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

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

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera. View Publication

Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesonephros are hypothesized to colonize the fetal liver,but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here,we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach,we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1,there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos,while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos,culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5,Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired inter-action with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis. 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

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet,it is unknown whether this loss of HSCs is an irreversible process. In this study,we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations,namely,HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors,we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression,replenishes the HSC pool,restores stem cell properties,and corrects platelet production. In some mice,megakaryocyte counts were atypically high,accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential,with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal. View Publication

Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors,which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here,we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA,synthesized siRNA and transcribed shRNA,reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction,however,was sufficient to increase transduction efficiency to two- to four-fold in cell cultures,and followed by a seven- to eight-fold increase in mice. The RNAi treated,lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together,both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus,RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy. View Publication

The main limitations of hematopoietic cord blood (CB) transplantation,viz,low cell dosage and delayed reconstitution,can be overcome by ex vivo expansion. CB expansion under conventional culture causes rapid cell differentiation and depletion of hematopoietic stem and progenitor cells (HSPCs) responsible for engraftment. In this study,we use combinatorial cell culture technology (CombiCult{\textregistered}) to identify medium formulations that promote CD133+ CB HSPC proliferation while maintaining their phenotypic characteristics. We employed second-generation CombiCult screens that use electrospraying technology to encapsulate CB cells in alginate beads. Our results suggest that not only the combination but also the order of addition of individual components has a profound influence on expansion of specific HSPC populations. Top protocols identified by the CombiCult screen were used to culture human CD133+ CB HSPCs on nanofiber scaffolds and validate the expansion of the phenotypically defined CD34+CD38lo/-CD45RA-CD90+CD49f+ population of hematopoietic stem cells and their differentiation into defined progeny. View Publication

Pre-clinical research on B and NK cell development relies on murine stromal cell-based systems with reduced physiological relevance and clinical applicability. A serum-free,fully humanized co-culture system utilizing human bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed to differentiate CB-CD34+ cells towards B and NK cell lineages. Differentiation dynamics were monitored via flow cytometry,with immunophenotypic analysis tracking progression from progenitors to mature cells. The system generated CD19+ IgM+ immature B cells and CD56+ CD16+ NK cells,recapitulating fetal stages of human lymphopoiesis. Serum-free media conditions ensured reproducibility and high overall yield of CD19+ B (35 ± 5.32%) and CD56+ NK (28.46 ± 7.01%) cell progenitors. Flow cytometry identified distinct population peaks,confirming temporal control over differentiation. This clinically relevant platform addresses the limitations of traditional models by providing a more physiologically accurate human microenvironment. The serum-free system supports applications in disease modeling,genotoxic compound screening,and mutational studies of hematopoiesis. By enabling scalable production of B and NK cells it aims to accelerate translational research for immunodeficiencies,cancer immunotherapy,and hematopoietic disorders. View Publication

Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However,the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34+ cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells,and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential,which was elevated compared with control stromal cells,as indicated by the colony-forming units in culture (CFU-Cs) (26-fold +/- 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's],63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-beta2m-/- (NOD/SCID-beta2-/-) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results,and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-beta2m-/- RCs proliferated efficiently on Ihh stromal cells,compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells. View Publication