rise to novel pathways into regenerative medicine research. For many years, stem cells have attracted attention as a potentially unlimited cell source for cellular therapy in neurodegenerative disorders, cardiovascular diseases, and spinal cord injuries, for example. In these studies, adult stem cells were insufficient; therefore, many attempts were made to obtain PSCs by other means. This review discusses key issues concerning the techniques of pluripotent cell acquisition. Technical and ethical issues hindered the medical use of somatic cell nuclear transfer and embryonic stem cells. Therefore, induced PSCs (iPSCs) emerged as a powerful technique with great potential for clinical applications, patient-specific disease modelling and pharmaceutical studies. The replacement of viral vectors or the administration of analogous proteins or chemical compounds during cell reprogramming are modifications designed to reduce tumorigenesis risk and to augment the procedure efficiency. Intensified analysis of new PSC lines revealed other barriers to overcome, such as epigenetic memory, disparity between human and mouse pluripotency, and variable response to differentiation of some iPSC lines. Thus, multidimensional verification must be conducted to fulfil strict clinical-grade requirements. Nevertheless, the first clinical trials in patients with spinal cord injury and macular dystrophy were recently carried out with differentiated iPSCs, encouraging alternative strategies for potential autologous cellular therapies.

  18. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  19. Tip cell-derived RTK signaling initiates cell movements in the Drosophila stomatogastric nervous system anlage.

    Science.gov (United States)

    González-Gaitán, M; Jäckle, H

    2000-10-01

    The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

  20. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy.

    Science.gov (United States)

    Osada, Hironari; Ogawa, Misato; Hasegawa, Ayana; Nagai, Makoto; Shirai, Junsuke; Sasaki, Kazuaki; Shimoda, Minoru; Itoh, Hiroshi; Kondo, Hirotaka; Ohmori, Keitaro

    2017-02-28

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE.

  1. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, pstem-cell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  2. Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats

    Directory of Open Access Journals (Sweden)

    Xing Yujie

    2012-01-01

    Full Text Available Abstract Background To explore the feasibility of constructing engineered myocardial tissues (EMTs in vivo, using polylactic acid -co-glycolic acid (PLGA for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs for seeded cells. Methods BMMSCs were isolated from femur and tibia of Sprague-Dawley (SD rats by density-gradient centrifugation. The third passage cells were treated with 10 μmol/L 5-azacytidine (5-aza and 0.1 μmol/L angiotensin II (Ang II for 24 h, followed by culturing in complete medium for 3 weeks to differentiated into cardiomyocyte-like cells. The cardiomyocyte-like cells were seeded into PLGA scaffolds to form the grafts. The grafts were cultured in the incubator for three days and then implanted into the peritoneal cavity of SD rats. Four weeks later, routine hematoxylin-eosin (HE staining, immunohistochemical staining for myocardium-specific cardiac troponin I (cTnI, scanning electron microscopy and transmission electron microscopy were used to analyze the morphology and microconstruction of the EMTs in host rats. Results HE staining showed that the cardiomyocyte-like cells distributed equally in the PLGA scaffold, and the nuclei arranged in the spindle shape. Immunohistochemical staining revealed that majority of engrafted cells in the PLGA -Cardiomyocyte-like cells group were positive for cTnI. Scanning electron microscopy showed that the inoculated cells well attached to PLGA and grew in 3 dimensions in construct. Transmission electron microscopy showed that the EMTs contained well arranged myofilaments paralleled to the longitudinal cell axis, the cells were rich in endoplasmic reticulum and mitochondria, while desmosomes, gap junction and Z line-like substances were also can be observed as well within the engrafted cells. Conclusion We have developed an in vivo method to construct engineered myocardial tissue. The in vivo microenvironment helped engrafted cells/tissue survive and

  3. Human iPS cell-derived insulin producing cells form vascularized organoids under the kidney capsules of diabetic mice.

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    Sudhanshu P Raikwar

    Full Text Available Type 1 diabetes (T1D is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.

  4. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

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    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  5. Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

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    Tomohisa Seki

    Full Text Available Induced pluripotent stem cells (iPSCs have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.

  6. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs.

  7. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

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    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  8. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

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    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.

  9. Impaired SIRT1 promotes the migration of vascular smooth muscle cell-derived foam cells.

    Science.gov (United States)

    Zhang, Ming-Jie; Zhou, Yi; Chen, Lei; Wang, Xu; Pi, Yan; Long, Chun-Yan; Sun, Meng-Jiao; Chen, Xue; Gao, Chang-Yue; Li, Jing-Cheng; Zhang, Li-Li

    2016-07-01

    The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. The previous study demonstrated that vascular smooth muscle cells (VSMCs) contain a much larger burden of the excess cholesterol in comparison with monocyte-derived macrophages in human coronary atherosclerosis, as the main origin of foam cells. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxidized low-density lipoprotein (oxLDL). In conclusion, we provide evidence that oxLDL induces the VSMC-derived foam cells formation with increased migration ability and MMP-9 expression, which were partly attributed to the impaired SIRT1 and enhanced nuclear factor-kappa B (NF-κB) activity. As activation of transient receptor potential vanilloid type 1 (TRPV1) has been reported to have anti-atherosclerotic effects, we investigated its role in oxLDL-treated VSMC migration. It is found that activating TRPV1 by capsaicin inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. The present study provides evidence that SIRT1 may be a promising intervention target of atherosclerosis, and raises the prospect of TRPV1 in prevention and treatment of atherosclerosis.

  10. Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    Han-Tso Lin; Shih-Hwa Chiou; Chung-Lan Kao; Yi-Ming Shyr; Chien-Jen Hsu; Yih-Wen Tarng; Larry L-T Ho; Ching-Fai Kwok; Hung-Hai Ku

    2006-01-01

    AIM: To isolate putative pancreatic stem cells (PSCs)from human adult tissues of pancreas duct using serumfree, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of β-cell differentiation in these PSCs were evaluated as well.METHODS: By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The MatrigelTM was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS)was used to detect the phenotypic markers of putative PSCs.RESULTS: A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin.They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29,CD44, CD49, CD50, CD51, CD62E, PDGFR-α, CD73 (SH2),CD81, CD105(SH3).CONCLUSION: In this study, we successfully isolated PSCs from adult human pancreatic duct by using serumfree medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

  11. Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

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    Busadee Pratumvinit

    2013-01-01

    due to difficulties in isolation, cell heterogeneity, lack of specific markers to identify myocardial endothelial cells, and inadequate conditions to maintain long-term cultures. Herein, we developed a method for isolation, characterization, and expansion of cardiac endothelial cells applicable to study endothelial cell biology and clinical applications such as neoangiogenesis. First, we dissociated the cells from murine heart by mechanical disaggregation and enzymatic digestion. Then, we used flow cytometry coupled with specific markers to isolate endothelial cells from murine hearts. CD45+ cells were gated out to eliminate the hematopoietic cells. CD31+/Sca-1+ cells were isolated as endothelial cells. Cells isolated from atrium grew faster than those from ventricle. Cardiac endothelial cells maintain endothelial cell function such as vascular tube formation and acetylated-LDL uptake in vitro. Finally, cardiac endothelial cells formed microvessels in dorsal matrigel plug and engrafted in cardiac microvessels following intravenous and intra-arterial injections. In conclusion, our multicolor flow cytometry method is an effective method to analyze and purify endothelial cells from murine heart, which in turn can be ex vivo expanded to study the biology of endothelial cells or for clinical applications such as therapeutic angiogenesis.

  12. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence

    Science.gov (United States)

    Wang, Zhe; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-01-01

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 106 cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  13. Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

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    Kati Juuti-Uusitalo

    Full Text Available Retinal pigment epithelial (RPE cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP, the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC and RPE derived from the hESC (hESC-RPE. Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

  14. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  15. Reinnervation of hair cells by neural stem cell-derived neurons

    Institute of Scientific and Technical Information of China (English)

    Yuan Yasheng; Wang Yang; Chi Fanglu

    2014-01-01

    Background Replacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss.However,the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored.The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro.Methods NSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor.Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane.Results At Day 3,the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia.After 9-day co-culture,neurites of NSC-derived neurons predominantly elongated toward hair cells.Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells,indicating the formation of new synaptic connections.After 14-day culture,triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle),indicating the formation of new ribbon synapse.Conclusions NSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration.Whether the newly forming synapse is functional merits further electrophysiological study.

  16. Amnion-Epithelial-Cell-Derived Exosomes Demonstrate Physiologic State of Cell under Oxidative Stress.

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    Samantha Sheller

    Full Text Available At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i exosomes act as carriers of signals in utero-placental compartments and ii exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC. We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H 3, heat shock protein (HSP 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK (P-p38 MAPK co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05. Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined

  17. Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

    Institute of Scientific and Technical Information of China (English)

    TAN YuZhen; ZHAO Yao; WANG HaiJie; ZHOU LiangFu; MAO Ying; LIU Rui; SHU Jia; WANG YongFei

    2008-01-01

    Human cerebral cavernous malformation (CM) is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type Ⅰ gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like struc-tures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to re-ceptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.

  18. Transition of mesenchymal stem/stromal cells to endothelial cells

    NARCIS (Netherlands)

    M. Crisan (Mihaela)

    2013-01-01

    textabstractMesenchymal stem/stromal cells (MSCs) are heterogeneous. A fraction of these cells constitute multipotent cells that can self-renew and mainly give rise to mesodermal lineage cells such as adipocytes, osteocytes and chondrocytes. The ability of MSCs to differentiate into endothelial cell

  19. Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration

    Directory of Open Access Journals (Sweden)

    Kenneth R. Boheler

    2011-01-01

    Full Text Available Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction.

  20. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells.

    Science.gov (United States)

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    2017-01-04

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC-RPE and performs better than often used biodegradable poly(d,l-lactide) (PDLLA) film. Human ESC-RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE-specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC-RPE monolayer in serum-free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC-RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.

  1. The role of tumor cell-derived connective tissue growth factor (CTGF/CCN2) in pancreatic tumor growth

    DEFF Research Database (Denmark)

    Bennewith, Kevin L; Huang, Xin; Ham, Christine M

    2009-01-01

    Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2...... adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms...

  2. Epigenetic Rejuvenation of Mesenchymal Stromal Cells Derived from Induced Pluripotent Stem Cells

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    Joana Frobel

    2014-09-01

    Full Text Available Standardization of mesenchymal stromal cells (MSCs remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns—particularly of DNAm patterns associated with tissue type and aging.

  3. Paracrine Regulation of Steroidogenesis in Theca Cells by Granulosa Cells Derived from Mouse Preantral Follicles.

    Science.gov (United States)

    Liu, Xiaoqiang; Qiao, Pengyun; Jiang, Aifang; Jiang, Junyi; Han, Haiyan; Wang, Li; Ren, Chune

    2015-01-01

    Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development. Androgen secreted by theca cells (TCs) can initiate follicle development and ovulation and provide precursor materials for estrogen synthesis. Therefore, studies on ovarian microenvironment will not only lead to better understanding of the steroidogenesis but also have clinical significance for ovarian endocrine abnormalities such as hyperandrogenism in polycystic ovary syndrome (PCOS). This study applied the Transwell coculture model to investigate if the interaction between granulosa and theca cells may affect androgen production in theca cells. Concentrations of testosterone and androstenedione in the spent medium were measured by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The results show that the coculture with granulosa cells (GCs) increases steroidogenesis in TCs. In addition, testosterone and androstenedione productions in response to LH stimulation were also increased in the coculture model. Significantly increased mRNA expressions of steroidogenic enzymes (Star, Cyp11a1, Cyp17a1, and Hsd3b2) were observed in the cocultured TCs. Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs. This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.

  4. Mesenchymal Stem Cell-Derived Exosomes: New Opportunity in Cell-Free Therapy

    Science.gov (United States)

    Pashoutan Sarvar, Davod; Shamsasenjan, Karim; Akbarzadehlaleh, Parvin

    2016-01-01

    Mesenchymal stromal/stem cells (MSCs) are involved in tissue homeostasis through direct cell-to-cell interaction, as well as secretion of soluble factors. Exosomes are the sort of soluble biological mediators that obtained from MSCs cultured media in vitro. MSC-derived exosomes (MSC-DEs) which produced under physiological or pathological conditions are central mediators of intercellular communications by conveying proteins, lipids, mRNAs, siRNA, ribosomal RNAs and miRNAs to the neighbor or distant cells. MSC-DEs have been tested in various disease models, and the results have revealed that their functions are similar to those of MSCs. They have the supportive functions in organisms such as repairing tissue damages, suppressing inflammatory responses, and modulating the immune system. MSC-DEs are of great interest in the scope of regenerative medicine because of their unique capacity to the regeneration of the damaged tissues, and the present paper aims to introduce MSC-DEs as a novel hope in cell-free therapy.

