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

Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development,disease modeling,drug discovery,and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined,growth factor- and serum-free system by temporal modulation of Wnt/$$-catenin signaling via small molecules. We demonstrate a 10-day,two-stage process that recapitulates endothelial cell development,in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. View Publication

The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However,generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)-derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons,which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology,we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure,function,and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders. View Publication

Aging is known to be the single most important risk factor for multiple diseases. Sirtuin 6,or SIRT6,has recently been identified as a critical regulator of transcription,genome stability,telomere integrity,DNA repair,and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging,we demonstrated that human dermal fibroblasts (HDFs) from older human subjects were more resistant to reprogramming by classic Yamanaka factors than those from younger human subjects,but the addition of SIRT6 during reprogramming improved such efficiency in older HDFs substantially. Despite the importance of SIRT6,little is known about the molecular mechanism of its regulation. We show,for the first,time posttranscriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop,which has implications for the modulation of SIRT6 expression in reprogramming of aging cells. View Publication

Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore,we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics,BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm),yet,24 hr later,suppressed endoderm and induced mesoderm. At lineage bifurcations,cross-repressive signals separated mutually exclusive fates; TGF-?? and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations,thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of pre-enhancer" states before activation� View Publication

Incurable neurological disorders such as Parkinson's disease (PD),Huntington's disease (HD),and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases,we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor,valproic acid (VPA),and inhibitor of p160-Rho associated coiled-coil kinase (ROCK),Y-27632,after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology,cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542,inhibitors of the SMAD signaling pathway,HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction,neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture,which had the ability to differentiate further into NPCs and neurons,as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View Publication

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.backslashnbackslashnFINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.backslashnbackslashnCONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic. View Publication

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy,reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative,but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC,comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated,through enrichment analysis,their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally,we report an unprecedented coverage of MSC CD markers,as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. View Publication

Coronary arteriogenesis is a central step in cardiogenesis,requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present,it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro,and contribute extensively to coronary-like vessels in vivo,forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates,and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo. View Publication

Human embryonic stem cells (hESCs) are used as platforms for disease study,drug screening and cell-based therapy. To facilitate these applications,it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However,the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However,certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site,probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein,LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. View Publication

Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here,we report the use of chitin-alginate 3D microfibrous scaffolds,previously developed for hPSC propagation,to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid,the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons,as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore,flow cytometry confirmed that textgreater90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines,a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons,without tumorigenesis,suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications. View Publication