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

Airway epithelial cells are of great interest for research on lung development,regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation,and full maturation of the cells in air-liquid interface cultures occurs in textless5 weeks. This protocol can be used for drug discovery,tissue regeneration or disease modeling. View Publication

G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth,death,movement,transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study,we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol,we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially,the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted,whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4,knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together,our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine. View Publication

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages,necessary to understand the species specific pathogenic effects of HCMV,has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells,iPS-derived neural stem cells (NSCs),neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection,i.e.,they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate. View Publication

Activation of thermogenic beige adipocytes has recently emerged as a promising therapeutic target in obesity and diabetes. Relevant human models for beige adipocyte differentiation are essential to implement such therapeutic strategies. We report a straightforward and efficient protocol to generate functional human beige adipocytes from human induced pluripotent stem cells (hiPSCs). Without overexpression of exogenous adipogenic genes,our method recapitulates an adipogenic developmental pathway through successive mesodermal and adipogenic progenitor stages. hiPSC-derived adipocytes are insulin sensitive and display beige-specific markers and functional properties,including upregulation of thermogenic genes,increased mitochondrial content,and increased oxygen consumption upon activation with cAMP analogs. Engraftment of hiPSC-derived adipocytes in mice produces well-organized and vascularized adipose tissue,capable of β-adrenergic-responsive glucose uptake. Our model of human beige adipocyte development provides a new and scalable tool for disease modeling and therapeutic screening. View Publication

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods,it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous,nonsense or splice variants,and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies,whereas the rest occurred during or after reprogramming. Thus,hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use. View Publication

Deficiency of the nuclear factor-kappa-B essential modulator (NEMO) is a rare X-linked disorder that presents in boys as hypohydrotic ectodermal dysplasia with immunodeficiency due to defective nuclear factor-κB activation. Here we report on the generation of 2 human embryonic stem cell lines from discarded in vitro fertilization (IVF) embryos ascertained via preimplantation genetic diagnosis. We have derived two human embryonic stem cell lines that carry a T458G hypomorphic mutation in exon 4 of the NEMO (or IKBKG) gene. One of the lines is diploid male; the other is diploid female but has clonally inactivated the X-chromosome that harbors the wild-type IKBKG gene. We show that both lines are pluripotent,have the capacity to differentiate into hematopoietic progenitors,and have defective inhibitor of nuclear factor kappa-B kinase activity. These NEMO deficiency hES cell lines provide an unlimited source for differentiated cell types and may serve as a unique tool to study NEMO deficiency and potentially lead to the development of new therapies for this disease. View Publication

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes,cells from the corneal stroma,may have the potential for restoration of vision in cell therapy and biomedical engineering applications,but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells,maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. View Publication

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs 1 – 3 . Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast,restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness,such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses,whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover,sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion. Subject terms: Haematopoietic stem cells,Self-renewal,Stem-cell niche,Endocytosis,Growth factor signalling View Publication