  5. Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence.

    Science.gov (United States)

    Ibiza, Sales; García-Cassani, Bethania; Ribeiro, Hélder; Carvalho, Tânia; Almeida, Luís; Marques, Rute; Misic, Ana M; Bartow-McKenney, Casey; Larson, Denise M; Pavan, William J; Eberl, Gérard; Grice, Elizabeth A; Veiga-Fernandes, Henrique

    2016-07-21

    Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed, but how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glial–ILC3–epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably, ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit, revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals.

  6. Paracrine Regulation of Steroidogenesis in Theca Cells by Granulosa Cells Derived from Mouse Preantral Follicles

    Directory of Open Access Journals (Sweden)

    Xiaoqiang Liu

    2015-01-01

    Full Text Available Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development. Androgen secreted by theca cells (TCs can initiate follicle development and ovulation and provide precursor materials for estrogen synthesis. Therefore, studies on ovarian microenvironment will not only lead to better understanding of the steroidogenesis but also have clinical significance for ovarian endocrine abnormalities such as hyperandrogenism in polycystic ovary syndrome (PCOS. This study applied the Transwell coculture model to investigate if the interaction between granulosa and theca cells may affect androgen production in theca cells. Concentrations of testosterone and androstenedione in the spent medium were measured by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The results show that the coculture with granulosa cells (GCs increases steroidogenesis in TCs. In addition, testosterone and androstenedione productions in response to LH stimulation were also increased in the coculture model. Significantly increased mRNA expressions of steroidogenic enzymes (Star, Cyp11a1, Cyp17a1, and Hsd3b2 were observed in the cocultured TCs. Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs. This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.

  7. In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer

    Science.gov (United States)

    Veluchamy, John P.; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A. M.; Verheul, Henk M. W.; Di Santo, James P.; de Gruijl, Tanja D.; van der Vliet, Hans J.

    2017-01-01

    Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RASwt metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR− RASwt, EGFR+ RASmut, and EGFR+ BRAFmut cells, A-PBNK were able to initiate lysis of EGFR+ colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR+ colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR−RASwt (42 ± 8 versus 67 ± 7%), EGFR+ RASmut (20 ± 2 versus 37 ± 6%), and EGFR+ BRAFmut (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR+ RASmut colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered. PMID:28220124

  8. Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons.

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    Linn Wicklund

    Full Text Available Cognitive impairment in Alzheimer's disease (AD patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs. Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES cells with nerve growth factor (NGF as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1-40 increased the number of functional neurons, whereas oligomeric Aβ1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1-40 and Aβ1-42 induced gliogenesis. These findings indicate that Aβ1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.

  9. TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells.

    Science.gov (United States)

    Haruta, M; Tomita, Y; Yuno, A; Matsumura, K; Ikeda, T; Takamatsu, K; Haga, E; Koba, C; Nishimura, Y; Senju, S

    2013-05-01

    We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

  10. Immunotherapy against Metastatic Melanoma with Human iPS Cell-Derived Myeloid Cell Lines Producing Type I Interferons.

    Science.gov (United States)

    Miyashita, Azusa; Fukushima, Satoshi; Nakahara, Satoshi; Kubo, Yosuke; Tokuzumi, Aki; Yamashita, Junji; Aoi, Jun; Haruta, Miwa; Senju, Satoru; Nishimura, Yasuharu; Jinnin, Masatoshi; Ihn, Hironobu

    2016-03-01

    In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAF(V600E) kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell-derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma.

  11. Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cell Seeding on Biofunctionalized Calcium Phosphate Cements

    Institute of Scientific and Technical Information of China (English)

    WahWah TheinHan; Jun Liu; Minghui Tang; Wenchuan Chen; Linzhao Cheng; Hockin H. K. Xu

    2013-01-01

    Induced pluripotent stem cells (iPSCs) have great potential due to their proliferation and differentiation capability. The objectives of this study were to generate iPSC-derived mesenchymal stem cells (iPSC-MSCs), and investigate iPSC-MSC proliferation and osteogenic differentiation on calcium phosphate cement (CPC) containing biofunctional agents for the first time. Human iPSCs were derived from marrow CD34+ cells which were reprogrammed by a single episomal vector. iPSCs were cultured to form embryoid bodies (EBs), and MSCs migrated out of EBs. Five biofunctional agents were incorporated into CPC:RGD (Arg-Gly-Asp) peptides, fibronectin (Fn), fibronectin-like engineered polymer protein (FEPP), extracellular matrix Geltrex, and platelet concentrate. iPSC-MSCs were seeded on five biofunctionalized CPCs:CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. iPSC-MSCs on biofunctional CPCs had enhanced proliferation, actin fiber expression, osteogenic differentiation and mineralization, compared to control. Cell proliferation was greatly increased on biofunctional CPCs. iPSC-MSCs underwent osteogenic differentiation with increased alkaline phosphatase, Runx2 and collagen-I expressions. Mineral synthesis by iPSC-MSCs on CPC-Platelets was 3-fold that of CPC control. In conclusion, iPSCs showed high potential for bone engineering. iPSC-MSCs on biofunctionalized CPCs had cell proliferation and bone mineralization that were much better than traditional CPC. iPSC-MSC-CPC constructs are promising to promote bone regeneration in craniofacial/orthopedic repairs.

  12. siRNA targeting stathmin inhibits invasion and enhances chemotherapy sensitivity of stem cells derived from glioma cell lines.

    Science.gov (United States)

    Song, Yuwen; Mu, Luyan; Han, Xuezhe; Liu, Xiaoqian; Fu, Songbin

    2014-12-01

    Glioma is one of the most highly angiogenic tumors, and glioma stem cells (GSCs) are responsible for resistance to chemotherapy and radiotherapy, as well as recurrence after operation. Stathmin is substantial for mitosis and plays an important role in proliferation and migration of glioma-derived endothelial cells. However, the relationship between stathmin and GSCs is incompletely understood. Here we isolated GSCs from glioma cell lines U87MG and U251, and then used siRNA targeting stathmin for silencing. We showed that silencing of stathmin suppressed the proliferation, increased the apoptosis rate, and arrested the cell cycle at G2/M phase in GSCs. Silencing of stathmin in GSCs also resulted in inhibited the migration/invasion as well as the capability of vasculogenic mimicry. The susceptibility of GSCs to temozolomide was also enhanced by stathmin silencing. Our findings suggest stathmin as a potential target in GSCs for glioma treatment.

  13. Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality.

    Science.gov (United States)

    Nakagawa, Kiyotaka; Kiko, Takehiro; Kuriwada, Satoko; Miyazawa, Taiki; Kimura, Fumiko; Miyazawa, Teruo

    2011-01-01

    It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.

  14. Adhesion of endothelial cells and endothelial progenitor cells on peptide-linked polymers in shear flow.

    Science.gov (United States)

    Wang, Xin; Cooper, Stuart

    2013-05-01

    The initial adhesion of human umbilical vein endothelial cells (HUVECs), cord blood endothelial colony-forming cells (ECFCs), and human blood outgrowth endothelial cells (HBOECs) was studied under radial flow conditions. The surface of a variable shear-rate device was either coated with polymer films or covered by synthetic fibers. Spin-coating was applied to produce smooth polymer films, while fibrous scaffolds were generated by electrospinning. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate (PEGMA), and CGRGDS peptide. The peptide was incorporated into the polymer system by coupling to an acrylate-PEG-N-hydroxysuccinimide comonomer. A shear-rate-dependent increase of the attached cells with time was observed with all cell types. The adhesion of ECs increased on RGD-linked polymer surfaces compared to polymers without adhesive peptides. The number of attached ECFCs and HBOECs are significantly higher than that of HUVECs within the entire shear-rate range and surfaces examined, especially on RGD-linked polymers at low shear rates. Their superior adhesion ability of endothelial progenitor cells under flow conditions suggests they are a promising source for in vivo seeding of vascular grafts and shows the potential to be used for self-endothelialized implants.

  15. Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.

    Science.gov (United States)

    Feng, Jun-Feng; Liu, Jing; Zhang, Xiu-Zhen; Zhang, Lei; Jiang, Ji-Yao; Nolta, Jan; Zhao, Min

    2012-02-01

    Small direct current (DC) electric fields (EFs) guide neurite growth and migration of rodent neural stem cells (NSCs). However, this could be species dependent. Therefore, it is critical to investigate how human NSCs (hNSCs) respond to EF before any possible clinical attempt. Aiming to characterize the EF-stimulated and guided migration of hNSCs, we derived hNSCs from a well-established human embryonic stem cell line H9. Small applied DC EFs, as low as 16 mV/mm, induced significant directional migration toward the cathode. Reversal of the field polarity reversed migration of hNSCs. The galvanotactic/electrotactic response was both time and voltage dependent. The migration directedness and distance to the cathode increased with the increase of field strength. (Rho-kinase) inhibitor Y27632 is used to enhance viability of stem cells and has previously been reported to inhibit EF-guided directional migration in induced pluripotent stem cells and neurons. However, its presence did not significantly affect the directionality of hNSC migration in an EF. Cytokine receptor [C-X-C chemokine receptor type 4 (CXCR4)] is important for chemotaxis of NSCs in the brain. The blockage of CXCR4 did not affect the electrotaxis of hNSCs. We conclude that hNSCs respond to a small EF by directional migration. Applied EFs could potentially be further exploited to guide hNSCs to injured sites in the central nervous system to improve the outcome of various diseases.

  16. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

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    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

  17. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  18. A subpopulation of large granular von Willebrand Ag negative and CD105 positive endothelial cells, isolated from abdominal aortic aneurysms, overexpress ICAM-1 and Fas antigen.

    Science.gov (United States)

    Páez, Araceli; Archundia, Abel; Méndez Cruz, René; Rodríguez, Emma; López Marure, Rebeca; Masso, Felipe; Aceves, José Luis; Flores, Leopoldo; Montaño, Luis F

    2002-01-01

    The aim of this work was to determine whether there is a pre-established basal condition of the endothelial cells isolated from aortic abdominal aneurysm that might augment immune effector mechanisms and thus provide us an insight into the possible causes of aneurysm rupture. Endothelial cells isolated from saccular aortic aneurysm fragments were analyzed by cytofluorometry for the expression of different immune response-related molecules. Our results showed that there is a subpopulation of granule-rich, CD105 positive and von Willebrand antigen negative endothelial cells that have an enhanced basal expression of ICAM-1, and Fas antigen, but, interestingly, no apoptotic bodies were detected. Control endothelial cells derived from healthy areas of the same abdominal aortas did not show such enhanced expression. We conclude that in the endothelium that lines abdominal aorta aneurysms there is, at least, one endothelial cell subpopulation with an apparent inhibition of programmed cell death and in a proinflammatory activation status.

  19. The Effect of the LysoPC-induced Endothelial Cell Conditioned Medium on Proliferating Cell Nuclear Antigen Expression of the Calf Thoracic Aorta Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    周洪莲; 姚济华; 余枢

    2002-01-01

    In order to study the effect of and mechanism of lysophosphatidylcholine (LysoPC) on proliferation of the calf thoracic aorta smooth muscle cells (ASMCs), the ASMCs were used to observe the effects of LysoPC-induced endothelial cell conditioned medium on the DNA content and proliferating cell nuclear antigen (PCNA) expression in the calf thoracic ASMCs by flow cytometry and Western Blot technique. It was found that LysoPC-induced endothelial cell conditioned medium could significantly promote PCNA expression of the calf ASMCs, induce the converting of ASMCs from G0/G1 phase to S phase of DNA synthesis, and increase the tyrosine phosphorylation protein expression. Tyrosine protein kinase inhibitor (TPKi) RG50864 could obviously inhibit proliferation of LysoPC-induced ASMCs in a dose-dependence manner. The results indicated that the effect of LysoPC promoting the proliferation of ASMCs is partly evoked by endothelial cell derived growth factors such as PDGF and so on.

  20. Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

    Science.gov (United States)

    Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

    2016-01-01

    Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

  1. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.

