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

Pluripotency is a unique state in which cells can self-renew indefinitely but also retain the ability to differentiate into other cell types upon receipt of extracellular cues. Although it is clear that stem cells have a distinct transcriptional program,little is known about how alterations in post-transcriptional mechanisms,such as mRNA turnover,contribute to the achievement and maintenance of pluripotency. Here we have assessed the rates of decay for the majority of mRNAs expressed in induced pluripotent stem (iPS) cells and the fully differentiated human foreskin fibroblasts (HFFs) they were derived from. Comparison of decay rates in the two cell types led to the discovery of three independent regulatory mechanisms that allow coordinated turnover of specific groups of mRNAs. One mechanism results in increased stability of many histone mRNAs in iPS cells. A second pathway stabilizes a large set of zinc finger protein mRNAs,potentially through reduced levels of miRNAs that target them. Finally,a group of transcripts bearing 3' UTR C-rich sequence elements,many of which encode transcription factors,are significantly less stable in iPS cells. Intriguingly,two poly(C)-binding proteins that recognize this type of element are reciprocally expressed in iPS and HFF cells. Overall,our results highlight the importance of post-transcriptional control in pluripotent cells and identify miRNAs and RNA-binding proteins whose activity may coordinately control expression of a wide range of genes in iPS cells. View Publication

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential,however,depends on the availability of culture methods that are robust,scalable,and use chemically defined materials. Despite significant advances in hiPSC technologies,the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts,such as Matrigel,which raises safety concerns over the use of these products. In this work,we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined,xeno-free synthetic peptide substrate,i.e. Corning Synthemax(®) Surface. We demonstrated that the Synthemax Surface supports the attachment,spreading,and proliferation of hiPSCs,as well as hiPSCs' lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α(v)β(5) integrins to mediate attachment to the substrate,whereas multiple integrins are involved in cell attachment to Matrigel. Finally,hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials. 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

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

The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However,how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells,however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs,suggesting it is a suppressed,bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression,and ectopic expression of p21 in hESCs triggered their differentiation. Further,we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner,whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes,thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals,while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs. 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

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work,we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks,and the process was optimized using a factorial design approach,involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures,that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that,after replating,retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled,automated and reproducible large-scale bioreactor culture systems. View Publication

Human pluripotent stem cells (hPSCs) - including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) - are very promising candidates for cell therapies,tissue engineering,high throughput pharmacology screens,and toxicity testing. These applications require large numbers of high quality cells; however,scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple,efficient,fully defined,scalable,and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here,we describe additional design rationale and characterization of this system. For instance,we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast,using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally,hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally,there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny. View Publication

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However,transplantation of pig organs results in an acute immune rejection,leading to death of the organ within minutes. The $\$-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection,yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g.,gene targeting) that often require highly robust and proliferative cells. In this report,we generated GALT-KO piPSCs by the overexpression of POU5F1,SOX2,NANOG,LIN28,KLF-4,and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics,expressing integrated reprogramming genes in addition to the pluripotent markers AP,SSEA1,and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation,somatic cell nuclear transfer,or chimera formation. View Publication

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

Current protocols for human pluripotent stem cell (hPSC) expansion require feeder cells or matrices from animal sources that have been the major obstacle to obtain clinical grade hPSCs due to safety issues,difficulty in quality control,and high expense. Thus,feeder-free,chemically defined synthetic platforms have been developed,but are mostly confined to typical polystyrene culture plates. Here,we report a chemically defined,material-independent,bio-inspired surface coating allowing for feeder-free expansion and maintenance of self-renewal and pluripotency of hPSCs on various polymer substrates and devices. Polydopamine (pDA)-mediated immobilization of vitronectin (VN) peptides results in surface functionalization of VN-dimer/pDA conjugates. The engineered surfaces facilitate adhesion,proliferation,and colony formation of hPSCs via enhanced focal adhesion,cell-cell interaction,and biophysical signals,providing a chemically defined,xeno-free culture system for clonal expansion and long-term maintenance of hPSCs. This surface engineering enables the application of clinically-relevant hPSCs to a variety of biomedical systems such as tissue-engineering scaffolds and medical devices. View Publication