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

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons,muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood,pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres,a hallmark of dyskeratosis congenita,impair tissue stem cell function in mouse models,indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state,iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT,the telomerase reverse transcriptase,a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast,mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53),telomerase catalytic activity is unperturbed,yet the ability of telomerase to lengthen telomeres is abrogated,because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal,indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics. View Publication

Neurogenin3+ (Ngn3+) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach,where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors,but the lineage and regulators of pancreatic gastrin+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin+ cells in the developing pancreas co-express glucagon,ghrelin or pancreatic polypeptide,but many gastrin+ cells do not express any other islet hormone. Pancreatic gastrin+ cells express the transcription factors Nkx6.1,Nkx2.2 and low levels of Pdx1,and derive from Ngn3+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3,Nkx2.2,NeuroD1 and Arx,but not Pax4 or Pax6. Finally,gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus,gastrin+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells. View Publication

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However,such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs),which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods,we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene,which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening. View Publication

INTRODUCTION The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. METHODS Expression of total YAP,its paralogue transcriptional co-activator with PDZ-binding motif (TAZ),and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis,C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally,YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. RESULTS YAP,but not TAZ,was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants,however,was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1,Id2 and Id3 in response to BMP-2. In developing mouse limbs,Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage,suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. CONCLUSIONS Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology. View Publication

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However,no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work,we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF,EGF and PDGF. These cells are SSEA4(+),OCT3/4(+),NANOG(+),SOX2(+),LIN28(+),CD13(+),CD105(+),CD34(-),CD45(-),CD90(+),CD29(+),CD73(+),STRO1(+) and CD146(-),and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly,DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm,endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4,GATA6,MIXL1,NANOG,OCT3/4,SOX1 and SOX2 to determine the degree of similarity between DPPSCs,EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs,hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages,they represent an easily accessible source of stem cells,which opens a range of new possibilities for regenerative medicine. View Publication

textlessptextgreater Hundreds of textlessitalictextgreaterL1CAMtextless/italictextgreater gene mutations have been shown to be associated with congenital hydrocephalus,severe intellectual disability,aphasia,and motor symptoms. How such mutations impair neuronal function,however,remains unclear. Here,we generated human embryonic stem (ES) cells carrying a conditional textlessitalictextgreaterL1CAMtextless/italictextgreater loss-of-function mutation and produced precisely matching control and textlessitalictextgreaterL1CAMtextless/italictextgreater -deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones,we found that deletion of textlessitalictextgreaterL1CAMtextless/italictextgreater dramatically impaired axonal elongation and,to a lesser extent,dendritic arborization. Unexpectedly,we also detected an ∼20–50% and ∼20–30% decrease,respectively,in the levels of ankyrinG and ankyrinB protein,and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM,but not of the L1CAM point mutants R1166X and S1224L,rescued the decrease in ankyrin levels. Importantly,we found that the textlessitalictextgreaterL1CAMtextless/italictextgreater mutation selectively decreased activity-dependent Na textlesssuptextgreater+textless/suptextgreater -currents,altered neuronal excitability,and caused impairments in action potential (AP) generation. Thus,our results suggest that the clinical presentations of textlessitalictextgreaterL1CAMtextless/italictextgreater mutations in human patients could be accounted for,at least in part,by cell-autonomous changes in the functional development of neurons,such that neurons are unable to develop normal axons and dendrites and to generate normal APs. textless/ptextgreater View Publication

While in vitro liver tissue engineering has been increasingly studied during the last several years,presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver,but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly,generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions,and has been inefficient so far. Towards generating a fully functional liver containing biliary system,we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver,EpCAM,is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can,not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes),in a 2D differentiation condition,but also form functional ductal structures in a 3D condition. Importantly,this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition,we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues,which may facilitate engineering of complete and functional liver tissue in the future. View Publication

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX,a putative homolog of the Xenopus xvent2 gene,is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34(+) leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations,with the highest expression in CD33(+) myeloid cells. Notably,expression levels of VENTX were negligible in normal CD34(+)/CD38(-) or CD34(+) human progenitor cells. In contrast to this,leukemic CD34(+)/CD38(-) cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34(+) cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34(+) human progenitor cells perturbs normal hematopoietic development,promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together,these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis. View Publication