    Science.gov (United States)

    Böcker, Wolfgang; Yin, Zhanhai; Drosse, Inga; Haasters, Florian; Rossmann, Oliver; Wierer, Matthias; Popov, Cvetan; Locher, Melanie; Mutschler, Wolf; Docheva, Denitsa; Schieker, Matthias

    2008-08-01

    Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

  2. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    Science.gov (United States)

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

    2016-01-01

    The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

  3. Optimization of the tissue source, malignancy, and initial substrate of tumor cell-derived matrices to increase cancer cell chemoresistance against 5-fluorouracil.

    Science.gov (United States)

    Hoshiba, Takashi; Tanaka, Masaru

    2015-02-13

    The low chemoresistance of in vitro cancer cells inhibits the development of new anti-cancer drugs. Thus, development of a new in vitro culture system is required to increase the chemoresistance of in vitro cancer cells. Tumor cell-derived matrices have been reported to increase the chemoresistance of in vitro cancer cells. However, it remains unclear how tissue sources and the malignancy of cells used for the preparation of matrices affect the chemoresistance of tumor cell-derived matrices. Moreover, it remains unclear how the initial substrates used for the preparation of matrices affect the chemoresistance. In this study, we compared the effects of tissue sources and the malignancy of tumor cells, as well as the effect of the initial substrates on chemoresistance against 5-fluorouracil (5-FU). The chemoresistance of breast and colon cancer cells against 5-FU increased on matrices prepared with cells derived from the corresponding original tissues with higher malignancy. Moreover, the chemoresistance against 5-FU was altered on matrices prepared using different initial substrates that exhibited different characteristics of protein adsorption. Taken together, these results indicated that the appropriate selection of tissue sources, malignancy of tumor cells, and initial substrates used for matrix preparation is important for the preparation of tumor cell-derived matrices for chemoresistance assays.

  4. Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

    Science.gov (United States)

    Fuerstenau-Sharp, Maya; Zimmermann, Martina E; Stark, Klaus; Jentsch, Nico; Klingenstein, Melanie; Drzymalski, Marzena; Wagner, Stefan; Maier, Lars S; Hehr, Ute; Baessler, Andrea; Fischer, Marcus; Hengstenberg, Christian

    2015-01-01

    Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

  5. Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Maya Fuerstenau-Sharp

    Full Text Available Induced pluripotent stem (iPS cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V. In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

  6. Effector cells derived from naive T cells used in tumor immunotherapy of mice bearing B16 melanoma

    Institute of Scientific and Technical Information of China (English)

    Wen Ming; Xu Weili; Ren Lili; Gao Fei; Cui Naipeng; Wen Junye; Li Xinjiang

    2014-01-01

    Background Adoptive cell transfer (ACT) immunotherapy has been used clinically for years to treat malignancies.Improving the killing efficiency of effector cells,such as tumor-specific cytotoxic T lymphocytes (CTLs),is an important component for enhancing the clinical response of cancer immunotherapy.Hence,we explored a novel method for preparing cancer-specific CTLs using naive T lymphocytes.Methods C57BL/6 mice bearing B16 melanoma tumors were pretreated with cyclophosphamide (CTX) by peritoneal injection.The immunosuppressive influence of CTX on tumor regression and the tumor microenvironment was assessed.Naive T cells and T cell pools were isolated via negative selection using immunomagnetic beads.The proliferative potential and cytokine production of different T cell subpopulations were evaluated in vitro.Tumor-specific CTLs derived from naive T cells (naive CD4+ T cells:naive CD8+ T cells=2:1) and pooled T cells were generated in vitro,respectively.B16 melanoma-bearing C57BL/6 mice were pretreated with CTX,followed by ACT immunotherapy using dendritic cell-induced CTLs.The homing abilities of the effector cells and interleukin-2 (IL-2),interferon-y,granzyme B,and perforin mRNA levels in tumor tissues were evaluated,and the change in tumor volume was measured.Results Mice receiving CTX peritoneal pretreatment injections did not display tumor regression compared with control mice.However,a significant downregulation of splenic Tregs and tumor growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) serum levels was observed (P <0.05).Naive T cells showed a stronger proliferative capacity and elevated cytokine production than did pooled T cells (P <0.05).In addition,effector cells generated from naive T cells displayed more potent antitumor activity in vivo than those derived from pooled T cells (P <0.05).Conclusion Effector cells derived from the naive T cells possess a stronger proliferative potential,homing capacity,and enhanced cytokine production

  7. Phenotypic and functional characterization of cytokine-induced killer cells derived from preterm and term infant cord blood.

    Science.gov (United States)

    Zhang, Qian; Wang, Lili; Luo, Chenghan; Shi, Zanyang; Cheng, Xinru; Zhang, Zhen; Yang, Yi; Zhang, Yi

    2014-11-01

    Cord blood has gradually become an important source for hematopoietic stem cell transplantation (HSCT) in the human, particularly in pediatric patients. Adoptive cellular immunotherapy of patients with hematologic malignancies after umbilical cord blood transplant is crucial. Cytokine‑induced killer (CIK) cells derived from cord blood are a new type of antitumor immune effector cells in tumor prevention and treatment and have increasingly attracted the attention of researchers. On the other hand, it has been suggested that preterm infant cord blood retains an early differentiation phenotype suitable for immunotherapy. Therefore, we determined the phenotypic and functional characterization of CIK cells derived from preterm infant cord blood (PCB-CIK) compared with CIK cells from term infant cord blood (TCB-CIK). Twenty cord blood samples were collected and classified into two groups based on gestational age. Cord blood mononuclear cells (CBMCs) were isolated, cultured and induced to CIK cells in vitro. We used flow cytometry to detect cell surface markers, FlowJo software to analyze the proliferation profile and intracellular staining to test the secretion of cytokines. Finally, we evaluated the antitumor activity of CIK cells against K562 in vitro. Compared with TCB-CIK, PCB-CIK cells demonstrated faster proliferation and higher expression of activated cell surface markers. The secretion of IL-10 was lower in PCB-CIK cells while the expression of perforin and CD107a had no significant difference between the two cell groups. PCB-CIK cells exhibited a high proliferation rate while the cytotoxic activity had no difference between the PCB-CIK and TCB-CIK cells. Hence preterm infant cord blood may be a potential source for immunotherapy.

  8. Cobalt protoporphyrin pretreatment protects human embryonic stem cell-derived cardiomyocytes from hypoxia/reoxygenation injury in vitro and increases graft size and vascularization in vivo.

    Science.gov (United States)

    Luo, Jun; Weaver, Matthew S; Cao, Baohong; Dennis, James E; Van Biber, Benjamin; Laflamme, Michael A; Allen, Margaret D

    2014-06-01

    Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can regenerate infarcted myocardium. However, when implanted into acutely infarcted hearts, few cells survive the first week postimplant. To improve early graft survival, hESC-CMs were pretreated with cobalt protoporphyrin (CoPP), a transcriptional activator of cytoprotective heme oxygenase-1 (HO-1). When hESC-CMs were challenged with an in vitro hypoxia/reoxygenation injury, mimicking cell transplantation into an ischemic site, survival was significantly greater among cells pretreated with CoPP versus phosphate-buffered saline (PBS)-pretreated controls. Compared with PBS-pretreated cells, CoPP-pretreated hESC-CM preparations exhibited higher levels of HO-1 expression, Akt phosphorylation, and vascular endothelial growth factor production, with reduced apoptosis, and a 30% decrease in intracellular reactive oxygen species. For in vivo translation, 1 × 10(7) hESC-CMs were pretreated ex vivo with CoPP or PBS and then injected intramyocardially into rat hearts immediately following acute infarction (permanent coronary ligation). At 1 week, hESC-CM content, assessed by quantitative polymerase chain reaction for human Alu sequences, was 17-fold higher in hearts receiving CoPP- than PBS-pretreated cells. On histomorphometry, cardiomyocyte graft size was 2.6-fold larger in hearts receiving CoPP- than PBS-pretreated cells, occupying up to 12% of the ventricular area. Vascular density of host-perfused human-derived capillaries was significantly greater in grafts composed of CoPP- than PBS-pretreated cells. Taken together, these experiments demonstrate that ex vivo pretreatment of hESC-CMs with a single dose of CoPP before intramyocardial implantation more than doubled resulting graft size and improved early graft vascularization in acutely infarcted hearts. These findings open the door for delivery of these, or other, stem cells during acute interventional therapy following myocardial infarction or ischemia.

  9. Cervical cancer cell-derived interleukin-6 impairs CCR7-dependent migration of MMP-9-expressing dendritic cells.

    Science.gov (United States)

    Pahne-Zeppenfeld, Jennifer; Schröer, Nadine; Walch-Rückheim, Barbara; Oldak, Monika; Gorter, Arko; Hegde, Subramanya; Smola, Sigrun

    2014-05-01

    Cervical carcinogenesis is a consequence of persistent infection with high-risk human papillomaviruses (HPVs). Recent studies indicate that HPV-transformed cells actively instruct their microenvironment to promote carcinogenesis. Here, we demonstrate that cervical cancer cells activate monocytes to produce their own CCL2 for further monocyte recruitment and reprogram their function during differentiation and maturation to dendritic cells (DCs). Our data show that cervical cancer cells suppress the induction of the chemokine receptor CCR7 in phenotypically mature DCs and impair their migration toward a lymph node homing chemokine, required to initiate adaptive immune responses. We confirmed the presence of CD83(+)CCR7(low) DCs in cancer biopsies. The second factor essential for DC migration, matrix-metalloproteinase MMP-9, which also has vasculogenic and protumorigenic properties, is not suppressed but upregulated in immature as well as mature DCs. We identified interleukin-6 (IL-6) as a crucial cervical cancer cell-derived mediator and nuclear factor kappaB (NF-jB) as the central signaling pathway targeted in DCs. Anti-IL-6 antibodies reverted not only NF-jB inhibition and restored CCR7-dependent migration but also blocked MMP-9 induction. This is the first report demonstrating the dissociation of CCR7 and MMP-9 expression in phenotypically mature CD83(+) DCs by cancer cells. Our results show that cervical cancer cells actively shape the local microenvironment. They induce the accumulation of myeloid cells and skew their function from immune activation to local production of protumorigenic MMP-9. Neutralizing anti-IL-6 antibodies can counteract this functional dysbalance and should therefore be considered for adjuvant cervical cancer therapy.

  10. A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy

    Directory of Open Access Journals (Sweden)

    Miki Ando

    2015-10-01

    Full Text Available The discovery of induced pluripotent stem cells (iPSCs has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9 into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

  11. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    Science.gov (United States)

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.

  12. Endothelial progenitor cells with Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    KONG Xiao-dong; ZHANG Yun; LIU Li; SUN Ning; ZHANG Ming-yi; ZHANG Jian-ning

    2011-01-01

    Background Endothelial dysfunction is thought to be critical events in the pathogenesis of Alzheimer's disease (AD).Endothelial progenitor cells (EPCs) have provided insight into maintaining and repairing endothelial function. To study the relation between EPCs and AD, we explored the number of circulating EPCs in patients with AD.Methods A total of 104 patients were recruited from both the outpatients and inpatients of the geriatric neurology department at General Hospital, rianjin Medical University. Consecutive patients with newly diagnosed AD (n=30),patients with vascular dementia (VaD, n=34), and healthy elderly control subjects with normal cognition (n=40) were enrolled after matching for age, gender, body mass index, medical history, current medication and Mini Mental State Examination. Middle cerebral artery flow velocity was examined with transcranial Doppler. Endothelial function was evaluated according to the level of EPCs, and peripheral blood EPCs was counted by flow cytometry.Results There were no significant statistical differences of clinical data in AD, VaD and control groups (P >0.05). The patients with AD showed decreased CD34-positive (CD34+) or CD133-positive (CD133+) levels compared to the control subjects, but there were no significant statistical differences in patients with AD. The patients with AD had significantly lower CD34+CD133+ EPCs(CD34 and CD133 double positive endothelial progenitor cells) than the control subjects (P <0.05). In the patients with AD, a lower CD34+CD133+ EPCs count was independently associated with a lower Mini-Mental State Examination score (r=0.514, P=0.004). Patients with VaD also showed a significant decrease in CD34+CD133+ EPCs levels, but this was not evidently associated with the Mini-Mental State Examination score. The changes of middle cerebral artery flow velocity were similar between AD and VaD. Middle cerebral artery flow velocity was decreased in the AD and VaD groups and significantly lower than

  13. Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering.

    Science.gov (United States)

    Lowenthal, Justin; Gerecht, Sharon

    2016-05-01

    Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine - including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering - have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies.

  14. Human embryonic stem cell-derived oligodendrocyte progenitors aid in functional recovery of sensory pathways following contusive spinal cord injury.

