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

Here,we show that as human embryonic stem cells (ESCs) exit the pluripotent state,NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here,we find that FGF2,acting through the MEK-ERK pathway,switches BMP4-induced human ESC differentiation outcome to mesendoderm,characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation,that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm,and that knockdown of NANOG greatly reduces T induction. Together,our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions,the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (ptextless0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC,E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2),at concentrations that selectively block I(K1) (50-100 µM),failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied,but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data,RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells,but robust expression of I(Kr.) In contrast to recently reported studies,our data point to major deficiencies of hiPSC-CM,with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus,efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications. View Publication

Reduced expression 1 (REX1) is a widely used pluripotency marker,but little is known about its roles in pluripotency. Here,we show that REX1 is functionally important in the reacquisition and maintenance of pluripotency. REX1-depleted human pluripotent stem cells (hPSCs) lose their self-renewal capacity and full differentiation potential,especially their mesoderm lineage potential. Cyclin B1/B2 expression was found to parallel that of REX1. REX1 positively regulates the transcriptional activity of cyclin B1/B2 through binding to their promoters. REX1 induces the phosphorylation of DRP1 at Ser616 by cyclin B/CDK1,which leads to mitochondrial fission and appears to be important for meeting the high-energy demands of highly glycolytic hPSCs. During reprogramming to pluripotency by defined factors (OCT4,SOX2,KLF4,and c-MYC),the reprogramming kinetics and efficiency are markedly improved by adding REX1 or replacing KLF4 with REX1. These improvements are achieved by lowering reprogramming barriers (growth arrest and apoptosis),by enhancing mitochondrial fission,and by conversion to glycolytic metabolism,dependent on the cyclin B1/B2-DRP1 pathway. Our results show that a novel pluripotency regulator,REX1,is essential for pluripotency and reprogramming. View Publication

Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here,human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes,and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay,the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay,phenotypic changes in mitochondrial membrane potential,calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes,albumin/urea secretion,and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition,the cell viability of SC-iHeps and p-Heps was increased by ketoconazole,a CYP3A4 inhibitor. Collectively,SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore,SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells. View Publication

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However,limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However,limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence,in this study,we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPs(GFP+) stably expressed high levels of GFP,CD73,CD90,and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1,RUNX2,OSTERIX,and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin,alkaline phosphatase,and collagen-I. In conclusion,we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future,these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration,safety,and therapeutic efficacy. View Publication

Rett syndrome (RTT) is an X-linked,neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene,which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated,its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis,we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs,we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel,we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation,respectively,and ameliorated the observed alterations in neuronal differentiation. Moreover,overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together,our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.Molecular Psychiatry advance online publication,25 April 2017; doi:10.1038/mp.2017.86. View Publication

Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. View Publication

BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication

Imatinib is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome-positive leukemias and other malignancies. Side effects are mostly moderate; however,a dose-dependent hematologic toxicity affecting all hematopoietic lineages is observed clinically. The aim of this study was to investigate the effect of imatinib on normal hematopoietic stem and progenitor cells in vitro. A dose-dependent decrease in proliferation potential was found when CD34+ cells were expanded in serum-free medium supplemented with 6 growth factors and imatinib. Functionally,a decrease in colony-forming capacity was observed under increasing doses of imatinib. However,no such effect on more primitive cobblestone area-forming cells was detectable. Both withdrawal of stem cell factor from our expansion cultures or functional inhibition of c-kit led to a similar degree of inhibition of expansion,whereas the effect of imatinib was substantially greater at all dose levels tested. These data suggest a significant inhibitory effect of imatinib on normal CD34+ progenitor (but not stem) cells that is largely independent of c-kit signaling. View Publication

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless,the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however,conflicting data generated from different biological sources prevent conclusions being drawn. In this work,we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate,an adjunctive medication for schizophrenia,brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders. ?? 2014 Elsevier B.V. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication