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

The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently,knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here,we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies,due to downstream suppression of p21,without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns,displayed normal karyotypes,contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion,transient p53 suppression increases reprogramming efficiency without affecting genomic stability,rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation. View Publication

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries,but reprogrammed neurons are dissociated and spatially disorganized during transplantation,rendering poor cell survival,functionality and engraftment in vivo. Here,we present the design of three-dimensional (3D) microtopographic scaffolds,using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming,neural network establishment and support neuronal engraftment into the brain. Scaffold-supported,reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices,showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells,and allowed delivery of multiple neuronal subtypes. Thus,3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance. View Publication

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived,patient-specific pluripotent cells for use in cell-based regenerative therapies. However,current methods of cell culture are tedious and expensive,and the mechanisms underlying cell proliferation are not understood. In this study,we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion,survival,and proliferation. We show that iPSC lines generated using Oct-3/4,Sox-2,Nanog,and Lin-28 express a repertoire of integrins similar to that of hESCs,with prominent expression of subunits alpha5,alpha6,alphav,beta1,and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin,in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel,as shown by immunological blockade experiments. On vitronectin,the integrin alphavbeta5 was required for initial attachment,but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore,iPSCs cultured on vitronectin for 9 passages retained normal karyotype,pluripotency marker expression,and capacity to differentiate in vitro. These studies suggest that vitronectin,or derivatives thereof,might substitute for Matrigel in a more defined system for iPSC culture. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

During human brain development,multiple signaling pathways generate diverse cell types with varied regional identities. Here,we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor,neuronal,and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together,these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. View Publication

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines,we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene,and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines,even in the absence of patient tissue. View Publication

Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche,where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation,dendritic cell maturation,and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9,in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease. View Publication

Patients with dyskeratosis congenita (DC),a disorder of telomere maintenance,suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity,which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state,that several telomerase components are targeted by pluripotency-associated transcription factors,and that in autosomal dominant DC,transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase,and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However,the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4,Sox2,c-Myc,and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog,TDGF,and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction,small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology,gene expression,long-term self-renewal ability,and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation. ?? 2008 Elsevier B.V. All rights reserved. View Publication

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting,X-chromosome inactivation and genomic architecture. In this study,we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes,including NANOG,SOX2,cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation,but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs,CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors,such as Cohesin,differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly,the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology. View Publication

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had textgreater1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization,two iPSC lines from each subject were selected,differentiated into postmitotic neurons,and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs,iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover,repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism. View Publication