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

Infection with Anaplasma phagocytophilum,a gram-negative,lipopolysaccharide (LPS)-negative,obligate intracellular bacterium,results in multiple peripheral blood cytopenias. We hypothesized that infection with this organism would result in decreased bone marrow (BM) function and shifts in hematopoietic progenitor cells (HPCs) and lineage-committed cells in a well-established murine model of infection. HPCs and lineage-committed progenitors were enumerated in the BM and spleen during acute infection. BM cytokine production and BM CXCL12 expression were determined. Infection resulted in peripheral blood bicytopenia,marked decreases in the number of lineage-committed HPCs in the BM along with concurrent increases in the number of lineage-committed HPCs in the spleen,and a mixed,predominantly myelosuppressive BM cytokine environment. There was significant downregulation of CXCL12 in BM cells that may have been partially responsible for changes in HPC trafficking observed. Changes occurred in the absence of direct pathogen infection of BM cells. Hematopoietic lineage assessment demonstrated that there was loss of erythrocytes and B lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to lymphoid hyperplasia and increased hematopoiesis,most notably erythropoiesis. These changes largely mimic well-described inflammation and endotoxin-mediated effects on the BM and spleen; however,the numbers of peripheral blood neutrophils appear to be independently modulated as granulocytic hyperplasia does not result in neutrophilia. Our findings highlight a well-conserved series of events that we demonstrate can be instigated by an LPS-negative pathogen in the absence of an endotoxin-mediated acute proinflammatory response. View Publication

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation,we cultured mouse ES cells on 3D,highly porous,biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however,unlike 2D differentiation,these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly,the efficiency of hematopoietic precursor cell (HPC) generation on 3D,as indicated by the expression of various HPC-specific surface markers (CD34,Sca-1,Flk-1,and c-Kit) and colony-forming cell (CFC) assays,was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication

The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however,the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here,we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore,we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests,including comprehensive tumorigenicity assays and genomic integrity validation,should be rigorously executed before the clinical application of HiPSCs. In addition,HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability. View Publication

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate,proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs,there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis,graft vasculopathy and hyperlipidemia-induced atherosclerosis,bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably,purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs,and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization,homing,differentiation and proliferation of bone marrow-derived vascular progenitor cells. View Publication

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),an endocrine disrupting chemical (EDC),can cause carcinogenesis,immunosuppression,and teratogenesis,through a ligand-activated transcription factor,the aryl hydrocarbon receptor (AhR). Despite remarkable recent advances in stem cell biology,the influence of TCDD on hematopoietic stem cells (HSCs),which possess the ability to reconstitute long-term multilineage hematopoiesis,has not been well investigated. In this study we examined the influence of TCDD on HSCs enriched for CD34(-),c-kit(+),Sca-1(+),lineage negative (CD34-KSL) cells. The number of the CD34-KSL cells was found to be increased about four-fold upon a single oral administration of TCDD (40 micro g/kg body weight). Surprisingly,we found that these TCDD-treated cells almost lost long-term reconstitution activity. This defect was not present in AhR(-/-) mice. These findings suggest that modulation of AhR/ARNT system activity may have an effect on HSC function or survival. View Publication

Vascular endothelial growth factor (VEGF) and its receptors play an essential role in the formation and maintenance of the hematopoietic and vascular compartments. The VEGF receptor-2 (VEGFR-2) is expressed on a population of hematopoietic cells,although its role in hematopoiesis is still unclear. In this report,we have utilized a strategy to selectively activate VEGFR-2 and study its effects in primary bone marrow cells. We found that VEGFR-2 can maintain the hematopoietic progenitor population in mouse bone marrow cultured in the absence of exogenous cytokines. Maintenance of the hematopoietic progenitor population is due to increased cell survival with minimal effect on proliferation. Progenitor survival is mainly mediated by activation of the phosphatidylinositol 3'-kinase/Akt pathway. Although VEGFR-2 also activated Erk1/2 mitogen-activated protein kinase,it did not induce cell proliferation,and blockade of this pathway only partially decreased VEGFR-2-mediated survival of hematopoietic progenitors. Thus,the role of VEGFR-2 in hematopoiesis is likely to maintain survival of hematopoietic progenitors through the activation of antiapoptotic pathways. View Publication

The lipocalin mouse 24p3 has been implicated in diverse physiological processes,including apoptosis,iron trafficking,development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However,a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3,we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid,myeloid,and erythroid cells,which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead,apoptotic defects were unique to many mature hematopoietic cell types,including neutrophils,cytokine-dependent mast cells,thymocytes,and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells,thus explaining their resistance to the ensuing cell death. The results of these studies,in conjunction with those of previous studies,reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly,these functions are limited to relatively mature blood cell compartments. View Publication

BACKGROUND: Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC),glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS). METHODOLOGY/PRINCIPAL FINDINGS: We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC,glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable,expressed mature and subtype-specific neuronal markers,and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC,glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations. View Publication

During hematopoietic differentiation of human embryonic stem cells (hESCs),early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays,their phenotype has not been defined. Here,using hESC differentiation in coculture with OP9 stromal cells,we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45,persisted on differentiating hematopoietic cells,and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore,we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)),expressed KDR,VE-cadherin,and CD105 endothelial proteins,and expressed GATA-2,GATA-3,RUNX1,C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile. View Publication

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen,we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure,while cell surface marker analyses revealed a VE-cadherin+CD31+CD34+KDR+CD43???putative endothelial progenitor population. Furthermore,molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL+multi-cellular modules and a VEGFR3+sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation. View Publication

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here,through the sequential in vitro targeting of selected signaling pathways,we have developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex,as an extracellular matrix,could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP,SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL,and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells,1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally,ES-DBCs were responsive to high glucose in static incubation and perifusion studies,and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion,targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs,small molecules or genes that may have potential to influence beta-cell function. View Publication