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

Clonal analysis is important for many areas of hematopoietic stem cell research,including in vitro cell expansion,gene therapy,and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle,they generally provide a low-resolution,biased,and incomplete assessment of clonality. To overcome those limitations,we labeled retroviral vectors with random sequence tags or barcodes." On integration� View Publication

The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However,this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g.,LMPPs),implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here,we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP),whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model. View Publication

BACKGROUND The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application. METHODS Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium,Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated. RESULTS We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF,respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF,respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium,for two cryopreservation systems (Ptextless0.05). CONCLUSION We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated,maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies. View Publication

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing,multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion. 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

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

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway,suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however,their role in the hematopoietic development in vivo remains unknown. Here,we identify a subpopulation of NG2 + Runx1 + perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2 + cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs,endothelial cells and hematopoietic cells in the murine AGM. Importantly,this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental,as NG2 + Runx1 + cells were not detected in the adult bone marrow,demonstrating the existence of a specialised pericyte population in the HSC-generating niche,unique to the embryo. Subject terms: Cell biology,Haematopoiesis,Cardiovascular biology View Publication

Human embryonic stem cells,because of their unique combination of long-term self-renewal properties and pluripotency,are providing new avenues of investigation of stem cell biology and human development and show promise in providing a new source of human cells for transplantation therapies and pharmaceutical testing. Current methods of propagating these cells using combinations of mouse fibroblast feeder cultures and bovine serum components are inexpensive and,in general,useful. However,the systematic investigation of the regulation of self-renewal and the production of safer sources of cells for transplantation depends on the elimination of animal products and the use of defined culture conditions. Both goals are served by the development of serum-free culture methods for human embryonic stem cells. View Publication

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals,thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide,the use of human cell-teratogenicity assays has just started to be explored. Herein,a human pluripotent stem cell test (hPST) for identifying teratogens is described,benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically,71 drug-like compounds with known in vivo effects,including thalidomide,were examined in the hPST. A threshold of 5μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore,15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally,to assess the suitability of the hPST for high-throughput screens,a small library of 300 kinase inhibitors was tested,demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus,the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models. View Publication

Although extracellular nucleotides support a wide range of biologic responses of mature blood cells,little is known about their effect on blood cell progenitor cells. In this study,we assessed whether receptors for extracellular nucleotides (P2 receptors [P2Rs]) are expressed on human hematopoietic stem cells (HSCs),and whether activation by their natural ligands,adenosine triphosphate (ATP) and uridine triphosphate (UTP),induces HSC proliferation in vitro and in vivo. Our results demonstrated that CD34(+) HSCs express functional P2XRs and P2YRs of several subtypes. Furthermore,stimulation of CD34(+) cells with extracellular nucleotides caused a fast release of Ca(2+) from intracellular stores and an increase in ion fluxes across the plasma membrane. Functionally,ATP and,to a higher extent,UTP acted as potent early acting growth factors for HSCs,in vitro,because they strongly enhanced the stimulatory activity of several cytokines on clonogenic CD34(+) and lineage-negative CD34(-) progenitors and expanded more primitive CD34(+)-derived long-term culture-initiating cells. Furthermore,xenogenic transplantation studies showed that short-term preincubation with UTP significantly expanded the number of marrow-repopulating HSCs in nonobese diabetic/severe combined immunodeficiency mice. Our data suggest that extracellular nucleotides may provide a novel and powerful tool to modulate HSC functions. View Publication

A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders,we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months,mice developed polycythemia,macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months,polycythemia regressed,abnormally shaped red blood cells and platelets were seen in circulation,and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia,thrombocytopenia,high neutrophilia,and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders. View Publication

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment,proliferation,and differentiation. Given its critical functions,c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay,we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic,functionality of binding was shown. The mimic decreased c-Myb expression,and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest,the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally,in studies using normal human CD34(+) cells,we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation,and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate,these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis. View Publication