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

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However,the generation methods reported so far vary greatly in quality and efficiency. Here,we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs� View Publication

The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs,heterogeneity,and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs,but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques,such as coculture,physical manipulation,sorting,or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First,epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days,iPSCs showed upregulation of mesodermal genes (MSX2,NCAM,HOXA2) and downregulation of pluripotency genes (OCT4,LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes,reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype,expressed high levels of vimentin and N-cadherin,and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs,whereas adipogenic differentiation was limited,as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture,providing a robust,clinically applicable,and efficient system for generating MSCs from human iPSCs. View Publication

Human pluripotent stem cells (hPSCs) are proving to be a valuable source of endothelial cells (ECs),pericytes,and vascular smooth muscle cells (vSMCs). Although an increasing number of phenotypic markers are becoming available to determine the phenotypes of these cells in vitro,the ability to integrate and form functional vessels in the host organism,typically mouse,remains critical for the assessment of EC functional competence. However,current mouse models require relatively large numbers of cells that might be difficult to derive simultaneously from multiple hPSCs lines. Therefore,there is an urgent need for new functional assays that are robust and can be performed with small numbers of cells. Here we describe a novel zebrafish xenograft model to test functionality of hPSC-derived ECs. The assay can be performed in 10 days and requires only ˜100-400 human cells per embryo. Thus,the zebrafish xenograft model can be useful for the accurate and rapid assessment of functionality of hPSC-derived ECs in a lower vertebrate model that is widely viewed by regulatory authorities as a more acceptable alternative to adult mice. View Publication

Conventional generation of stem cells from human blastocysts produces a developmentally advanced,or primed,stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However,whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here,we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration,global gene expression,and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. View Publication

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-β[2-(di-n-butylamino)-6-naphthyl]butadienylquinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses,we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision,generating nearly identical values for AP duration (AP durations at 10%,50%,and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure,with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally,di-4-ANBDQBS allowed for moderate-throughput analyses,increasing throughput textgreater10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs. View Publication

textlessptextgreater Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases,thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics,but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover,the protooncogene textlessitalictextgreatermyctextless/italictextgreater showed the strongest expression in HEP-iPSC,compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as textlessitalictextgreatermyctextless/italictextgreater might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases. textless/ptextgreater View Publication

Neural stem cells (NSCs) possess immunosuppressive characteristics,but effects of NSCs on human dendritic cells (DCs),the most important antigen presenting cells,are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14+ monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line,as well as human bone marrow derived mesenchymal stem cells (MSCs),were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14+ monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a,whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However,when effects on the function of derived DCs were investigated,NSCs suppressed the elevation of the DC maturation marker CD83,although not the up-regulation of costimulatory molecules CD80,CD86 and CD40,and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs,these stem cells can still affect the function of DCs,ultimately regulating specific immune responses. View Publication

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here,we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4,SOX2,KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology,chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65,RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane,and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together,our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. View Publication

BACKGROUND:Emerging studies of human pluripotent stem cells (hPSCs) raise new prospects for neurodegenerative disease modeling and cell replacement therapies. Therefore,understanding the mechanisms underlying the commitment of neural progenitor cells (NPCs) is important for the application of hPSCs in neurodegenerative disease therapies. It has been reported that epigenetic modifications of histones play important roles in neural differentiation,but the exact mechanisms in regulating hPSC differentiation towards NPCs are not fully elucidated.RESULTS:We demonstrated that suppression of histone deacetylases (HDACs) promoted the differentiation of hPSCs towards NPCs. Application of HDAC inhibitors (HDACi) increased the expression of neuroectodermal markers and enhanced the neuroectodermal specification once neural differentiation was initiated,thereby leading to more NPC generation. Similarly,the transcriptome analysis showed that HDACi increased the expression levels of ectodermal markers and triggered the NPC differentiation related pathways,while decreasing the expression levels of endodermal and mesodermal markers. Furthermore,we documented that HDAC3 but not HDAC1 or HDAC2 was the critical regulator participating in NPC differentiation,and knockdown of HDAC3's cofactor SMRT exhibited a similar effect as HDAC3 on NPC generation.CONCLUSIONS:Our study reveals that HDACs,especially HDAC3,negatively regulate the differentiation of hPSCs towards NPCs at an earlier stage of neural differentiation. Moreover,HDAC3 might function by forming a repressor complex with its cofactor SMRT during this process. Thus,our findings uncover an important epigenetic mechanism of HDAC3 in the differentiation of hPSCs towards NPCs. View Publication

Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs),large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34,respectively,from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGF$$-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542(+) hESC-ECs,SB431542(-) hESC-ECs,and HUVECs showed similar permeability to 10,000 Da dextran,but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542(+) hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542(-) hESC-ECs and HUVECs responded differently to VEGF and bFGF,which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542(-) hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. This article is protected by copyright. All rights reserved. View Publication

The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover,these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology,positive staining for alkaline phosphatase,normal karyotype,and expression of hESC-like markers including Nanog,Rex1,Oct4,TRA-1-60,TRA-1-80,SSEA-3,and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers,and could as well be readily differentiated into functional motor neurons. Among other things,our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming,differentiation ability,stability of the resulting lineages,and the risk of associated abnormalities. View Publication