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

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However,functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC,a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover,strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα),were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors,and may contribute to the block in definitive hematopoiesis from hESC. View Publication

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here,we identify a newly discovered human repopulating cell,distinct from previously identified repopulating cells,that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells,or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR,Thy-1 and CD34). In contrast to CD34+ subfractions,Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations,providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation. View Publication

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function,little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages,to migrate toward stromal cell-derived factor 1α,and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However,the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast,adhesion to the extracellular matrix was inhibited by silencing of talin-1,but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity,independent of integrin functions. 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

Although hematopoiesis is known to originate in a population of very primitive cells with both lymphopoietic and myelopoietic potential,a procedure for enumerating such cells has to date not been available. We now describe a quantitative assay for long-term repopulating stem cells with the potential for reconstituting all hematopoietic lineages. This assay has two key features. The first is the use of competitive repopulation conditions that ensure not only the detection of a very primitive class of hematopoietic stem cells but also the survival of lethally irradiated mice transplanted with very low numbers of such cells. The second is the use of a limiting-dilution experimental design to allow stem cell quantitation. The assay involves transplanting limiting numbers of male test" cells into lethally irradiated syngeneic female recipients together with 1-2 x 10(5) syngeneic female marrow cells whose long-term repopulating ability has been compromised by two previous cycles of marrow transplantation. The proportion of assay recipients whose regenerated hematopoietic tissues are determined to contain greater than or equal to 5% cells of test cell origin (male) greater than or equal to 5 weeks later is then used to calculate the frequency of competitive repopulating units (CRU) in the original male test cell suspension (based on Poisson statistics). Investigation of this assay system has shown that all three potential sources of stem cells (test cells� View Publication

SHP2,a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene,plays a critical role in developmental hematopoiesis in the mouse,and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However,the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition,the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF,and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation,survival,and differentiation of human progenitor cells. View Publication

Efficient derivation and isolation of hematopoietic stem cells (HSCs) from human pluripotent stem cell (hPSC) populations remains a major goal in the field of developmental hematopoiesis. These enticing pluripotent stem cells (comprising both human embryonic stem cells and induced pluripotent stem cells) have been successfully used to generate a wide array of hematopoietic cells in vitro,from primitive hematoendothelial precursors to mature myeloid,erythroid,and lymphoid lineage cells. However,to date,PSC-derived cells have demonstrated only limited potential for long-term multilineage hematopoietic engraftment in vivo - the test by which putative HSCs are defined. Successful generation and characterization of HSCs from hPSCs not only requires an efficient in vitro differentiation system that provides insight into the developmental fate of hPSC-derived cells,but also necessitates an in vivo engraftment model that allows identification of specific mechanisms that hinder or promote hematopoietic engraftment. In this chapter,we will describe a method that utilizes firefly luciferase-expressing hPSCs and bioluminescent imaging to noninvasively track the survival,proliferation,and migration of transplanted hPSC-derived cells. Combined with lineage and functional analyses of engrafted cells,this system is a useful tool to gain insight into the in vivo potential of hematopoietic cells generated from hPSCs. View Publication

In the field of hematopoietic regeneration,deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells,remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines,UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here,using a serum-free,feeder-free,and chemically defined induction protocol,we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34(+)CD45(+) derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol,the derived hematopoietic progenitors failed to engraft in NOG mice,indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols,UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro. View Publication

BACKGROUND: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood,ex vivo transduction of the gene of interest into them,and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor,time and money,while enhancing HSCs viability,transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes,in which reverse transcription of viral DNA is not completed. METHODS: In the present study,we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors,based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction,we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector,developed in our laboratory,that allows targeted transduction to specific cell receptors via antibody recognition. RESULTS: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. CONCLUSIONS: Overall,the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. View Publication

Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation. View Publication

Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998),human embryonic stem cells (hESCs) have been derived from blastocysts,and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs,fibroblast feeder layers are often used at some phase in the culturing protocol. The feeders - often mouse embryonic fibroblasts (mEFs) - provide a substrate that increases plating efficiency,helps maintain pluripotency,and facilitates survival and growth of the stem cells. Various protocols for culturing embryonic stem cells from both species are available with newer trends moving toward feeder-free and serum-free culture. The purpose of this chapter is to provide basic protocol information on the isolation of mouse embryonic fibroblasts and establishment of feeder layers,the culture of mESCs on both mEFs and on gelatin in serum-containing medium,and the culture of hESCs in defined media on both mEFs (hESC culture medium) and Matrigel (mTeSR). These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant user's particular purpose. View Publication

To exploit the full potential of human pluripotent stem cells for regenerative medicine,developmental biology and drug discovery,defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed,but finding chemically defined,robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides,which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells. View Publication