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

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion,differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded,that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10 7 cells/ml); (ii) quick recovery of encapsulated cells (textless10min at 37°C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with textgreater17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype invitro and the ability to form derivatives of the three germ layers both invitro and invivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications. textcopyright 2011 Elsevier Ltd. View Publication

BACKGROUND Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling,drug screening,and heart repair. Here,we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS We systematically investigated cell composition,matrix,and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological,functional,and transcriptome analyses to benchmark maturation of EHM. RESULTS EHM demonstrated important structural and functional properties of postnatal myocardium,including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction,cardiomyocyte hypertrophy,cardiomyocyte death,and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition,we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined,serum-free conditions. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Precise,robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension,this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover,an intermediate population of neural precursor cells,which are routinely textgreater95% NCAM(pos) and Tra-1-60(neg) by FACS analysis,may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments. View Publication

G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth,death,movement,transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study,we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol,we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially,the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted,whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4,knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together,our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine. View Publication

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day,7-stage protocol. Cultures were ˜90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17,but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine,but not glucose in perifusion studies. Compared to adult human islets,hESC-derived cells expressed ˜10-fold higher levels of glucose transporter 1 (GLUT1) mRNA,but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However,GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells,whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays,hESC-derived cells displayed mild potassium channel activity,but no responsiveness to glucose,metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells,suggesting a lack of functional KATP channels at the cell surface. In addition,KATP channel subunit transcript levels were not at a 1:1 ratio,as would be expected (SUR1 levels were ˜5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells,and along with lack of GLUT1,may explain the absence of glucose-stimulated insulin secretion.?? 2013 Elsevier B.V. View Publication

Airway epithelial cells are of great interest for research on lung development,regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation,and full maturation of the cells in air-liquid interface cultures occurs in textless5 weeks. This protocol can be used for drug discovery,tissue regeneration or disease modeling. View Publication

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus,hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks,however,conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology,retained a normal karyotype,and expressed characteristic hESC markers (OCT4,SSEA3,SSEA4 and TRA-1-60). Moreover,the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus,the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. View Publication

Human embryonic stem cells (hESCs) are self-renewing,pluripotent cells derived from the inner cell mass of blastocysts,early-stage embryos,or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore,these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy,regenerative medicine,drug screening and discovery,and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable,and inefficient derivation of hESCs would be a terrible waste of human embryos. Here,we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies. View Publication

Human pluripotent stem cells (hPSCs) are sensitive to DNA damage and undergo rapid apoptosis compared to their differentiated progeny cells. Here,we explore the underlying mechanisms for the increased apoptotic sensitivity of hPSCs that helps to determine pluripotent stem cell fate. Apoptosis was induced by exposure to actinomycin D,etoposide,or tunicamycin,with each agent triggering a distinct apoptotic pathway. We show that hPSCs are more sensitive to all three types of apoptosis induction than are lineage-non-specific,retinoic-acid-differentiated hPSCs. Also,Bax activation and pro-apoptotic mitochondrial intermembrane space protein release,which are required to initiate the mitochondria-mediated apoptosis pathway,are more rapid in hPSCs than in retinoic-acid-differentiated hPSCs. Surprisingly,Bak and not Bax is essential for actinomycin-D-induced apoptosis in human embryonic stem cells. Finally,P53 is degraded rapidly in an ubiquitin-proteasome-dependent pathway in hPSCs at steady state but quickly accumulates and induces apoptosis when Mdm2 function is impaired. Rapid degradation of P53 ensures the survival of healthy hPSCs but avails these cells for immediate apoptosis upon cellular damage by P53 stabilization. Altogether,we provide an underlying,interconnected molecular mechanism that primes hPSCs for quick clearance by apoptosis to eliminate hPSCs with unrepaired genome alterations and preserves organismal genomic integrity during the early critical stages of human embryonic development. View Publication