搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets,the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study,we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation,cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating,even after cell sheets were detached from culture dishes. Furthermore,extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. View Publication

Human-induced pluripotent stem cells (iPSCs) generated from human adult somatic cells through reprogramming hold great promises for future regenerative medicine. However,exposure of human iPSCs to animal feeder and serum in the process of their generation and maintenance imposes risk of transmitting animal pathogens to human subjects,thus hindering the potential therapeutic applications. Here,we report the successful generation of human iPSCs in a feeder-independent culture system with defined factors. Two stable human iPSC lines were established from primary human dermal fibroblasts of two healthy volunteers. These human iPSCs expressed a panel of pluripotency markers including stage-specific embryonic antigen (SSEA)-4,tumor-rejection antigen (TRA)-1-60,TRA-1-81,and alkaline phosphatase,while maintaining normal karyotypes and the exogenous reprogramming factors being silenced. In addition,these human iPSCs can differentiate along lineages representative of the three embryonic germ layers upon formation of embryoid bodies,indicating their pluripotency. Furthermore,subcutaneous transplantation of these cells into immunodeficient mice resulted in teratoma formation in 6 to 8 weeks. Our findings are an important step toward generating patient-specific iPSCs in a more clinically compliant manner by eliminating the need of animal feeder cells and animal serum. View Publication

Hematopoiesis during development is a dynamic process,with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis,the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1),best known for its proinflammatory properties,has radioprotective effects on adult bone marrow HSCs,induces HSC mobilization,and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1,IL-1 receptors (IL-1Rs),and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM,and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic,endothelial,and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation,concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development,acting to limit the differentiation of some HSCs along the myeloid lineage. View Publication

Nano- and microscale topographical cues play critical roles in the induction and maintenance of various cellular functions,including morphology,adhesion,gene regulation,and communication. Recent studies indicate that structure and function at the heart tissue level is exquisitely sensitive to mechanical cues at the nano-scale as well as at the microscale level. Although fabrication methods exist for generating topographical features for cell culture,current techniques,especially those with nanoscale resolution,are typically complex,prohibitively expensive,and not accessible to most biology laboratories. Here,we present a tunable culture platform comprised of biomimetic wrinkles that simulate the heart's complex anisotropic and multiscale architecture for facile and robust cardiac cell alignment. We demonstrate the cellular and subcellular alignment of both neonatal mouse cardiomyocytes as well as those derived from human embryonic stem cells. By mimicking the fibrillar network of the extracellular matrix,this system enables monitoring of protein localization in real time and therefore the high-resolution study of phenotypic and physiologic responses to in-vivo like topographical cues. View Publication

UNLABELLED: Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated,there has been limited progress in iPSC-based drug screening/discovery for liver diseases,and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency,for which there is currently no drug or gene therapy available,we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay,based on our hepatic differentiation protocol,was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients,we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening,five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition,using the recently developed transcription activator-like effector nuclease technology,we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications.backslashnbackslashnCONCLUSIONS: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs,both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. View Publication

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However,successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models,the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary,we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury,KDR,CD31 and Lmo2 gene expression,while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development,consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs,and although simplified,results in an eightfold increase in cell yield. View Publication

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity,in contrast to CD133(+)G(1) cells. Here,we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%,respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures,the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (Ptextless0.001) throughout the culture period. Furthermore,a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols. View Publication

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties,we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR,a fluorescent substrate for ALDH,and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56(+) fraction in those cells,but,we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly,ALDH activity and Aldh1a1 expression levels were very low in mouse,rat,rabbit and non-human primate myoblasts. Using different approaches,from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde,an inhibitor of class I ALDH,to cell fractionation by flow cytometry using the ALDEFLUOR assay,we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H(2) O(2) )-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity,as a purification strategy,could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. View Publication

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only ∼ 44% of the seeded cells were rescued,but an optimized heat shock treatment combined with Ri significantly increased cell survival to ∼ 60%. Mechanistically,our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2,hES3) that were used at higher passages (textgreater 86). In contrast,rapid down regulation of Oct4,Tra-1-60,and SSEA4 was observed for ESI049,a clinically compliant line,used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless,our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. textcopyright 2010 Elsevier B.V. All rights reserved. View Publication

Although it has been observed that aggregate size affects cardiac development,an incomplete understanding of the cellular mechanisms underlying human pluripotent stem cell-derived cardiomyogenesis has limited the development of robust defined-condition cardiac cell generation protocols. Our objective was thus to elucidate cellular and molecular mechanisms underlying the endogenous control of human embryonic stem cell (hESC) cardiac tissue development,and to test the hypothesis that hESC aggregate size influences extraembryonic endoderm (ExE) commitment and cardiac inductive properties. hESC aggregates were generated with 100,1000,or 4000 cells per aggregate using microwells. The frequency of endoderm marker (FoxA2 and GATA6)-expressing cells decreased with increasing aggregate size during early differentiation. Cardiogenesis was maximized in aggregates initiated from 1000 cells,with frequencies of 0.49±0.06 cells exhibiting a cardiac progenitor phenotype (KDR(low)/C-KIT(neg)) on day 5 and 0.24±0.06 expressing cardiac Troponin T on day 16. A direct relationship between ExE and cardiac differentiation efficiency was established by forming aggregates with varying ratios of SOX7 (a transcription factor required for ExE development) overexpressing or knockdown hESCs to unmanipulated hESCs. We demonstrate,in a defined,serum-free cardiac induction system,that robust and efficient cardiac differentiation is a function of endogenous ExE cell concentration,a parameter that can be directly modulated by controlling hESC aggregate size. View Publication

Inefficient neural differentiation of human induced pluripotent stem cells (hiPSCs) motivates recent investigation of the influence of biophysical characteristics of cellular microenvironment,in particular nanotopography,on hiPSC fate decision. However,the roles of geometry and dimensions of nanotopography in neural lineage commitment of hiPSCs have not been well understood. The objective of this study is to delineate the effects of geometry,feature size and height of nanotopography on neuronal differentiation of hiPSCs. HiPSCs were seeded on equally spaced nanogratings (500 and 1000nm in linewidth) and hexagonally arranged nanopillars (500nm in diameter),each having a height of 150 or 560nm,and induced for neuronal differentiation in concert with dual Smad inhibitors. The gratings of 560nm height reduced cell proliferation,enhanced cytoplasmic localization of Yes-associated protein,and promoted neuronal differentiation (up to 60% βIII-tubulin(+) cells) compared with the flat control. Nanograting-induced cell polarity and cytoplasmic YAP localization were shown to be critical to the induced neural differentiation of hiPSCs. The derived neuronal cells express MAP2,Tau,glutamate,GABA and Islet-1,indicating the existence of multiple neuronal subtypes. This study contributes to the delineation of cell-nanotopography interactions and provides the insights into the design of nanotopography configuration for pluripotent stem cell neural lineage commitment. View Publication

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication