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

Human bone marrow (BM) cells contain low levels of the DNA repair protein,O6-alkylguanine-DNA alkyltransferase,which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor,interleukin-3,stem cell factor,the combination,or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition,O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU,resulting in a reduction in the dose of drug (termed the dose-modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since,unlike many other cell types,proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase,myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials. View Publication

While a third of the world carries the burden of tuberculosis,disease control has been hindered by a lack of tools,including a rapid,point-of-care diagnostic and a protective vaccine. In many infectious diseases,antibodies (Abs) are powerful biomarkers and important immune mediators. However,in Mycobacterium tuberculosis (Mtb) infection,a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach,we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses,such that Ltb infection is associated with unique Ab Fc functional profiles,selective binding to FcγRIII,and distinct Ab glycosylation patterns. Moreover,compared to Abs from Atb,Abs from Ltb drove enhanced phagolysosomal maturation,inflammasome activation,and,most importantly,macrophage killing of intracellular Mtb. Combined,these data point to a potential role for Fc-mediated Ab effector functions,tuned via differential glycosylation,in Mtb control. View Publication

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

We asked whether the hypoxia-regulated factor,insulin-like growth factor binding protein-3 (IGFBP3),could modulate stem cell factor receptor (c-kit+),stem cell antigen-1 (sca-1+),hematopoietic stem cell (HSC),or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies,GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration,protection of the developing vasculature from hyperoxia-induced regression,and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion,IGFBP3 mediates EPC migration,differentiation,and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions. View Publication

Cdx1,Cdx2,and Cdx4 comprise the caudal-like Cdx gene family in mammals,whose homologues regulate hematopoietic development in zebrafish. Previously,we reported that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells (ESCs). Here we compare the effect of ectopic Cdx1,Cdx2,and Cdx4 on the differentiation of murine ESC-derived hematopoietic progenitors. The 3 Cdx genes differentially influence the formation and differentiation of hematopoietic progenitors within a CD41(+)c-kit(+) population of embryoid body (EB)-derived cells. Cdx1 and Cdx4 enhance,whereas Cdx2 strongly inhibits,the hematopoietic potential of CD41(+)ckit(+) EB-derived cells,changes that are reflected by effects on hematopoietic lineage-specific and Hox gene expression. When we subject stromal cell and colony assay cultures of EB-derived hematopoietic progenitors to ectopic expression of Cdx genes,Cdx4 dramatically enhances,whereas Cdx1 and Cdx2 both inhibit hematopoietic activity,probably by blocking progenitor differentiation. These data demonstrate distinct effects of Cdx genes on hematopoietic progenitor formation and differentiation,insights that we are using to facilitate efforts at in vitro culture of hematopoietic progenitors from ESC. The behavior of Cdx genes in vitro suggests how derangement of these developmental regulators might contribute to leukemogenesis. View Publication

Irradiation with X-rays has been widely utilized in the clinical treatment of solid tumors and certain hematopoietic malignancies. However,this method fails to completely distinguish between malignant and normal cells. Prolonged or repeated exposure to radiation,whether due to occupational hazards or therapeutical interventions,can cause damage to normal tissues,particularly impacting the hematopoietic system. Therefore,it is important to investigate the effects of total body irradiation on the hematopoietic system of mice and to compare the inhibitory effects of various doses of irradiation on this system. In this study,we primarily employed flow cytometry to analyze mature lineage cells in the peripheral blood,as well as immature hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and spleen. Additionally,we evaluated the multilineage differentiation capacity of HSPCs through colony-forming cell assays. Our results indicated that peripheral B and T cells demonstrated increased sensitivity to irradiation,with significant cell death observed 1-day post-irradiation. Common lymphoid progenitor cells exhibited greater radiotolerance compared to other progenitor cell types,enabling them to maintain a certain population even at elevated doses. Moreover,notable differences were observed between intramedullary and extramedullary hematopoietic stem cells and common lymphoid progenitor cells regarding the extent of damage and recovery rate following irradiation. The multilineage differentiation capacity of HSPCs was also compromised during radiation exposure. In conclusion,different types of mature blood cells,along with immature HSPCs,exhibited varying degrees of sensitivity and tolerance to irradiation,resulting in distinct alterations in cell percentages and numbers. View Publication

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche,but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor,RET,at the cell surface of HSCs,in mediating sustained cellular growth,resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex,GDNF/GFRa1,show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally,we demonstrate that RET drives a multi-faceted intracellular signalling pathway,including key signalling intermediates AKT,ERK1/2,NFkB and p53,responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions. View Publication

Realizing the potential that human embryonic stem cells (hESCs) hold,both for the advancement of biomedical science and the development of new treatments for many human disorders,will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard,it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay,thus,provides a valuable tool for monitoring the quality of hESC cultures,and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies),or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm,mesoderm,and ectoderm lineages. View Publication