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

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive,insulin-producing beta cells from self-renewing,pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol,hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17,and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor,basic fibroblast growth factor,and noggin. Soon thereafter,expression of Ptf1a and Ngn3 was detected,indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development,and the final population contained representatives of the ductal,exocrine,and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells,as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs,representing levels higher than that of human fetal islets. In addition,the hESC-derived ILCs contained numerous secretory granules,as determined by electron microscopy,and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling,the dysregulation of which causes several pathologies,such as congenital defects and cancer,is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells,such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis,but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells,the maturation process,but not the early steps of human mesenchymal stem cell differentiation,is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression,whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes,adipogenesis appears dramatically impaired,with reduced lipid accumulation,a decrease in adipocyte-specific markers,and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells. View Publication

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids,pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction,the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step,3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors,and they proliferate and expand over 1-3 months to give rise to intestinal tissue,complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date,this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro. View Publication

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet,acquiring donor cells is,in most instances,an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances,as the isolation of urinary cells is simple (30 ml of urine are sufficient),cost-effective and universal (can be applied to any age,gender and race). Moreover,the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential,and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases,including those affecting the kidney. View Publication

T-cell genome engineering holds great promise for cell-based therapies for cancer,HIV,primary immune deficiencies,and autoimmune diseases,but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types� View Publication

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are,in part,controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis,we have compared gene expression profiles of human erythroblasts,megakaryocytes,B cells,cytotoxic and helper T cells,natural killer cells,granulocytes,and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors,immunoglobulin superfamily members,and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude,ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition,we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg,GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data,which are freely accessible,will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. View Publication

Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres,referred to as mEBs,were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling pathways were hallmarks of 10-day differentiated mEBs. We then generated mammary-like organoids from 10-day mEBs using 3D floating mixed gel culture and a three-stage differentiation procedure. These organoids expressed common breast tissue,luminal,and basal markers,including estrogen receptor,and could be induced to produce milk protein. These results demonstrate that human iPSCs can be directed in vitro toward mammary lineage differentiation. Our findings provide an iPSC-based model for studying regulation of normal mammary cell fate and function as well as breast disease development. View Publication

In the adult human,mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages,including cartilage. In this study,culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days,cells secreted an extracellular matrix incorporating type II collagen,aggrecan,and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine,the withdrawal of TGF-beta(3),and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues,their maintenance,and their regeneration. View Publication

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However,protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation. View Publication

Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However,isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients,and repetitive isolations may be required. To overcome these difficulties,we have examined alternative methods for MSC production. Here,we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology,formed three-dimensional spheroid structures,and expressed characteristic mesenchymal stem cell surface markers such as CD29,CD33,CD73,CD90,and CD105. Moreover,iMSCs were capable of differentiating into adipogenic,osteogenic,and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels,and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice,and suggests that iPSCs are a viable alternate source for deriving MSCs as needed. textcopyright 2014 Elsevier Inc. View Publication

Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34(+) progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201- restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34(+) progenitor cells isolated from patients with chronic myeloid leukemia (CML),whereas colony formation by normal CD34(+) progenitor cells is unaffected. Thus,the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo. View Publication

The extensive molecular characterization of human pluripotent stem cells (hPSCs),human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) is required before they can be applied in the future for personalized medicine and drug discovery. Despite the efforts that have been made with kinome analyses,we still lack in-depth insights into the molecular signatures of receptor tyrosine kinases (RTKs) that are related to pluripotency. Here,we present the first detailed and distinct repertoire of RTK characteristic for hPSC pluripotency by determining both the expression and phosphorylation profiles of RTKs in hESCs and hiPSCs using reverse transcriptase-polymerase chain reaction with degenerate primers that target conserved tyrosine kinase domains and phospho-RTK array,respectively. Among the RTKs tested,the up-regulation of EPHA1,ERBB2,FGFR4 and VEGFR2 and the down-regulation of AXL,EPHA4,PDGFRB and TYRO3 in terms of both their expression and phosphorylation levels were predominantly related to the maintenance of hPSC pluripotency. Notably,the specific inhibition of AXL was significantly advantageous in maintaining undifferentiated hESCs and hiPSCs and for the overall efficiency and kinetics of hiPSC generation. Additionally,a global phosphoproteomic analysis showed that ∼30% of the proteins (293 of 970 phosphoproteins) showed differential phosphorylation upon AXL inhibition in undifferentiated hPSCs,revealing the potential contribution of AXL-mediated phosphorylation dynamics to pluripotency-related signaling networks. Our findings provide a novel molecular signature of AXL in pluripotency control that will complement existing pluripotency-kinome networks. View Publication