    Directory of Open Access Journals (Sweden)

    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

  15. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, p<0.02) resulting in greater left-ventricular mass (1.24 [0.29] vs 1.57 [0.27] g) and better cardiac function (shortening fraction 9.2 [4.9] vs 17.2 [4.2]%, n=8 per group, p<0.05). INTERPRETATION: These findings show that SDF-1 is sufficient to induce therapeutic stem-cell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  16. The Role of Tumor Cell-Derived Connective Tissue Growth Factor (CTGF/CCN2) in Pancreatic Tumor Growth

    Science.gov (United States)

    Bennewith, Kevin L.; Huang, Xin; Ham, Christine M.; Graves, Edward E.; Erler, Janine T.; Kambham, Neeraja; Feazell, Jonathan; Yang, George P.; Koong, Albert

    2009-01-01

    Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted subcutaneously. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by PET imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed co-localization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer. PMID:19179545

  17. Angiogenesis and collagen type IV expression in different endothelial cell culture systems.

    Science.gov (United States)

    Bahramsoltani, M; Slosarek, I; De Spiegelaere, W; Plendl, J

    2014-04-01

    In vitro angiogenesis assays constitute an important tool for studying the mechanisms of angiogenesis and for identification of pro- and anti-angiogenic substances. Therefore, endothelial cell and media systems used for in vitro angiogenesis assays are required to mimic the angiogenic process in vivo including endothelial capability to express collagen type IV as a component of the basement membrane. In this study, the expression of collagen type IV and its α chains (α1-6) was investigated in different endothelial cell culture systems in vitro qualitatively and quantitatively. These systems included four different batches of microvascular endothelial cells derived from the human skin, heart and lung, from which only two batches were found to be angiogenic and two batches were classified as non-angiogenic. Distribution of the transcripts of the α chains of collagen type IV was similar in all cell and media systems investigated. However, secretion and deposition of a stable extracellular network of collagen type IV could only be observed in the angiogenic cultures. In conclusion, the consecutive steps of the angiogenic cascade in vivo as well as in vitro depend on an increasing secretion and subsequent extracellular deposition of collagen type IV.

  18. Endothelial cells, tissue factor and infectious diseases

    Directory of Open Access Journals (Sweden)

    Lopes-Bezerra L.M.

    2003-01-01

    Full Text Available Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

  19. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  20. Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation

    DEFF Research Database (Denmark)

    Nielsen, Christoffer T; Østergaard, Ole; Stener, Line

    2012-01-01

    To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters.......To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters....

  1. Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord.

    Science.gov (United States)

    Zhang, Haoqian; Trivedi, Alpa; Lee, Jung-Uek; Lohela, Marja; Lee, Sang Mi; Fandel, Thomas M; Werb, Zena; Noble-Haeusslein, Linda J

    2011-11-01

    The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

  2. Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.

    Science.gov (United States)

    Donegan, J J; Tyson, J A; Branch, S Y; Beckstead, M J; Anderson, S A; Lodge, D J

    2016-08-02

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that the stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia.Molecular Psychiatry advance online publication, 2 August 2016; doi:10.1038/mp.2016.121.

  3. Structural differentiation, proliferation, and association of human embryonic stem cell-derived cardiomyocytes in vitro and in their extracardiac tissues.

    Science.gov (United States)

    Cui, Li; Johkura, Kohei; Takei, Shunsuke; Ogiwara, Naoko; Sasaki, Katsunori

    2007-06-01

    The proliferation, structural differentiation, and capacity of association of human ES cell-derived cardiomyocytes were assessed in culture and in extracardiac graft tissues. Embryoid body (EB) outgrowths having cardiomyocytes, and their transplants in mice retroperitoneum or renal subcapsular region were analyzed mainly by immunochemistry. During the culture of EB outgrowths, colonies of cardiomyocytes grew in size exhibiting synchronized beatings. Subcellular structures of those cardiomyocytes involved in the contraction, hormone production, and intercellular integration differentiated with distinct immunoreactivity for constituent proteins/peptides. Judging from PCNA staining, proliferation potential was maintained in part for more than 70 days. In teratoma tissues on post-transplantation Day 7, cardiomyocytes maintained their integration with connexin 43 and cadherin at their junctions. They partly exhibited strong PCNA reactivity. On Day 28, large part of the cardiomyocytes lost their association, dispersing among non-cardiac cells without discernible cadherin reactivity. Proliferation potential was generally low irrespective of their tissue diversity. From these results, structural differentiation and active proliferation of human ES cell-derived cardiomyocytes occurred in vitro, maintaining their association. When developed in extracardiac tissues, however, the cardiomyocytes showed low proliferation potential and reduced cellular integration. This leads to the proposal that some procedure will be necessary to accelerate or maintain the proliferation of cardiomyocytes in vivo.

  4. Mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs induce immune modulatory profile in monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Fernando de Sá Silva

    Full Text Available BACKGROUND: Mesenchymal stem cells have prominent immune modulatory properties, which may have clinical applications; however their major source, bone marrow, is of limited availability. On the other hand, mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs are readily accessible, but their immune regulatory properties have not been completely investigated. This study was designed, therefore, to evaluate the SHEDs influence on DCs differentiation, maturation, ability to activate T cells and to expand CD4(+Foxp3(+ T cells. METHODOLOGY/PRINCIPAL FINDINGS: The experiments were based in cellular co-culture during differentiation and maturation of monocyte derived-DCs (moDCs, with, or not, presence of SHEDs. After co-culture with SHEDs, (moDCs presented lower expression of BDCA-1 and CD11c, in comparison to DC cultivated without SHEDs. CD40, CD80, CD83 and CD86 levels were also decreased in mature DCs (mDCs after co-cultivation with SHEDs. To assess the ability of SHEDs-exposed moDCs to modulate T cell responses, the former were separated from SHEDs, and co-cultured with peripheral blood lymphocytes. After 5 days, the proliferation of CD4(+ and CD8(+ T cells was evaluated and found to be lower than that induced by moDCs cultivated without SHEDs. In addition, an increase in the proportion of CD4(+Foxp3(+IL-10(+ T cells was observed among cells stimulated by mature moDCs that were previously cultivated with SHEDs. Soluble factors released during co-cultures also showed a reduction in the pro-inflammatory cytokines (IL-2, TNF-α and IFN-γ, and an increase in the anti-inflammatory molecule IL-10. CONCLUSION/SIGNIFICANCE: This study shows that SHEDs induce an immune regulatory phenotype in moDCs cells, evidenced by changes in maturation and differentiation rates, inhibition of lymphocyte stimulation and ability to expand CD4(+Foxp3(+ T cells. Further characterization and validation of this phenomenon could support the use of SHEDs

  5. Endothelial Progenitor Cells Enter the Aging Arena.

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    Kate eWilliamson

    2012-02-01

    Full Text Available Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells.In this review, we discuss the current understanding of the cells termed ‘EPCs’, examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

  6. Inhibitory Effects of Fenofibrate on Plasminogen Activator Inhibitor-1 Expression in Human Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    DONG Chunxia; HU Yu; WANG Huafang; SUN Chunyan; WANG Yadan; HE Wenjuan; ZHANG Xiaoping

    2006-01-01

    The effects of fenofibrate on plasminogen activator inhibitor-1 (PAI-1) expression in human umbilical endothelial cell-derived transformed cell line-ECV 304 cells were investigated. ECV 304 cells were incubated with different concentrations of fenofibrate (0, 10, 50, 100 μmol/L) for 24 h. PAI-1 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Westernblot respectively. PAI-1 antigenic content of endothelial cells was measured by using ELISA. Fenofibrate could inhibit the PAI-1 mRNA and protein expression and reduce PAI-1 antigenic content dependently. After treatment with fenofibrate (10 μmol/L), the expression levels of PAI-1 mRNA and protein were 0.65±0.05 and 0.96±0.11 respectively, significantly lower than in the control group (0.78±0.03 and 1.21±0.15, respectively, P<0.05). PAI-1 antigenie contents (24.52±8.39) in ECV304 cells treated with 10 μmol/L fenofibrate were significantly lower than those in the control group (6.98±5.12, P<0.05). It was concluded that fenofibrate inhibited the expression of PAI-1 mRNA in ECV304 cells, and reduce the protein expression and the antigenic content of PAI-1, suggesting that fenofibrate may have an antiatherosclerotic effect on endothelial cells by PAI-1 pathway.

  7. Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain.

    Science.gov (United States)

    Gauthier, Sebastien A; Sahoo, Susmita; Jung, Sonia S; Levy, Efrat

    2012-01-01

    The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic "hill and valley" morphology and are immunoreactive for the smooth muscle cell marker α-actin. Endothelial cells display a "cobblestone" pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.

  8. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

    Science.gov (United States)

    Salomon, Carlos; Torres, Maria Jose; Kobayashi, Miharu; Scholz-Romero, Katherin; Sobrevia, Luis; Dobierzewska, Aneta; Illanes, Sebastian E; Mitchell, Murray D; Rice, Gregory E

    2014-01-01

    Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6-12 weeks), second (ST, 22-24 weeks) and third (TT, 32-38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (pexosomes present in maternal plasma increased significantly with gestational age by more that two-fold (pExosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

  9. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

    Directory of Open Access Journals (Sweden)

    Carlos Salomon

    Full Text Available Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group were obtained from non-pregnant and pregnant women in the first (FT, 6-12 weeks, second (ST, 22-24 weeks and third (TT, 32-38 weeks trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP, respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte. Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001. During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001. Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

  10. Enhancing endothelial progenitor cell for clinical use

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Circulating endothelial progenitor cells (EPCs) havebeen demonstrated to correlate negatively with vascularendothelial dysfunction and cardiovascular risk factors.However, translation of basic research into the clinicalpractice has been limited by the lack of unambiguousand consistent definitions of EPCs and reduced EPCcell number and function in subjects requiring them forclinical use. This article critically reviews the definitionof EPCs based on commonly used protocols, their valueas a biomarker of cardiovascular risk factor in subjectswith cardiovascular disease, and strategies to enhanceEPCs for treatment of ischemic diseases.

  11. Role of mast cell- and non-mast cell-derived inflammatory mediators in immunologic induction of synaptic plasticity

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    A.A.C. Albuquerque

    1997-07-01

    Full Text Available We have previously discovered a long-lasting enhancement of synaptic transmission in mammal autonomic ganglia caused by immunological activation of ganglionic mast cells. Subsequent to mast cell activation, lipid and peptide mediators are released which may modulate synaptic function. In this study we determined whether some mast cell-derived mediators, prostaglandin D2 (PGD2; 1.0 µM, platelet aggregating factor (PAF; 0.3 µM and U44619 (a thromboxane analogue; 1.0 µM, and also endothelin-1 (ET-1; 0.5 µM induce synaptic potentiation in the guinea pig superior cervical ganglion (SCG, and compared their effects on synaptic transmission with those induced by a sensitizing antigen, ovalbumin (OVA; 10 µg/ml. The experiments were carried out on SCGs isolated from adult male guinea pigs (200-250 g actively sensitized to OVA, maintained in oxygenated Locke solution at 37oC. Synaptic potentiation was measured through alterations of the integral of the post-ganglionic compound action potential (CAP. All agents tested caused long-term (LTP; duration ³30 min or short-term (STP; <30 min potentiation of synaptic efficacy, as measured by the increase in the integral of the post-ganglionic CAP. The magnitude of mediator-induced potentiation was never the same as the antigen-induced long-term potentiation (A-LTP. The agent that best mimicked the antigen was PGD2, which induced a 75% increase in CAP integral for LTP (antigen: 94% and a 34% increase for STP (antigen: 91%. PAF-, U44619-, and ET-1-induced increases in CAP integral ranged for LTP from 34 to 47%, and for STP from 0 to 26%. These results suggest that the agents investigated may participate in the induction of A-LTP

  12. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

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    Jan David Kijlstra

    2015-12-01

    Full Text Available The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

  13. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  14. A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes

    Directory of Open Access Journals (Sweden)

    Iwamuro Masaya

    2012-12-01

    Full Text Available Abstract Background Bioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells. Methods Mouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 μm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope. Results At the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture

  15. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    Science.gov (United States)

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  16. Meet the inlaws: Embryonic stem cell derivatives meet the immune system

    Institute of Scientific and Technical Information of China (English)

    William B Tabayoyong; Nicholas Zavazava

    2009-01-01

    @@ Since the derivation of embryonic stem (ES) cell lines from human blasto-cysts in 1998 [1], ES cells have emerged as a potential source of cells and tissues that could be used for cell replacement therapy of incurable degenerative diseases. This is due to their remarkable pluripotency, which enables them to differentiate into any adult cell type of the three embryonal germ layers.

