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

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

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4,SOX2,NANOG,and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes,express telomerase activity,express cell surface markers and genes that characterize human ES cells,and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development,as well as for applications in transplantation medicine,once technical limitations (for example,mutation through viral integration) are eliminated. View Publication

While enucleation is a critical step in the terminal differentiationbackslashnof human red blood cells,the molecular mechanisms underlying thisbackslashnunique process remain unclear. To investigate erythroblast enucleationbackslashnwe studied the erythroid differentiation of human embryonic stembackslashncells (hESCs),which provide a unique model for deeper understandingbackslashnof the development and differentiation of multiple cell types. Firstly,backslashnusing a two-step protocol,we demonstrated that terminal erythroidbackslashndifferentiation from hESCs is directly dependent on the age of thebackslashnembryoid bodies. Secondly,by choosing hESCs in two extreme conditionsbackslashnof erythroid culture,we obtained an original differentiation modelbackslashnwhich allows one to study the mechanisms underlying the enucleationbackslashnof erythroid cells by analyzing the gene and miRNA (miR) expressionbackslashnprofiles of cells from these two culture conditions. Thirdly,usingbackslashnan integrated analysis of mRNA and miR expression profiles,we identifiedbackslashn5 miRs potentially involved in erythroblast enucleation. Finally,backslashnby selective knockdown of these 5 miRs we found miR-30a to be a regulatorbackslashnof erythroblast enucleation in hESCs. This article is protected bybackslashncopyright. All rights reserved. View Publication

Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation,but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify $\$-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore,treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous $\$-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing. View Publication

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here,we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol,which induced their differentiation into intermediate mesoderm,then into nephron progenitors and,finally,into mature podocytes. After differentiation,cells expressed the main podocyte markers,such as synaptopodin,WT1,α-Actinin-4,P-cadherin and nephrin at the protein and mRNA level,and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall,these findings demonstrate the establishment of a robust protocol that,mimicking developmental stages,makes it possible to derive functional podocytes in vitro. View Publication

Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1,12,17,and 20. Thus,hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes,there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding,qPCR,fluorescence in situ hybridization,and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods,a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine. View Publication

Human pluripotent stem (hPS) cells such as human embryonic stem (hES) and induced pluripotent stem (hiPS) cells are vulnerable under single cell conditions,which hampers practical applications; yet,the mechanisms underlying this cell death remain elusive. In this paper,we demonstrate that treatment with a specific inhibitor of non-muscle myosin II (NMII),blebbistatin,enhances the survival of hPS cells under clonal density and suspension conditions,and,in combination with a synthetic matrix,supports a fully defined environment for self-renewal. Consistent with this,genetically engineered mouse embryonic stem cells lacking an isoform of NMII heavy chain (NMHCII),or hES cells expressing a short hairpin RNA to knock down NMHCII,show greater viability than controls. Moreover,NMII inhibition increases the expression of self-renewal regulators Oct3/4 and Nanog,suggesting a mechanistic connection between NMII and self-renewal. These results underscore the importance of the molecular motor,NMII,as a novel target for chemically engineering the survival and self-renewal of hPS cells. View Publication

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells,including fibroblasts,keratinocytes and peripheral blood cells,with variable reprogramming efficiencies and kinetics. Here,we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells,with similar efficiencies to keratinocytes,which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure. View Publication

In the present study,the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal,(iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media,keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®. (ii) When hSKP were subsequently exposed to NeuroCult® media,cells underwent a weak neuro-ectodermal differentiation expressing nestin,myelin binding protein (MBP),vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin(+)/vimentin(+)/MBP(-)/AFP(-)). (iii) Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone,small lipid droplets were observed,suggesting their differentiation potential towards adipocyte-like cells. (iv) Finally,after sequential exposure to hepatogenic growth factors and cytokines,an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach. In summary,a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition,we demonstrate here for the first time that,in a suitable keratinocyte stimulating micro-environment,hSKP can generate keratinocyte-like progeny in vitro. View Publication

Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones,three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation,but it does not require culture preadaptation,use of microcarriers or any other matrices. Over a time course of 4-7 d,hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly,hPSCs maintain pluripotency and karyotype stability for more than ten passages. View Publication

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However,the lack of simple,robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells,2) significantly increases viability and replating efficiency,3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation,we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%,with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions,and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs. View Publication

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression,could be maintained in culture for several weeks,expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice,the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease View Publication