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

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups,previously introduced by nitrogen atmospheric pressure nonequilibrium plasma,are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1,a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and,in the swollen state,are translucent,soft,and pliable,yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant,since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds,the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions. View Publication

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However,mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study,we investigated the effects of two nuclear receptors,liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter,while GCNF strongly suppressed CR-1 expression in these cells. In addition,the CR-1 promoter was unmethylated in NTERA-2 cells,while T47D,ZR75-1,and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells,promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors,suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively,these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer. View Publication

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study,we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2,lipoprotein lipase,and adipsin increased during adipocytogenesis of hMSCs. Simultaneously,the expression of canonical WNT2,10B,13,and 14 decreased,whereas non-canonical WNT4 and 11 increased,and WNT5A was unchanged. A small molecule WNT mimetic,SB-216763,increased accumulation of β-catenin protein,inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast,knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells. View Publication

BACKGROUND AIMS: Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently,stem cell culture has been carried out using feeder cells,and culture media,that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells,essential conditions for the use of stem cells for clinical purposes. To date,however,there has been limited success in achieving this aim. METHODS,RESULTS AND CONCLUSIONS: Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington's disease and myotonic dystrophy 1) genomically stable hES cell lines,and their adaptation to feeder-free culture,all under xeno-free conditions. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC) show great promise as autologous donor cells to treat heart disease. A major technical obstacle to this approach is that available induction methods often produce heterogeneous cell population with low percentage of cardiomyocytes. Here we describe a cardiac enrichment approach using non-integrating adeno-associated virus (AAV). We first examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes. Result showed that AAV1 demonstrated the highest in vitro transduction efficiency among seven widely used serotypes. Next differentiated iPSC derivatives were transduced with drug-selectable AAV1 expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to 57% in two weeks. Compared to other enrichment strategies such as integrative genetic selection,mitochondria labeling or surface marker cell sorting,this simple AAV method described herein bypasses antibody or dye labeling. These findings provide proof-of-concept for large-scale cardiomyocyte enrichment by exploiting AAV's intrinsic tissue tropism. View Publication

Transformed,oncogenic precursors,possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours,have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs),amongst which BMP4 elicits the strongest effect,trigger a significant reduction in the stem-like,tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly,in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation,and increased expression of markers of neural differentiation,with no effect on cell viability. The concomitant reduction in clonogenic ability,in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating,stem-like cells from GBMs and the results also identify BMP4 as a novel,non-cytotoxic therapeutic effector,which may be used to prevent growth and recurrence of GBMs in humans. View Publication

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs),hESCs normally undergo high rates of spontaneous apoptosis and differentiation,making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However,despite the accumulation of p53,it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types,including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation. View Publication

Conventionally,researchers remove spontaneously differentiated areas in human pluripotent stem cell (hPSC) colonies by using a finely drawn glass pipette or a commercially available syringe needle. However,when extreme differentiation occurs,it is inefficient to purify the remaining undifferentiated cells,as these undifferentiated areas are too small to be isolated completely with the mechanical method. Antibodies can be utilized to purify the rare undifferentiated cells; however,this type of purification cannot be used in xeno-free culture systems. To avoid the loss of valuable hPSCs,we developed a novel method to isolate undifferentiated hPSCs from extremely differentiated colonies that could be easily adapted to xeno-free culture conditions. This protocol involves dissecting away differentiated areas,dissociating the remaining colony into clumps,seeding small clumps into new dishes,and picking undifferentiated colonies for expansion. Using this method,we routinely achieve completely undifferentiated colonies in one passage without the use of antibody-based purification. View Publication

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising can- didate cell sources for regenerative medicine. However,despite the common ability of hiPSCs and hESCs to dif- ferentiate into all 3 germ layers,their functional equivalence at the single cell level remains to be demonstrated. Moreover,single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here,we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs,suggesting that hiPSCs occupy an alternate,less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exer- cised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs,particularly when producing differentiated cells for regenerative medicine aims. View Publication

It has been postulated that during human fetal development,all cells of the lung epithelium derive from embryonic,endodermal,NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However,this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity,these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support,this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively,when recombined with fetal mouse lung mesenchyme,the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved,stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted,patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine. View Publication

Human induced pluripotent stem cells (hiPSCs) are being considered for use in understanding haematopoietic disorders and as a potential source of in vitro manufactured red cells. Here,we show that hiPSCs are able to recapitulate various stages of developmental erythropoiesis. We show that primitive erythroblasts arise first,express CD31(+) with CD235a(+),embryonic globins and red cell markers,but fail to express the hallmark red cell transcripts of adult erythropoiesis. When hiPSC-derived CD45(+) CD235a(-) haematopoietic progenitors are isolated on day 12 and further differentiated on OP9 stroma,they selectively express CD36(+) and CD235a(+),adult erythroid transcripts for transcription factors (e.g.,BCL11A,KLF1) and fetal/adult globins (HBG1/2,HBB). Importantly,hiPSC- and cord-derived CD36(+) CD235a(+) erythroblasts show a striking homology by transcriptome array profiling (only 306 transcripts with a 2Log fold change<1textperiodcentered5- or 2textperiodcentered8-fold). Phenotypic and transcriptome profiling of CD45(+) CD117(+) CD235a(+) pro-erythroblasts and terminally differentiated erythroblasts is also provided,including evidence of a HbF (fetal) to HbA (adult) haemoglobin switch and enucleation,that mirrors their definitive erythroblast cord-derived counterparts. These findings provide a molecular roadmap of developmental erythropoiesis from hiPSC sources at several critical stages,but also helps to inform on their use for clinical applications and modelling human haematopoietic disease. View Publication