  17. Pluripotency of adult stem cells derived from human and rat pancreas

    Science.gov (United States)

    Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

    Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

  18. In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

    Science.gov (United States)

    Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

    2012-05-01

    In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

  19. Are globoseries glycosphingolipids SSEA-3 and -4 markers for stem cells derived from human umbilical cord blood?

    Institute of Scientific and Technical Information of China (English)

    Heli Suila; Jari Natunen; Saara Laitinen; Leena Valmu; Virve Pitk(a)nen; Tia Hirvonen; Annamari Heiskanen; Heidi Anderson; Anita Laitinen; Suvi Natunen; Halina Miller-Podraza; Tero Satomaa

    2011-01-01

    Umbilical cord blood (UCB) is an efficient and valuable source of hematopoietic stem cells (HSCs) for transplantation. In addition to HSCs it harbours low amounts of mesenchymal stem cells (MSCs). No single marker to identify cord blood-derived stem cells, or to indicate their multipotent phenotype, has been characterized so far. SSEA-3 and -4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells, where SSEA-3 rapidly disappears when the cells start to differentiate. Lately SSEA-3 and -4 have also been observed in MSCs. As there is an ongoing discussion and variation of stem-cell markers between laboratories, we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB. We have performed complementary analysis using gene expression analysis, mass spectrometry and immunochemical methods, including both flow cytometry and immunofluoresence microscopy. SSEA-4, but not SSEA-3, was expressed on MSCs but absent from HSCs. Our findings indicate that SSEA-3 and/or -4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood, as their expression may be altered by cell-culture conditions.

  20. Metastases of Renal Cell Carcinoma to the Thyroid Gland with Synchronous Benign and Malignant Follicular Cell-Derived Neoplasms

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    Carlos Zamarrón

    2013-01-01

    Full Text Available Clear cell renal cell carcinoma (CCRCC is the most common origin for metastasis in the thyroid. A 51-year-old woman was referred to our hospital for a subcarinal lesion. Ten years before, the patient had undergone a nephrectomy for CCRCC. Whole-body fluorodeoxyglucose positron emission tomography revealed elevated values in the thyroid gland, while the mediastinum was normal. An endoscopic ultrasonography-guided fine-needle aspiration biopsy of the mediastinal mass was consistent with CCRCC, and this was confirmed after resection. The thyroidectomy specimen also revealed lymphocytic thyroiditis, nodular hyperplasia, one follicular adenoma, two papillary microcarcinomas, and six foci of metastatic CCRCC involving both thyroid lobes. Curiously two of the six metastatic foci were located inside two adenomatoid nodules (tumor-in-tumor. The metastatic cells were positive for cytokeratins, CD10, epidermal growth factor receptor, and vascular endothelial growth factor receptor 2. No BRAF gene mutations were found in any of the primary and metastatic lesions. The patient was treated with sunitinib and finally died due to CCRCC distant metastases 6 years after the thyroidectomy. In CCRCC patients, a particularly prolonged survival rate may be achieved with the appropriate therapy, in contrast to the ominous prognosis typically found in patients with thyroid metastases from other origins.

  1. Increased stromal-cell-derived factor 1 enhances the homing of bone marrow derived mesenchymal stem cells in dilated cardiomyopathy in rats

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yan-li; Michael Fu; ZHANG Hai-feng; LI Xin-li; DI Ruo-min; YAO Wen-ming; LI Dian-fu; FENG Jian-lin; HUANG Jun; CAO Ke-jiang

    2010-01-01

    Background Stem cell transplantation has been shown to have beneficial effects on dilated cardiomyopathy. However,mechanism for stem cell homing to cardiac tissue in dilated cardiomyopathy has not yet been elucidated.Methods Mesenchymal stem cells were obtained from rat bone marrow, expanded in vitro, and labeled with 99mTc.Cardiomyopathy model was induced by doxorubicin in rats. 99mTc labeled cells were infused into the left ventricles in cardiomyopathy and control rats. Sixteen hours after injection, animals were sacrificed and different tissues were harvested to measure specific radioactivity. By use of real-time polymerase chain reaction and immunohistochemistry,Mrna and protein expressions for stromal-cell-derived factor 1 in cardiac tissue were measured.Results Labeling efficiency of mesenchymal stem cells was (70.0±11.2)%. Sixteen hours after mesenchymal stem cell transplantation, the heart-to-muscle radioactivity ratio was increased significantly in cardiomyopathy hearts as compared to control hearts. Both Mrna and rotein expressions of stromal-cell-derived factor 1 were up-regulated in cardiomyopathy hearts as compared with control hearts.Conclusion In dilated cardiomyopathy induced by doxorubicin up-regulated expression of stromal-cell-derived factor 1in heart may induce mesenchymal stem cells home to the heart.

  2. Human embryonic stem cell-derived hematopoietic cells maintain core epigenetic machinery of the polycomb group/Trithorax Group complexes distinctly from functional adult hematopoietic stem cells.

    Science.gov (United States)

    Schnerch, Angelique; Lee, Jung Bok; Graham, Monica; Guezguez, Borhane; Bhatia, Mickie

    2013-01-01

    Hematopoietic cells derived from human embryonic stem cells (hESCs) have a number of potential utilities, including the modeling of hematological disorders in vitro, whereas the use for cell replacement therapies has proved to be a loftier goal. This is due to the failure of differentiated hematopoietic cells, derived from human pluripotent stem cells (hPSCs), to functionally recapitulate the in vivo properties of bona fide adult hematopoietic stem/progenitor cells (HSPCs). To better understand the limitations of differentiation programming at the molecular level, we have utilized differential gene expression analysis of highly purified cells that are enriched for hematopoietic repopulating activity across embryonic, fetal, and adult human samples, including in vivo explants of human HSPCs 8-weeks post-transplantation. We reveal that hESC-derived hematopoietic progenitor cells (eHPCs) fail to express critical transcription factors which are known to govern self-renewal and myeloid/lymphoid development and instead retain the expression of Polycomb Group (PcG) and Trithorax Group (TrxG) factors which are more prevalent in embryonic cell types that include EZH1 and ASH1L, respectively. These molecular profiles indicate that the differential expression of the core epigenetic machinery comprising PcGs/TrxGs in eHPCs may serve as previously unexplored molecular targets that direct hematopoietic differentiation of PSCs toward functional HSPCs in humans.

  3. Epigenetic regulation of cardiac progenitor cells marker c-kit by stromal cell derived factor-1α.

    Directory of Open Access Journals (Sweden)

    Zhongpu Chen

    Full Text Available BACKGROUND: Cardiac progenitor cells (CPCs have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF, are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α could enhance the expression of c-kit. However, the mechanism is unknown. METHODS AND RESULTS: CPCs were isolated from adult mouse hearts, c-kit(+ and c-kit(- CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+CPCs, made c-kit(-CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+ and c-kit(- CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+ and c-kit(- CPCs. CONCLUSIONS: SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+CPCs and make c-kit(-CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT

  4. 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.

  5. B cell-derived transforming growth factor-β1 expression limits the induction phase of autoimmune neuroinflammation

    Science.gov (United States)

    Bjarnadóttir, Kristbjörg; Benkhoucha, Mahdia; Merkler, Doron; Weber, Martin S.; Payne, Natalie L.; Bernard, Claude C. A.; Molnarfi, Nicolas; Lalive, Patrice H.

    2016-01-01

    Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-β1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-β1 expression in B cells (B–TGF-β1−/−) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B–TGF-β1−/− mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B–TGF-β1−/− mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-β1–deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-β1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-β1, findings that may be relevant to B cell-targeted therapies. PMID:27708418

  6. From fetus towards adult : maturation and functional analysis of pluripotent stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Catarino, Ribeiro M.

    2016-01-01

    This thesis describes research about the differentiation of human stem cells into cardiomyocytes (heart cells). During the differentiation process the stem cells become contractile myocytes that resemble the native heart cells. Nevertheless, the phenotype of these cardiomyocytes is comparable to a s

  7. Expression of hyaluronan and the hyaluronan-binding proteoglycans neurocan, aggrecan, and versican by neural stem cells and neural cells derived from embryonic stem cells.

    Science.gov (United States)

    Abaskharoun, Mary; Bellemare, Marie; Lau, Elizabeth; Margolis, Richard U

    2010-04-23

    We have examined the expression and localization patterns of hyaluronan and hyaluronan-binding chondroitin sulfate proteoglycans in neural stem cells and differentiated neural cells derived from mouse embryonic stem cells. Expression of proteoglycans and hyaluronan was weak in the SSEA1-positive embryonic stem cells but increased noticeably after retinoic acid induction to nestin-positive neural stem cells. After subsequent plating, the hyaluronan-binding chondroitin sulfate proteoglycans aggrecan, neurocan, and versican are expressed by cells in both the astrocytic and neuronal lineages. During the time period that hyaluronan was present, it co-localized with each of the hyaluronan-binding proteoglycans studied and was found to be clearly associated with beta-III tubulin-expressing neurons and oligodendrocytes expressing the O4 sulfatide marker. Although proteoglycan expression levels increased to varying degrees following neural differentiation, they did not change noticably during the following 2 weeks in culture, but there was a significant decrease in hyaluronan expression. Our studies therefore demonstrate the expression by neural stem cells and neural cells derived from them of hyaluronan and its associated proteoglycans, thereby providing a necessary foundation for integrating their specific properties into developing strategies for therapeutic applications.

  8. Stem cells derived from testis show promise for treating a wide variety of medical conditions

    Institute of Scientific and Technical Information of China (English)

    Karim Nayernia

    2007-01-01

    @@ The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of germline testis stem cells,the spermatogonial stem cells.These are single cells situated on the basal membrane of the seminiferous epithelium.Only 0.03%of all germ cells are spermatogonial stem cells(SSCs)[1-3].To maintain spermatogenesis,the processes of self-renewal and differentiation of SSCS must be precisely regulated by intrinsic gene expression in the stem cells and extrinsic signals,including soluble factors or adhesion molecules from the surrounding microenvironment,the stem cell niche.

  9. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    Science.gov (United States)

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  10. Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

    Science.gov (United States)

    Zahanich, Ihor; Sirenko, Syevda G; Maltseva, Larissa A; Tarasova, Yelena S; Spurgeon, Harold A; Boheler, Kenneth R; Stern, Michael D; Lakatta, Edward G; Maltsev, Victor A

    2011-01-01

    There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be

  11. Melanoma affects the composition of blood cell-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Nina Koliha

    2016-07-01

    Full Text Available Extracellular vesicles are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell and extracellular vesicles in melanoma patient’s plasma could be indicative for the tumor. Likewise, extracellular vesicles might influence tumor progression by regulating immune responses. We performed a broad protein characterization of extracellular vesicles from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer cells, monocytes, monocyte-derived dendritic cells and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on extracellular vesicles. Hierarchal clustering of protein intensity patterns grouped extracellular vesicles according to their originating cell type. The analysis of extracellular vesicles from stimulated B cells and monocyte-derived dendritic cells revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of extracellular vesicles from platelets, antigen presenting cells and natural cells as shown by platelet markers, costimulatory proteins, and a natural killer cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers indicating a changed vesicle secretion or protein loading of extracellular vesicles by platelets and a lower CD8 signal that might be associated with a diminished activity of natural killer cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of extracellular vesicles in melanoma plasma, but rather argue

  12. Optical Investigations of Endothelial Cell Motility

    DEFF Research Database (Denmark)

    Rossen, Ninna Struck

    of tissues and holds great promises for treatments and regenerative therapies. It faces an important obstacle before such promises can be realized, the engineered tissues needs to be of a size large enough to function and to relieve the damaged bodily functions. The current state of the art in tissue......A monolayer of endothelial cells lines the entire circulatory system and create a barrier between the circulatory system and the tissues. To create and maintain an intact barrier, the individual cells have to connect tightly with their neighbors, which causes a highly correlated motion between...... are fascinating from a biophysical point of view. The vasculature also plays a signi cant role in many pathologies. In diabetic blindness or ischemic diseases the ow of blood is insucient to sustain certain tissues or whole limbs. The creation of new blood vessels can relieve or treat such diseases. In other...

