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

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates,cells were cocultured for 10 days with the stem cell supporting cell line AFT024,fibronectin (FN),or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times,a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time,we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC),this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential. View Publication

Type 1 interferon-producing cells (IPCs),also known as plasmacytoid dendritic cell (DC) precursors,represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection,autoimmune SLE,and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3),IL-7,stem cell factor (SCF),macrophage-colony-stimulating factor (M-CSF),and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture,they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L,inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system,combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors,permits the generation of more than 10(9) IPCs from a single blood donor. View Publication

Dysregulated innate immune responses underlie multiple inflammatory diseases,but clinical translation of preclinical innate immunity research in mice is hampered by the difficulty of studying human inflammatory reactions in an in vivo context. We therefore sought to establish in vivo human inflammatory responses in NSG-QUAD mice that express four human myelopoiesis transgenes to improve engraftment of a human innate immune system. We reconstituted NSG-QUAD mice with human hematopoietic stem and progenitor cells (HSPCs),after which we evaluated human myeloid cell development and subsequent human responses to systemic and local lipopolysaccharide (LPS) challenges. NSG-QUAD mice already displayed engraftment of human monocytes,dendritic cells and granulocytes in peripheral blood,spleen and liver at 6 weeks after HSPC reconstitution,in which both classical,intermediate and non-classical monocytes were present. These huNSG-QUAD mice responded to intraperitoneal and intranasal LPS challenges with production of NF-κB-dependent human cytokines,a human type I interferon response,as well as inflammasome-mediated production of human IL-1β and IL-18. The latter were specifically abrogated by the NLRP3 inhibitor MCC950,while LPS-induced human monocyte death was not altered. Besides providing proof-of-principle for small molecule testing of human inflammatory reactions in huNSG-QUAD mice,this observation suggests that LPS-induced in vivo release of human NLRP3 inflammasome-generated cytokines occurs in a cell death-independent manner. HuNSG-QUAD mice are competent for the NF-κB,interferon and inflammasome effectors of human innate immunity,and can thus be utilized to investigate signaling mechanisms and pharmacological targeting of human inflammatory responses in an in vivo setting. 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

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