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

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells,although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need,we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and,in some cases,lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3),IL-6,and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean,27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage),as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418,averaged 11% and 12%,respectively,in 6 experiments. These values could be increased to 100% and 77%,respectively,by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%,respectively,in the CD34+HSA- cells. In addition,the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population was textless or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells,ie,those expressing little or no CD38,could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition,such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry. View Publication

Human stem cells are promising sources for bladder regeneration. Among several possible sources,pluripotent stem cells are the most fascinating because they can differentiate into any cell type,and proliferate limitlessly in vitro. Here,we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE),and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes,but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs,they notably expressed urothelium-specific genes such as UPIb,UPII,UPIIIa,P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII,CK8/18 and P63 as well as tight junction molecules,E-CADHERIN and ZO-1. Additionally,hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay,indicating BUCs possessed the functional units of barrier on their surfaces. However,BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary,we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro. View Publication

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast,we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34,lineage markers,CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells),endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo,CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials,characterized by the expression of c-Kit and CXCR4 as well as EphB4,EphB2,and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life. View Publication

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for textgreater10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media,the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR),HEScGRO,and KnockOut media,mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions,cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4,stage-specific embryonic antigens 4 (SSEA-4),and Tra-1-60. In addition,hESC maintained the expression of podocalyxin-like protein-1 (PODXL),an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills textgreater90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies,derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal,endodermal,and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%,higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications. View Publication

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro,as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study,we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal,neonatal,and adult fibroblasts through reprogramming with POU5F1,SOX2,NANOG,and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC,H1,H7,H9,H13,and H14). Similar to hESCs,all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors,iPSC-derived primitive blood cells formed all types of hematopoietic colonies,including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic),lin(-)CD34(+)CD43(+)CD45(-) (multipotent),and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs,the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal,neonatal,or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes,patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks. View Publication

Monocytic lineage cells (monocytes,macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established,these methods depend on the use of xenogeneic materials and,therefore,have a relatively poor-reproducibility. Here,we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine,disease-specific iPSC studies and drug discovery. View Publication

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus,the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here,we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by textgreater10 000-fold for both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also,we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore,a TAT-SALL4B fusion rapidly expanded CD34(+) cells,and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs. View Publication