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

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation,gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here,we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood,mobilized peripheral blood,and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic,cytochemical,immunophenotypical,clonogenic,and gene expression analyses. Notably,valproic acid highly preserves the CD34 positivity after 1 week (range,40-89%) or 3 weeks (range,21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover,valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4,a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133,a recognized marker gene for stem cell populations. Overall,our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro. View Publication

Resveratrol (trans-3,5,4'-trihydroxystilbene) (RSV) is a natural polyphenol with protective effects over cardiac tissues and can affect cell survival and differentiation in cardiac stem cells transplantation. However,whether this agent can affect cardiomyocytes (CMs) differentiation of induced pluripotent stem cells (iPSCs) is not yet clear. This study explored whether RSV can affect CMs differentiation of human iPSCs. Under embryoid bodies (EBs) condition,the effect of RSV on the change of pluripotent markers,endoderm markers,mesoderm markers,and ectoderm markers was measured using qRT-PCR. Under CM differentiation culture,the effect of RSV on CM specific markers was also measured. The regulative role of RSV over canonical Wnt signal pathway and serum response factor- (SRF-) miR-1 axis and the functions of these two axes were further studied. Results showed that RSV had no effect on the self-renewal of human iPSCs but could promote mesoderm differentiation. Under CM differentiation culture,RSV could promote CM differentiation of human iPSCs through suppressing canonical Wnt signal pathway and enhancing SRF-miR-1 axis. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology,gene therapy,and clinical transplantation. Here,we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor,thrombopoietin,Flt-3 ligand,interleukin (IL)-3,and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition,we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice,which showed myeloid,B,T,and natural killer cells as well as CD133(+)CD34(+) cells,and hematopoietic reconstitution in the secondary recipients. Interestingly,the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation. View Publication

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays,knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells,respectively,as well as a 2- to 4-fold increase in SRC after 4 d of culture. However,after 9 d of culture,all SRC were lost,despite further increases in total cells,CFC content,and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation,and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells. View Publication

Hematopoiesis is dependent upon the bone marrow microenvironment,which is comprised of multiple mesenchymal cell types,including fibroblasts,endothelial cells,osteoblasts,and stroma progenitors. The canonical Wnt signaling pathway,which relies on the beta-catenin protein to mediate its signal,is necessary for the normal development of mesenchymal tissue. We hypothesized that canonical Wnt signaling regulates the cellular composition and function of the bone marrow microenvironment. We observed that a beta-catenin-deficient bone marrow microenvironment maintained hematopoietic stem cells but exhibited a decreased capacity to support primitive hematopoietic cells. These results correlated with decreased numbers of osteoblasts and with decreased production of basic fibroblast growth factor,stem cell factor,and vascular cell adhesion molecule-1. From these data,we propose a model in which beta-catenin in the microenvironment is required noncell autonomously for long-term maintenance of hematopoietic progenitors. View Publication

A microfluidic human pluripotent stem cell (hPSC) array has been developed for robust and reproducible hPSC culture methods to assess chemically defined serum- and feeder-free culture conditions. This microfluidic platform,combined with image cytometry,enables the systematic analysis of multiple simultaneously detected marker expression in individual cells,for screening of various chemically defined media across hPSC lines,and the study of phenotypic responses. View Publication

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade,and two clinical trials have been recently approved. iPSCs were created only recently,and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However,there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review,we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine. View Publication

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However,current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation,which is required to generate endodermal,ectodermal,and mesodermal cell lineages. We report a new,straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs,which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1,GAG,and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats,magnetic resonance imaging studies showed gradual engraftment,and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs,which could improve cartilage regeneration outcomes in arthritic joints. View Publication

We have developed a simple protocol for inducing the myocardial differentiation of human induced pluripotent stem (iPS) cells. Human iPS cell-derived embryonic bodies (EBs) were treated with a combination of activin-A,bone morphogenetic protein-4 and wnt-3a for one day in serum-free suspension culture,and were subsequently treated with noggin for three days. Thereafter,the EBs were subjected to adherent culture in media with 5% serum. All EBs were differentiated into spontaneously beating EBs,which were identified by the presence of striated muscles in transmission electron microscopy and the expression of the specific cardiomyocyte markers,NKX2-5 and TNNT2. The beating rate of the beating EBs was decreased by treatment with a rapidly activating delayed rectifier potassium current (Ikr) channel blocker,E-4031,an Ikr trafficking inhibitor,pentamidin,and a slowly activating delayed rectifier potassium current (Iks) channel blocker,chromanol 293B,and was increased by treatment with a beta-receptor agonist,isoproterenol. At a low concentration,verapamil,a calcium channel blocker,increased the beating rate of the beating EBs,while a high concentration decreased this rate. These findings suggest that the spontaneously beating EBs were myocardial cell clusters. This simple protocol for myocardial differentiation would be useful in providing a sufficient number of the beating myocardial cell clusters for studies requiring human myocardium. View Publication

Human tumor vessels express tumor vascular markers (TVM),proteins that are not expressed in normal blood vessels. Antibodies targeting TVMs could act as potent therapeutics. Unfortunately,preclinical in vivo studies testing anti-human TVM therapies have been difficult to do due to a lack of in vivo models with confirmed expression of human TVMs. We therefore evaluated TVM expression in a human embryonic stem cell-derived teratoma (hESCT) tumor model previously shown to have human vessels. We now report that in the presence of tumor cells,hESCT tumor vessels express human TVMs. The addition of mouse embryonic fibroblasts and human tumor endothelial cells significantly increases the number of human tumor vessels. TVM induction is mostly tumor-type-specific with ovarian cancer cells inducing primarily ovarian TVMs,whereas breast cancer cells induce breast cancer specific TVMs. We show the use of this model to test an anti-human specific TVM immunotherapeutics; anti-human Thy1 TVM immunotherapy results in central tumor necrosis and a three-fold reduction in human tumor vascular density. Finally,we tested the ability of the hESCT model,with human tumor vascular niche,to enhance the engraftment rate of primary human ovarian cancer stem-like cells (CSC). ALDH(+) CSC from patients (n = 6) engrafted in hESCT within 4 to 12 weeks whereas none engrafted in the flank. ALDH(-) ovarian cancer cells showed no engraftment in the hESCT or flank (n = 3). Thus,this model represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology. View Publication