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

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC,we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular,and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells,although present at normal frequencies within the CD34(+) subset,were therefore absolutely decreased. In contrast,even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased,and the telomere lengths of these cells were also markedly reduced. Nevertheless,the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs. View Publication

OBJECTIVE: We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) : n the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP),a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation. RESEARCH DESIGN AND METHODS: We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies. RESULTS: In control platelets,CO selectively promotes phosphorylation at VASP Ser-157,whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239,with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets,neither agent resulted in VASP phosphorylation. In nondiabetic EPCs,NO and CO increased phosphorylation at Ser-239 and Ser-157,respectively,but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions,both CO and NO induced phosphorylation at Ser-157 and Ser-239; however,this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose,VASP was redistributed to filopodia-like structures following CO or NO exposure; however,redistribution was dramatically attenuated under high glucose conditions. CONCLUSIONS: Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation,and in diabetes,blunted responses to these agents may lead to reduced vascular repair and tissue perfusion. View Publication

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly,purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic,adipocytic,and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore,mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models. View Publication

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4,Sox2,Klf4,and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs,we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first,followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene,marking fully reprogrammed cells,was only observed late in the process. Importantly,the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming. View Publication

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium,and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity,related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast,they are markedly enriched for transcripts encoding Sca1,Sdf1,c-Met,Nestin,and Sox9,markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing pancreatospheres" in suspension culture� View Publication

In the field of hematopoietic regeneration,deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells,remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines,UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here,using a serum-free,feeder-free,and chemically defined induction protocol,we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34(+)CD45(+) derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol,the derived hematopoietic progenitors failed to engraft in NOG mice,indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols,UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro. 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

BACKGROUND: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood,ex vivo transduction of the gene of interest into them,and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor,time and money,while enhancing HSCs viability,transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes,in which reverse transcription of viral DNA is not completed. METHODS: In the present study,we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors,based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction,we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector,developed in our laboratory,that allows targeted transduction to specific cell receptors via antibody recognition. RESULTS: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. CONCLUSIONS: Overall,the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. View Publication

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

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However,transferred TCR chains can pair with endogenous TCR chains,resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity,because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice,TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study,we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner. View Publication

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. View Publication