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

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such,hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However,for their full potential to be realized,both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however,they suffer from some major limitations including lack of definition,xenobiotic nature,batch-to-batch variation,and labor-intensive production. Therefore,hESC culture definition is essential if hESC lines,and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state,for over 30 passages using MT and SP. Additionally,we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture,contributing to the standardization of hESC in vitro models and ultimately their application. 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

OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases. View Publication

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH,however,lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore,other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs),characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming,and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation,the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View Publication

Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However,transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here,we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore,Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore,we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery,thus providing therapeutic benefits for patients after clinical myelosuppressive treatment. View Publication

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells,its physiological function(s) remain unknown,and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations,followed by competitive bone marrow reconstitution assays,shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent,the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore,PrP is a marker for HSCs and supports their self-renewal. 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

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

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples,as well as in wild-type versus Hoxa9-deficient marrow,using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors,being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells,indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1,an important regulator of myelopoiesis,was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein,HOXA9 did not appear to modulate Pu.1 expression in murine BM cells. View Publication