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

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors,we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive,insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells,we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded small hairpin RNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells,release C-peptide in response to secretagogues and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes. View Publication

Since the derivation of human embryonic stem (hES) cells,their translation to clinical therapies has been met with several challenges,including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We,along with other groups,have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence,flow cytometry,and teratoma formation assays,we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly,the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices,which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies. View Publication

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH),a DA neuron marker,and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth. View Publication

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However,it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines,along with methylomes of ES cells,somatic cells,and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability,including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation,and differences in CG methylation and histone modifications. Lastly,differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency,providing an iPSC reprogramming signature that is maintained after differentiation. View Publication

Human embryonic stem (hES) cells have the dual ability to self-renew and differentiate into specialized cell types. However,in order to realize the full potential of these cells it is important to understand how the genes responsible for their unique characteristics are regulated. In this study we examine the regulation of the tropomyosin-related kinase (TRK) genes which encode for receptors important in hES cell survival and self-renewal. Although the TRK genes have been studied in many neuronal cell types,the regulation of these genes in hES cells is unclear. Our study demonstrates a novel regulatory relationship between the TRKC gene and the transcription factor SOX2. Our results found that hES cells highly express full-length and truncated forms of the TRKC gene. However,examination of the related TRKB gene showed a lower overall expression of both full-length and truncated forms. Through RNA interference,we knocked down expression levels of SOX2 in hES cells and examined the expression of TRKC,as well as TRKB. Upon loss of SOX2 we found that TRKC mRNA levels were significantly downregulated but TRKB levels remained unchanged,demonstrating an important regulatory dependence on SOX2 by TRKC. We also found that TRKC protein levels were also decreased after SOX2 knock down. Further analysis found the regulatory region of TRKC to be highly conserved among many mammals with potential SOX binding motifs. We confirmed a specific binding motif as a site that SOX2 utilizes to directly interact with the TRKC regulatory region. In addition,we found that SOX2 drives expression of the TRKC gene by activating a luciferase reporter construct containing the TRKC regulatory region and the SOX binding motif. View Publication

The recent Zika virus (ZIKV) outbreak,which mainly affected Brazil and neighbouring states,demonstrated the paucity of information concerning the epidemiology of several flaviruses,but also highlighted the lack of available agents with which to treat such emerging diseases. Here,we show that heparin,a widely used anticoagulant,while exerting a modest inhibitory effect on Zika Virus replication,fully prevents virus-induced cell death of human neural progenitor cells (NPCs). View Publication

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes,we differentiated human induced pluripotent stem cells into pancreatic endocrine cells,including $\beta$ cells. Here,we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived $\beta$ cells,along with a reduced effect on $\alpha$ cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response. View Publication

A fast track Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement View Publication

Despite the high incidence of trophoblast-related diseases,the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study,we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs,the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally,we preliminarily indicated in vitro artificial" differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage as witnessed in vivo. In conclusion we provided an in vitro cellular model to study early trophoblast development for specific individual by using the feasible amnion. View Publication

Induced pluripotent stem cells (iPSCs) have the ability to differentiate in any specialized somatic cell type,which makes them an attractive tool for a wide variety of scientific approaches,including regenerative medicine. However,their pluripotent state and their growth in compact colonies render them difficult to access and,therefore,restrict delivery of specific agents for cell manipulation. Thus,our investigation focus was set on the evaluation of the capability of Layer-by-Layer (LbL) designed microcarriers to serve as a potential drug delivery system to iPSCs,as they offer several appealing advantages. Most notably,these carriers allow for the transport of active agents in a protected environment and for a rather specific delivery through surface modifications. As we could show,charge and mode of LbL carrier application as well as the size of the iPSC colonies determine the interaction with and the uptake rate by iPSCs. None of the examined conditions had an influence on iPSC colony properties such as colony morphology and size or maintenance of pluripotent properties. An overall interaction rate of LbL carriers with iPSCs of up to 20 % was achieved. Those data emphasize the applicability of LbL carriers for stem cell research. Additionally,the potential use of LbL carriers as a promising delivery tool for iPSCs was contrasted to viral particles and liposomes. The identified differences among those delivery tools have substantiated our major conclusion that LbL carrier uptake rate is influenced by characteristic features of the iPSC colonies (most notably colony size) in addition to their surface charges. View Publication

BACKGROUND: Malignant melanoma is an exceptionally aggressive,drug-resistant and heterogeneous cancer. Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common,but markers distinguishing such cells from cells lacking these abilities have not been identified. There is therefore no definite evidence that an exclusive cell subpopulation,i.e. cancer stem cells (CSC),exists in malignant melanoma. Rather,it is suggested that multiple cell populations are implicated in initiation and progression of the disease,making it of importance to identify subpopulations with elevated aggressive properties. METHODS AND FINDINGS: In several other cancer forms,Aldehyde Dehydrogenase (ALDH),which plays a role in stem cell biology and resistance,is a valuable functional marker for identification of cells that show enhanced aggressiveness and drug-resistance. Furthermore,the presence of ALDH(+) cells is linked to poor clinical prognosis in these cancers. By analyzing cell cultures,xenografts and patient biopsies,we showed that aggressive melanoma harboured a large,distinguishable ALDH(+) subpopulation. In vivo,ALDH(+) cells gave rise to ALDH(-) cells,while the opposite conversion was rare,indicating a higher abilities of ALDH(+) cells to reestablish tumour heterogeneity with respect to the ALDH phenotype. However,both ALDH(+) and ALDH(-) cells demonstrated similarly high abilities for clone formation in vitro and tumour initiation in vivo. Furthermore,both subpopulations showed similar sensitivity to the anti-melanoma drugs,dacarbazine and lexatumumab. CONCLUSIONS: These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells,implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma,and arguing against ALDH as a universal" marker. Besides� View Publication