  13. Epithelial cell-derived micro RNA-146a generates interleukin-10-producing monocytes to inhibit nasal allergy.

    Science.gov (United States)

    Luo, Xi; Han, Miaomiao; Liu, Jianqi; Wang, Yu; Luo, Xiangqian; Zheng, Jing; Wang, Shuai; Liu, Zhigang; Liu, Dabo; Yang, Ping-Chang; Li, Huabin

    2015-11-03

    The aberrant immunity plays an important role in the pathogenesis of allergic diseases. Micro RNAs (miR) are involved in regulating the immunity in the body. This study aims to test a hypothesis that miR-146a induces the expression of interleukin (IL)-10 in monocytes (Mos). In this study, the levels of miR-146a were determined by real time RT-PCR. The IL-10(+) Mos were evaluated by flow cytometry. The miR-146a-laden exosomes were generated with RPMI2650 cells (an airway epithelial cell line). An allergic rhinitis mouse model was developed. The results showed that nasal epithelial cells expressed miR-146a, which was markedly lower in the nasal epithelial cells of patients with nasal allergy than that in healthy controls. Exposure to T helper (Th)2 cytokines suppressed the levels of miR-146a in the nasal epithelial cells. The nasal epithelial cell-derived miR-146a up regulated the expression of IL-10 in Mos. The inducible IL-10(+) Mos showed an immune suppressor effect on the activities of CD4(+) effector T cells and the Th2 polarization in a mouse model of allergic rhinitis. In summary, nasal epithelial cells express miR-146a, the latter is capable of inducing IL-10 expression in Mos, which suppress allergic reactions in the mouse nasal mucosa.

  14. The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Heras-Bautista, Carlos O; Katsen-Globa, Alisa; Schloerer, Nils E; Dieluweit, Sabine; Abd El Aziz, Osama M; Peinkofer, Gabriel; Attia, Wael A; Khalil, Markus; Brockmeier, Konrad; Hescheler, Jürgen; Pfannkuche, Kurt

    2014-08-01

    Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates.

  15. Multipotent stromal cells derived from common marmoset Callithrix jacchus within alginate 3D environment: Effect of cryopreservation procedures.

    Science.gov (United States)

    Gryshkov, Oleksandr; Hofmann, Nicola; Lauterboeck, Lothar; Pogozhykh, Denys; Mueller, Thomas; Glasmacher, Birgit

    2015-08-01

    Multipotent stromal cells derived from the common marmoset monkey Callithrix jacchus (cjMSCs) possess high phylogenetic similarity to humans, with a great potential for preclinical studies in the field of regenerative medicine. Safe and effective long-term storage of cells is of great significance to clinical and research applications. Encapsulation of such cell types within alginate beads that can mimic an extra-cellular matrix and provide a supportive environment for cells during cryopreservation, has several advantages over freezing of cells in suspension. In this study we have analysed the effect of dimethyl sulfoxide (Me2SO, 2.5-10%, v/v) and pre-freeze loading time of alginate encapsulated cjMSCs in Me2SO (0-45 min) on the viability and metabolic activity of the cells after freezing using a slow cooling rate (-1°C/min). It was found that these parameters affect the stability and homogeneity of alginate beads after thawing. Moreover, the cjMSCs can be frozen in alginate beads with lower Me2SO concentration of 7.5% after 30 min of loading, while retaining high cryopreservation outcome. We demonstrated the maximum viability, membrane integrity and metabolic activity of the cells under optimized, less cytotoxic conditions. The results of this study are another step forward towards the application of cryopreservation for the long-term storage and subsequent applications of transplants in cell-based therapies.

  16. Plasticity of human menstrual blood stem cells derived from the endometrium

    Institute of Scientific and Technical Information of China (English)

    Jian LIN; Dennis XIANG; Jin-long ZHANG; Julie ALLICKSON; Charlie XIANG

    2011-01-01

    Stem cells can be obtained from women's menstrual blood derived from the endometrium. The cells display stem cell markers such as Oct-4, SSEA-4, Nanog, and c-kit (CD117), and have the potent ability to differentiate into various cell types, including the heart, nerve, bone, cartilage, and fat. There has been no evidence of teratoma,ectopic formation, or any immune response after transplantation into an animal model. These cells quickly regenerate after menstruation and secrete many growth factors to display recurrent angiogenesis. The plasticity and safety of the acquired cells have been demonstrated in many studies. Menstrual blood-derived stem cells (MenSCs) provide an alternative source of adult stem cells for research and application in regenerative medicine. Here we summarize the multipotent properties and the plasticities of MenSCs and other endometrial stem cells from recent studies conducted both in vitro and in vivo.

  17. Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme.

    Science.gov (United States)

    Sneddon, Julie B; Borowiak, Malgorzata; Melton, Douglas A

    2012-11-29

    One goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. To this end, the stepwise differentiation of stem cells into specialized derivatives has been successful for some cell types, but a major problem remains the inefficient conversion of cells from one stage of differentiation to the next. If specialized cells are to be produced in large numbers it will be necessary to expand progenitor cells, without differentiation, at some steps of the process. Using the pancreatic lineage as a model for embryonic-stem-cell differentiation, we demonstrate that this is a solvable problem. Co-culture with organ-matched mesenchyme permits proliferation and self-renewal of progenitors, without differentiation, and enables an expansion of more than a million-fold for human endodermal cells with full retention of their developmental potential. This effect is specific both to the mesenchymal cell and to the progenitor being amplified. Progenitors that have been serially expanded on mesenchyme give rise to glucose-sensing, insulin-secreting cells when transplanted in vivo. Theoretically, the identification of stage-specific renewal signals can be incorporated into any scheme for the efficient production of large numbers of differentiated cells from stem cells and may therefore have wide application in regenerative biology.

  18. Endothelial progenitor cells and integrins: adhesive needs

    Directory of Open Access Journals (Sweden)

    Caiado Francisco

    2012-03-01

    Full Text Available Abstract In the last decade there have been multiple studies concerning the contribution of endothelial progenitor cells (EPCs to new vessel formation in different physiological and pathological settings. The process by which EPCs contribute to new vessel formation in adults is termed postnatal vasculogenesis and occurs via four inter-related steps. They must respond to chemoattractant signals and mobilize from the bone marrow to the peripheral blood; home in on sites of new vessel formation; invade and migrate at the same sites; and differentiate into mature endothelial cells (ECs and/or regulate pre-existing ECs via paracrine or juxtacrine signals. During these four steps, EPCs interact with different physiological compartments, namely bone marrow, peripheral blood, blood vessels and homing tissues. The success of each step depends on the ability of EPCs to interact, adapt and respond to multiple molecular cues. The present review summarizes the interactions between integrins expressed by EPCs and their ligands: extracellular matrix components and cell surface proteins present at sites of postnatal vasculogenesis. The data summarized here indicate that integrins represent a major molecular determinant of EPC function, with different integrin subunits regulating different steps of EPC biology. Specifically, integrin α4β1 is a key regulator of EPC retention and/or mobilization from the bone marrow, while integrins α5β1, α6β1, αvβ3 and αvβ5 are major determinants of EPC homing, invasion, differentiation and paracrine factor production. β2 integrins are the major regulators of EPC transendothelial migration. The relevance of integrins in EPC biology is also demonstrated by many studies that use extracellular matrix-based scaffolds as a clinical tool to improve the vasculogenic functions of EPCs. We propose that targeted and tissue-specific manipulation of EPC integrin-mediated interactions may be crucial to further improve the usage of

  19. Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

    Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

  20. Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2016-02-01

    Full Text Available The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.

  1. Myocardial regeneration by transplantation of modified endothelial progenitor cells expressing SDF-1 in a rat model

    DEFF Research Database (Denmark)

    Schuh, A.; Kroh, A.; Konschalla, S.

    2012-01-01

    Cell based therapy has been shown to attenuate myocardial dysfunction after myocardial infarction (MI) in different acute and chronic animal models. It has been further shown that stromal-cell derived factor-1a (SDF-1a) facilitates proliferation and migration of endogenous progenitor cells...... into injured tissue. The aim of the present study was to investigate the role of exogenously applied and endogenously mobilized cells in a regenerative strategy for MI therapy. Lentivirally SDF-1a-infected endothelial progenitor cells (EPCs) were injected after 90 min. of ligation and reperfusion of the left...... anterior descending artery (LAD) intramyocardial and intracoronary using a new rodent catheter system. Eight weeks after transplantation, echocardiography and isolated heart studies revealed a significant improvement of LV function after intramyocardial application of lentiviral with SDF-1 infected EPCs...

  2. BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies.

    Science.gov (United States)

    Boyd, N L; Dhara, S K; Rekaya, R; Godbey, E A; Hasneen, K; Rao, R R; West, F D; Gerwe, B A; Stice, S L

    2007-06-01

    The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these

  3. Reverse-D-4F Increases the Number of Endothelial Progenitor Cells and Improves Endothelial Progenitor Cell Dysfunctions in High Fat Diet Mice.

    Science.gov (United States)

    Nana, Yang; Peng, Jiao; Jianlin, Zhang; Xiangjian, Zhang; Shutong, Yao; Enxin, Zhan; Bin, Li; Chuanlong, Zong; Hua, Tian; Yanhong, Si; Yunsai, Du; Shucun, Qin; Hui, Wang

    2015-01-01

    Although high density lipoprotein (HDL) improves the functions of endothelial progenitor cells (EPCs), the effect of HDL ApoAI mimetic peptide reverse-D-4F (Rev-D4F) on EPC mobilization and repair of EPC dysfunctions remains to be studied. In this study, we investigated the effects of Rev-D4F on peripheral blood cell subpopulations in C57 mice treated with a high fat diet and the mechanism of Rev-D4F in improving the function of EPCs impaired by tumor necrosis factor-α (TNF-α). The high fat diet significantly decreased the number of EPCs, EPC migratory functions, and the percentage of lymphocytes in the white blood cells. However, it significantly increased the number of white blood cells, the percentage of monocytes in the white blood cells, and the level of vascular endothelial growth factor (VEGF) and TNF-α in the plasma. Rev-D4F clearly inhibited the effect of the high fat diet on the quantification of peripheral blood cell subpopulations and cytokine levels, and increased stromal cell derived factor 1α (SDF-1α) in the plasma. We provided in vitro evidence that TNF-α impaired EPC proliferation, migration, and tube formation through inactive AKT and eNOS, which was restored by Rev-D4F treatment. In contrast, both the PI3-kinase (PI3K) inhibitor (LY294002) and AKT inhibitor (perifosine) obviously inhibited the restoration of Rev-4F on EPCs impaired by TNF-α. Our results suggested that Rev-D4F increases the quantity of endothelial progenitor cells through increasing the SDF-1α levels and decreasing the TNF-α level of peripheral blood in high fat diet-induced C57BL/6J mice, and restores TNF-α induced dysfunctions of EPCs partly through stimulating the PI3K/AKT signal pathway.

  4. Pluripotent stem cells derived from mouse and human white mature adipocytes.

    Science.gov (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R; Shahmirian, Laurine J; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A; Yao, Yucheng; Boström, Kristina I

    2014-02-01

    White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5-7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.

  5. Study on the induction and differentiation of megakaryocyte progenitor cell derived from umbilical cord blood

    Institute of Scientific and Technical Information of China (English)

    陈琳

    2014-01-01

    Objective To build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.Methods Red blood cells were precipitated by hydroxyethyl starch(HES).Mononuclear cells were obtained by density gradient centrifugation with Ficoll.The inducing efficiencies of megakaryocytes using different cytokine cocktails and culture media were analyzed.Results The best choice for erythrocyte sedimenta-

  6. ES cells derived from cloned embryos in monkey - a jump toward human therapeutic cloning

    Institute of Scientific and Technical Information of China (English)

    Xiangzhong Yang; Sadie L Smith

    2007-01-01

    @@ Therapeutic cloning refers to the derivation of embryonic stem cells (ntESC) from embryos derived from somatic cell nuclear transfer (SCNT) also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus (DNA) removed.

  7. [Application Prospect of Stem Cell-derived Microvesicles in Regeneration of Injured Tissues].

    Science.gov (United States)

    Yin, Huiqun; Jiang, Hong

    2015-06-01

    More and more evidence indicates that microvesicles (MVs) play a key role in cell-to-cell communication. The MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the cell surface membranes of most types. Components of donor cells are incorporated into MVs that contain bioactive lipids, proteins, genetic cargoes. MVs derived from stem cells may reprogram cells that survived in injury tissue and favor tissue regeneration by delivering their bioactive cargoes to influence the behaviors of recipient cells. Compared with mesenchymal stem cells (MSCs), MVs derived from MSCs were found to mimic the beneficial effects of these cells. These proregenerative effects mediated by MVs can be explained by the fact that MVs are enriched in bioactive lipids, anti-apoptotic and pro-stimulatory growth factors or cytokines, and deliver mRNAs, regulatory miRNAs and proteins that improve overall cell function. Therefore, it opens novel perspectives in exploiting these MVs in tissue regeneration and repair. In addition, the use of MVs instead of stem cells could represent a safe and potentially more advantageous alternative to cell-therapy approaches.

