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

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts,and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer,retaining a pre-inactivation X chromosome state,and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF,naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells,they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression,pronounced tendency for X chromosome inactivation in most female human ES cells,increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells,from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells,and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively,our findings establish new avenues for regenerative medicine,patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. View Publication

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized,the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here,using a short fragment of laminin 411 (LM411-E8),an ECM predominantly expressed in the vascular endothelial basement membrane,we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (textgreater95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. 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

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

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

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast,humans with NEUROG3 mutations are born with endocrine pancreas function,calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly,we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3(-/-) hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover,NEUROG3(-/-) hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1(+)/NKX6.1(+) pancreatic progenitors into mice. In contrast,a 75-90% knockdown of NEUROG3 caused a reduction,but not a loss,of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells. View Publication

Genome-wide association studies (GWASs) have increased our knowledge of loci associated with a range of human diseases. However,applying such findings to elucidate pathophysiology and promote drug discovery remains challenging. Here,we created isogenic human ESCs (hESCs) with mutations in GWAS-identified susceptibility genes for type 2 diabetes. In pancreatic beta-like cells differentiated from these lines,we found that mutations in CDKAL1,KCNQ1,and KCNJ11 led to impaired glucose secretion in vitro and in vivo,coinciding with defective glucose homeostasis. CDKAL1 mutant insulin+ cells were also hypersensitive to glucolipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-specific defects in vitro and in vivo by inhibiting the FOS/JUN pathway. Our approach of a proof-of-principle platform,which uses isogenic hESCs for functional evaluation of GWAS-identified loci and identification of a drug candidate that rescues gene-specific defects,paves the way for precision therapy of metabolic diseases. View Publication

Reduced-intensity conditioning regimens for transplant recipients have heightened awareness of immunologic resistance to allogeneic bone marrow transplants (BMT). Although T cell-mediated cytotoxicity has been assumed to play a role in the resistance against donor allogeneic hematopoietic stem and progenitor cell grafts,several studies have reported relatively unimpaired resistance by recipients who lack perforin,Fas ligand (FasL),and other cytotoxic mediators. This study compared the early kinetics of T cell-mediated resistance in B6 (H2b) cytotoxically normal versus deficient recipients after transplantation with major histocompatibility complex-matched,minor histocompatibility antigen (MiHA)-mismatched allogeneic marrow grafts. Wild-type B6 or cytotoxic double-deficient perforin-/-/ gld+/+ (B6-cdd) mice were sensitized against major histocompatibility complex-matched BALB.B or C3H.SW (H2b) MiHA and transplanted with a high dose (1 ?? 107) of T cell-depleted bone marrow. CD8 T memory cells were shown to be present in recipients before BMT,and anti-CD8 monoclonal antibody infusion abolished resistance,thus demonstrating that CD8 T cells are the host effector population. Donor-committed and high proliferative potential progenitor numbers were markedly diminished by 48 hours after transplantation in both wild-type B6 and B6-cdd anti-donor MiHA-sensitized recipients. These observations indicate that the resistance pathway used in the cytotoxic deficient mice was both potent and rapidly induced - consistent with a CD8 memory T-cell response. To examine the role of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)- and TL1A-mediated cytotoxicity in this strong resistance,newly generated monoclonal antibodies specific for these ligands were administered to B6-cdd recipients sensitized to donor antigens. Recipients of syngeneic B6-gfp bone marrow exhibited significant donor colony-forming unit numbers after BMT. In contrast,low or absent colony-forming unit levels were detected in allogeneic recipients,including those that lacked perforin and FasL and that received anti-TWEAK,anti-tumor necrosis factor-related apoptosis-inducing ligand,and anti-TL1A monoclonal antibodies. These findings extend previous observations by demonstrating the existence of a rapidly effected resistance pathway mediated by memory CD8 effector T cells independent of the 2 major pathways of cytotoxicity. Together with previous findings,these results support the notion that effector cells derived from memory CD8 T-cell populations can mediate strong resistance against donor allogeneic MiHA-disparate hematopoietic engraftment by using a mechanism that is independent of the contribution of perforin,FasL,and the known death ligand receptor pathways. ?? 2005 American Society for Blood and Marrow Transplantation. View Publication