  8. Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

    Science.gov (United States)

    Goldring, Christopher; Antoine, Daniel J; Bonner, Frank; Crozier, Jonathan; Denning, Chris; Fontana, Robert J; Hanley, Neil A; Hay, David C; Ingelman-Sundberg, Magnus; Juhila, Satu; Kitteringham, Neil; Silva-Lima, Beatriz; Norris, Alan; Pridgeon, Chris; Ross, James A; Young, Rowena Sison; Tagle, Danilo; Tornesi, Belen; van de Water, Bob; Weaver, Richard J; Zhang, Fang; Park, B Kevin

    2017-02-01

    Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).

  9. Stem cell-derived models to improve mechanistic understanding and prediction of human drug induced liver injury

    Science.gov (United States)

    Goldring, Christopher; Antoine, Daniel J.; Bonner, Frank; Crozier, Jonathan; Denning, Chris; Fontana, Robert J.; Hanley, Neil A.; Hay, David C.; Ingelman-Sundberg, Magnus; Juhila, Satu; Kitteringham, Neil; Silva-Lima, Beatriz; Norris, Alan; Pridgeon, Chris; Ross, James A.; Sison Young, Rowena; Tagle, Danilo; Tornesi, Belen; van de Water, Bob; Weaver, Richard J.; Zhang, Fang; Park, B. Kevin

    2016-01-01

    Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalised toxicology to determine inter-individual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury (DILI) means that no current single cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human DILI. Nevertheless, a single cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment, and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally-differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. PMID:27775817

  10. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components

    Science.gov (United States)

    Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  11. Advances of mesenchymal stem cells derived from bone marrow and dental tissue in craniofacial tissue engineering.

    Science.gov (United States)

    Yang, Maobin; Zhang, Hongming; Gangolli, Riddhi

    2014-05-01

    Bone and dental tissues in craniofacial region work as an important aesthetic and functional unit. Reconstruction of craniofacial tissue defects is highly expected to ensure patients to maintain good quality of life. Tissue engineering and regenerative medicine have been developed in the last two decades, and been advanced with the stem cell technology. Bone marrow derived mesenchymal stem cells are one of the most extensively studied post-natal stem cell population, and are widely utilized in cell-based therapy. Dental tissue derived mesenchymal stem cells are a relatively new stem cell population that isolated from various dental tissues. These cells can undergo multilineage differentiation including osteogenic and odontogenic differentiation, thus provide an alternative source of mesenchymal stem cells for tissue engineering. In this review, we discuss the important issues in mesenchymal stem cell biology including the origin and functions of mesenchymal stem cells, compare the properties of these two types of mesenchymal cells, update recent basic research and clinic applications in this field, and address important future challenges.

  12. Culture and Identification of Monoclonal Neural Stem Cells Derived from Cerebral Cortex

    Institute of Scientific and Technical Information of China (English)

    TAO Kaixiong; CHEN Jingbo; WANG Guobin; SHU Xiaogang

    2006-01-01

    To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

  13. Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

    Institute of Scientific and Technical Information of China (English)

    Chang Dong LI; Wei Yuan ZHANG; He Lian LI; Xiao Xia JIANG; Yi ZHANG; Pei Hsien TANG; Ning MAO

    2005-01-01

    Human placenta-derived mononuclear cells (MNC) were isolated by a Percoll density gradient and cultured in mesenchymal stem cell (MSC) maintenance medium.The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology,a large expansive potential,and cell cycle characteristics including a subset of quiescent cells.In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic,osteogenic and chondrogenic lineages.Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells,which uniformly expressed CD29,CD44,CD73,CD 105,CD166,laminin,fibronectin and vimentin while being negative for expression of CD31,CD34,CD45 and α-smooth muscle actin.Most importantly,immuno-phenotypic analyses demonstrated that these cells expressed class I major histocompatibility complex (MHC-Ⅰ),but they did not express MHC-Ⅱ molecules.Additionally these cells could suppress umbilical cord blood (UCB) lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli.This strongly implies that they may have potential application in allograft transplantation.Since placenta and UCB are homogeneous,the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells (HSC) from UCB to reduce the potential graft-versus-host disease (GVHD) in recipients.

  14. Engineered human embryonic stem cell-derived lymphocytes to study in vivo trafficking and immunotherapy.

    Science.gov (United States)

    Knorr, David A; Bock, Allison; Brentjens, Renier J; Kaufman, Dan S

    2013-07-01

    Human embryonic stem cell (hESC)-derived natural killer (NK) cells are a promising source of antitumor lymphocytes for immunotherapeutics. They also provide a genetically tractable platform well suited for the study of antitumor immunotherapies in preclinical models. We have previously demonstrated the potency of hESC-derived NK cells in vivo. Here we use both bioluminescent and fluorescent imaging to demonstrate trafficking of hESC-derived NK cells to tumors in vivo. Our dual-imaging approach allowed us to more specifically define the kinetics of NK cell trafficking to tumor sites. NK cell persistence and trafficking were further evaluated by flow cytometry and immunohistochemistry. This integrated approach provides a unique system to apply the use of human pluripotent stem cells to study the kinetics and biodistribution of adoptively transferred lymphocytes, advances broadly applicable to the field of immunotherapy.

  15. Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

    Directory of Open Access Journals (Sweden)

    Ivana Acimovic

    2014-01-01

    Full Text Available Human pluripotent stem cells (hPSCs, namely, embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs, with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs.

  16. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    Science.gov (United States)

    Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

    2014-01-01

    Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  17. Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway.

    Science.gov (United States)

    Kim, You-Sun; Kim, Ji-Young; Cho, RyeonJin; Shin, Dong-Myung; Lee, Sei Won; Oh, Yeon-Mok

    2017-01-13

    Cell therapy using stem cells has produced therapeutic benefits in animal models of COPD. Secretory mediators are proposed as one mechanism for stem cell effects because very few stem cells engraft after injection into recipient animals. Recently, nanovesicles that overcome the disadvantages of natural exosomes have been generated artificially from cells. We generated artificial nanovesicles from adipose-derived stem cells (ASCs) using sequential penetration through polycarbonate membranes. ASC-derived artificial nanovesicles displayed a 100 nm-sized spherical shape similar to ASC-derived natural exosomes and expressed both exosomal and stem cell markers. The proliferation rate of lung epithelial cells was increased in cells treated with ASC-derived artificial nanovesicles compared with cells treated with ASC-derived natural exosomes. The lower dose of ASC-derived artificial nanovesicles had similar regenerative capacity compared with a higher dose of ASCs and ASC-derived natural exosomes. In addition, FGF2 levels in the lungs of mice treated with ASC-derived artificial nanovesicles were increased. The uptake of ASC-derived artificial nanovesicles was inhibited by heparin, which is a competitive inhibitor of heparan sulfate proteoglycan that is associated with FGF2 signaling. Taken together, the data indicate that lower doses of ASC-derived artificial nanovesicles may have beneficial effects similar to higher doses of ASCs or ASC-derived natural exosomes in an animal model with emphysema, suggesting that artificial nanovesicles may have economic advantages that warrant future clinical studies.

  18. Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Parallel Human Differentiation Into Sensory Neural Subtypes.

    Science.gov (United States)

    Webb, Robin L; Gallegos-Cárdenas, Amalia; Miller, Colette N; Solomotis, Nicholas J; Liu, Hong-Xiang; West, Franklin D; Stice, Steven L

    2017-04-01

    The pig is the large animal model of choice for study of nerve regeneration and wound repair. Availability of porcine sensory neural cells would conceptually allow for analogous cell-based peripheral nerve regeneration in porcine injuries of similar severity and size to those found in humans. After recently reporting that porcine (or pig) induced pluripotent stem cells (piPSCs) differentiate into neural rosette (NR) structures similar to human NRs, here we demonstrate that pig NR cells could differentiate into neural crest cells and other peripheral nervous system-relevant cell types. Treatment with either bone morphogenetic protein 4 or fetal bovine serum led to differentiation into BRN3A-positive sensory cells and increased expression of sensory neuron TRK receptor gene family: TRKA, TRKB, and TRKC. Porcine sensory neural cells would allow determination of parallels between human and porcine cells in response to noxious stimuli, analgesics, and reparative mechanisms. In vitro differentiation of pig sensory neurons provides a novel model system for neural cell subtype specification and would provide a novel platform for the study of regenerative therapeutics by elucidating the requirements for innervation following injury and axonal survival.

  19. Mesenchymal stem cells derived from adipose tissue are not affected by renal disease.

    Science.gov (United States)

    Roemeling-van Rhijn, Marieke; Reinders, Marlies E J; de Klein, Annelies; Douben, Hannie; Korevaar, Sander S; Mensah, Fane K F; Dor, Frank J M F; IJzermans, Jan N M; Betjes, Michiel G H; Baan, Carla C; Weimar, Willem; Hoogduijn, Martin J

    2012-10-01

    Mesenchymal stem cells are a potential therapeutic agent in renal disease and kidney transplantation. Autologous cell use in kidney transplantation is preferred to avoid anti-HLA reactivity; however, the influence of renal disease on mesenchymal stem cells is unknown. To investigate the feasibility of autologous cell therapy in patients with renal disease, we isolated these cells from subcutaneous adipose tissue of healthy controls and patients with renal disease and compared them phenotypically and functionally. The mesenchymal stem cells from both groups showed similar morphology and differentiation capacity, and were both over 90% positive for CD73, CD105, and CD166, and negative for CD31 and CD45. They demonstrated comparable population doubling times, rates of apoptosis, and were both capable of inhibiting allo-antigen- and anti-CD3/CD28-activated peripheral blood mononuclear cell proliferation. In response to immune activation they both increased the expression of pro-inflammatory and anti-inflammatory factors. These mesenchymal stem cells were genetically stable after extensive expansion and, importantly, were not affected by uremic serum. Thus, mesenchymal stem cells of patients with renal disease have similar characteristics and functionality as those from healthy controls. Hence, our results indicate the feasibility of their use in autologous cell therapy in patients with renal disease.

  20. Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway

    Science.gov (United States)

    Kim, You-Sun; Kim, Ji-Young; Cho, RyeonJin; Shin, Dong-Myung; Lee, Sei Won; Oh, Yeon-Mok

    2017-01-01

    Cell therapy using stem cells has produced therapeutic benefits in animal models of COPD. Secretory mediators are proposed as one mechanism for stem cell effects because very few stem cells engraft after injection into recipient animals. Recently, nanovesicles that overcome the disadvantages of natural exosomes have been generated artificially from cells. We generated artificial nanovesicles from adipose-derived stem cells (ASCs) using sequential penetration through polycarbonate membranes. ASC-derived artificial nanovesicles displayed a 100 nm-sized spherical shape similar to ASC-derived natural exosomes and expressed both exosomal and stem cell markers. The proliferation rate of lung epithelial cells was increased in cells treated with ASC-derived artificial nanovesicles compared with cells treated with ASC-derived natural exosomes. The lower dose of ASC-derived artificial nanovesicles had similar regenerative capacity compared with a higher dose of ASCs and ASC-derived natural exosomes. In addition, FGF2 levels in the lungs of mice treated with ASC-derived artificial nanovesicles were increased. The uptake of ASC-derived artificial nanovesicles was inhibited by heparin, which is a competitive inhibitor of heparan sulfate proteoglycan that is associated with FGF2 signaling. Taken together, the data indicate that lower doses of ASC-derived artificial nanovesicles may have beneficial effects similar to higher doses of ASCs or ASC-derived natural exosomes in an animal model with emphysema, suggesting that artificial nanovesicles may have economic advantages that warrant future clinical studies. PMID:28082743

  1. Tolerance induction and reversal of diabetes in mice transplanted with human embryonic stem cell-derived pancreatic endoderm.

    Science.gov (United States)

    Szot, Gregory L; Yadav, Mahesh; Lang, Jiena; Kroon, Evert; Kerr, Justin; Kadoya, Kuniko; Brandon, Eugene P; Baetge, Emmanuel E; Bour-Jordan, Hélène; Bluestone, Jeffrey A

    2015-02-05

    Type 1 diabetes (T1D) is an autoimmune disease caused by T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans. In most cases, reversal of disease would require strategies combining islet cell replacement with immunotherapy that are currently available only for the most severely affected patients. Here, we demonstrate that immunotherapies that target T cell costimulatory pathways block the rejection of xenogeneic human embryonic-stem-cell-derived pancreatic endoderm (hESC-PE) in mice. The therapy allowed for long-term development of hESC-PE into islet-like structures capable of producing human insulin and maintaining normoglycemia. Moreover, short-term costimulation blockade led to robust immune tolerance that could be transferred independently of regulatory T cells. Importantly, costimulation blockade prevented the rejection of allogeneic hESC-PE by human PBMCs in a humanized model in vivo. These results support the clinical development of hESC-derived therapy, combined with tolerogenic treatments, as a sustainable alternative strategy for patients with T1D.

  2. Ape1/Ref-1 induces glial cell-derived neurotropic factor (GDNF) responsiveness by upregulating GDNF receptor alpha1 expression.

    Science.gov (United States)

    Kim, Mi-Hwa; Kim, Hong-Beum; Acharya, Samudra; Sohn, Hong-Moon; Jun, Jae Yeoul; Chang, In-Youb; You, Ho Jin

    2009-04-01

    Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor alpha1 (GFRalpha1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRalpha1 transcription through enhanced binding of NF-kappaB complexes to the GFRalpha1 promoter. GFRalpha1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRalpha1 expression and invasion in response to GNDF, while overexpression of GFRalpha1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from beta-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRalpha1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.

  3. Immunogenicity of in vitro maintained and matured populations: potential barriers to engraftment of human pluripotent stem cell derivatives.

    Science.gov (United States)

    Tang, Chad; Weissman, Irving L; Drukker, Micha

    2013-01-01

    The potential to develop into any cell type makes human pluripotent stem cells (hPSCs) one of the most promising sources for regenerative treatments. Hurdles to their clinical applications include (1) formation of heterogeneously differentiated cultures, (2) the risk of teratoma formation from residual undifferentiated cells, and (3) immune rejection of engrafted cells. The recent production of human isogenic (genetically identical) induced PSCs (hiPSCs) has been proposed as a "solution" to the histocompatibility barrier. In theory, differentiated cells derived from patient-specific hiPSC lines should be histocompatible to their donor/recipient. However, propagation, maintenance, and non-physiologic differentiation of hPSCs in vitro may produce other, likely less powerful, immune responses. In light of recent progress towards the clinical application of hPSCs, this review focuses on two antigen presentation phenomena that may lead to rejection of isogenic hPSC derivates: namely, the expression of aberrant antigens as a result of long-term in vitro maintenance conditions or incomplete somatic cell reprogramming, and the unbalanced presentation of receptors and ligands involved in immune recognition due to accelerated differentiation. Finally, we discuss immunosuppressive approaches that could potentially address these immunological concerns.

  4. N-butylidenephthalide attenuates Alzheimer's disease-like cytopathy in Down syndrome induced pluripotent stem cell-derived neurons.

    Science.gov (United States)

    Chang, Chia-Yu; Chen, Sheng-Mei; Lu, Huai-En; Lai, Syu-Ming; Lai, Ping-Shan; Shen, Po-Wen; Chen, Pei-Ying; Shen, Ching-I; Harn, Horng-Jyh; Lin, Shinn-Zong; Hwang, Shiaw-Min; Su, Hong-Lin

    2015-03-04

    Down syndrome (DS) patients with early-onset dementia share similar neurodegenerative features with Alzheimer's disease (AD). To recapitulate the AD cell model, DS induced pluripotent stem cells (DS-iPSCs), reprogrammed from mesenchymal stem cells in amniotic fluid, were directed toward a neuronal lineage. Neuroepithelial precursor cells with high purity and forebrain characteristics were robustly generated on day 10 (D10) of differentiation. Accumulated amyloid deposits, Tau protein hyperphosphorylation and Tau intracellular redistribution emerged rapidly in DS neurons within 45 days but not in normal embryonic stem cell-derived neurons. N-butylidenephthalide (Bdph), a major phthalide ingredient of Angelica sinensis, was emulsified by pluronic F127 to reduce its cellular toxicity and promote canonical Wnt signaling. Interestingly, we found that F127-Bdph showed significant therapeutic effects in reducing secreted Aβ40 deposits, the total Tau level and the hyperphosphorylated status of Tau in DS neurons. Taken together, DS-iPSC derived neural cells can serve as an ideal cellular model of DS and AD and have potential for high-throughput screening of candidate drugs. We also suggest that Bdph may benefit DS or AD treatment by scavenging Aβ aggregates and neurofibrillary tangles.

  5. BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies.

    Science.gov (United States)

    Conley, Brock J; Ellis, Sarah; Gulluyan, Lerna; Mollard, Richard

    2007-02-01

    Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, alpha-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.

  6. Comparative studies of different cryopreservation methods for mesenchymal stem cells derived from human fetal liver.

    Science.gov (United States)

    Todorov, Plamen; Hristova, Elena; Konakchieva, Rossitza; Michova, Antoaneta; Dimitrov, Josif

    2010-03-29

    Fetal stem cells possess some intriguing characteristics, which delineate them as promising cellular therapeutics. They are less immunogenic, at lower stage of differentiation and have higher potential for repopulation and migration. Furthermore, the fetal stem cells secrete a set of cytokines and growth factors, which stimulate the regeneration of the recipient tissue. The present study indicated that the adhesive fraction of human fetal liver cells possessed the morphological characteristics of mesenchymal stem cells, as well as potential to differentiate into adipocyte and osteoblast lineages. The immunophenotypic analysis showed that the cells expressed CD13, CD73, CD90 and CD105 (typical for mesenchymal stem cells) and lacked the haematopoietic lineage markers CD34 and CD45. Addressing the issue of the low-temperature storage of the human fetal liver cells, four different methods for cryopreservation were assessed: conventional slow freezing, program freezing and two vitrification protocols. The obtained results demonstrated that the cells were cryotolerant and maintained their properties and differentiation potential after thawing. Program freezing showed to be the most efficient method for cryopreservation of the investigated cells.

  7. Effect of F68 on cryopreservation of mesenchymal stem cells derived from human tooth germ.

    Science.gov (United States)

    Doğan, Ayşegül; Yalvaç, Mehmet Emir; Yılmaz, Aysu; Rizvanov, Albert; Sahin, Fikrettin

    2013-12-01

    The use of stem-cell-based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer's disease, diabetes, spinal cord injuries, and cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory, and angiogenic effects which are attributed to the restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Unlike bone-marrow-derived MSCs, HTGSCs can be easily isolated, expanded, and cryopreserved, which makes them an alternative stem cell source. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation, hindering their therapeutic capacity. Amelioration of the side effects of cryopreservation on MSCs seems to be a priority in order to maximize the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F68) on HTGSCs during long-term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long-term cryopreservation.

  8. Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Qiao Lu

    Full Text Available Human induced pluripotent stem cells (hiPSCs have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

  9. Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

    Science.gov (United States)

    Lu, Qiao; Yu, Meixing; Shen, Chongyang; Chen, Xiaoping; Feng, Ting; Yao, Yongchao; Li, Jinrong; Li, Hong; Tu, Wenwei

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

  10. Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

    Science.gov (United States)

    Lakowski, Jorn; Gonzalez-Cordero, Anai; West, Emma L; Han, Ya-Ting; Welby, Emily; Naeem, Arifa; Blackford, Samuel J I; Bainbridge, James W B; Pearson, Rachael A; Ali, Robin R; Sowden, Jane C

    2015-08-01

    Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.

  11. Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kim, Yoon Young; Ku, Seung-Yup; Huh, Yul; Liu, Hung-Ching; Kim, Seok Hyun; Choi, Young Min; Moon, Shin Yong

    2013-10-01

    Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

  12. Jin Fu Kang Oral Liquid Inhibits Lymphatic Endothelial Cells Formation and Migration

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    Hai-Lang He

    2016-01-01

    Full Text Available Lung cancer is the leading cause of cancer-related deaths worldwide. Jin Fu Kang (JFK, an oral liquid prescription of Chinese herbal drugs, has been clinically available for the treatment of non-small cell lung cancer (NSCLC. Lymphangiogenesis is a primary event in the process of cancer development and metastasis, and the formation and migration of lymphatic endothelial cells (LECs play a key role in the lymphangiogenesis. To assess the activity of stromal cell-derived factor-1 (SDF-1 and the coeffect of SDF-1 and vascular endothelial growth factor-C (VEGF-C on the formation and migration of LECs and clarify the inhibitory effects of JFK on the LECs, the LECs were differentiated from CD34+/VEGFR-3+ endothelial progenitor cells (EPCs, and JFK-containing serums were prepared from rats. SDF-1 and VEGF-C both induced the differentiation of CD34+/VEGFR-3+ EPCs towards LECs and enhanced the LECs migration. Couse of SDF-1 and VEGF-C displayed an additive effect on the LECs formation but not on their migration. JFK inhibited the formation and migration of LECs, and the inhibitory effects were most probably via regulation of the SDF-1/CXCR4 and VEGF-C/VEGFR-3 axes. The current finding suggested that JFK might inhibit NSCLC through antilymphangiogenesis and also provided a potential to discover antilymphangiogenesis agents from natural resources.

  13. Hematopoietic stem cell-derived adipocytes and fibroblastsin the tumor microenvironment

    Institute of Scientific and Technical Information of China (English)

    Ying Xiong; Lindsay T McDonald; Dayvia L Russell; Ryan R Kelly; Katie R Wilson; Meenal Mehrotra; Adam C Soloff; Amanda C LaRue

    2015-01-01

    The tumor microenvironment (TME) is complex andconstantly evolving. This is due, in part, to the crosstalkbetween tumor cells and the multiple cell types thatcomprise the TME, which results in a heterogeneouspopulation of tumor cells and TME cells. This reviewwill focus on two stromal cell types, the cancerassociatedadipocyte (CAA) and the cancer-associatedfibroblast (CAF). In the clinic, the presence of CAAsand CAFs in the TME translates to poor prognosis inmultiple tumor types. CAAs and CAFs have an activatedphenotype and produce growth factors, inflammatoryfactors, cytokines, chemokines, extracellular matrixcomponents, and proteases in an accelerated andaberrant fashion. Through this activated state, CAAs andCAFs remodel the TME, thereby driving all aspects oftumor progression, including tumor growth and survival,chemoresistance, tumor vascularization, tumor invasion,and tumor cell metastasis. Similarities in the tumorpromotingfunctions of CAAs and CAFs suggest that amultipronged therapeutic approach may be necessaryto achieve maximal impact on disease. While CAAsand CAFs are thought to arise from tissues adjacentto the tumor, multiple alternative origins for CAAs andCAFs have recently been identified. Recent studiesfrom our lab and others suggest that the hematopoieticstem cell, through the myeloid lineage, may serve asa progenitor for CAAs and CAFs. We hypothesize thatthe multiple origins of CAAs and CAFs may contributeto the heterogeneity seen in the TME. Thus, a betterunderstanding of the origin of CAAs and CAFs, howthis origin impacts their functions in the TME, and the temporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.

  14. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

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    Heiner Falkenberg

    2013-01-01

    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  15. Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood

    DEFF Research Database (Denmark)

    Berg, Lise Charlotte; Koch, Thomas Gadegaard; Heerkens, T.

    2009-01-01

    Objective: Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenthymal stromal cells (MSC) have been derived from multiple sources in the horse...

  16. Importance of Being Nernst: Synaptic Activity and Functional Relevance in Stem Cell-derived Neurons

    Science.gov (United States)

    2015-07-26

    Zhang C. Induced multilineage differen- tiation of chicken embryonic germ cells via embryoid body formation. Stem Cells Dev 2010; 19: 195-202 [PMID...Khademhosseini A, Kim JH, Lee SH. Controlled-size embryoid body formation in concave microwell arrays. Biomaterials 2010; 31: 4296-4303 [PMID: 20206991 DOI

  17. In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

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    Ilaria Turin

    2014-03-01

    Full Text Available Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

  18. Could a B-1 cell derived phagocyte "be one" of the peritoneal macrophages during LPS-driven inflammation?

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    Ana Flavia Popi

    Full Text Available The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP, and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/- mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11 that is often found in B-1 cells. These results strongly suggest that op/op((-/- peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of

  19. Silencing of directional migration in roundabout4 knockdown endothelial cells

    Directory of Open Access Journals (Sweden)

    Roberts David D

    2008-11-01

    Full Text Available Abstract Background Roundabouts are axon guidance molecules that have recently been iden