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

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5,originally isolated from a NOD mouse,has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy,leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge,this work is the first to use a hybrid insulin peptide,or any neoepitope,to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients. View Publication

The role of autophagy,a lysosomal degradation pathway which prevents cellular damage,in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis,malignant transformation of HSCs leads to leukemia. Therefore,mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study,we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions,a severe myeloproliferation,and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species,as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded,Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions,the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively,these data show that Atg7 is an essential regulator of adult HSC maintenance. View Publication

Nonsense mutations are present in 10{\%} of patients with CF,produce a premature termination codon in CFTR mRNA causing early termination of translation,and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study,we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels,demonstrates absence of CFTR function,and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly,CFTR restoration is observed in G542X intestinal organoids treated with G418,an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations. View Publication

Hematopoietic cells (HCs) promote blood vessel formation by producing various proangiogenic cytokines and chemokines and matrix metalloproteinases. We injected mouse colon26 colon cancer cells or human PC3 prostate adenocarcinoma cells into mice and studied the localization of HCs during tumor development. HCs were distributed in the inner tumor mass in all of the tumor tissues examined; however,the localization of HCs in the tumor tissue differed depending on the tumor cell type. In the case of colon26 tumors,as the tumor grew,many mature HCs migrated into the tumor mass before fine capillary formation was observed. On the other hand,although very few HCs migrated into PC3 tumor tissue,c-Kit+ hematopoietic stem/progenitor cells accumulated around the edge of the tumor. Bone marrow suppression induced by injection of anti-c-Kit neutralizing antibody suppressed tumor angiogenesis by different mechanisms according to the tumor cell type: bone marrow suppression inhibited the initiation of sprouting angiogenesis in colon26 tumors,while it suppressed an increase in the caliber of newly developed blood vessels at the tumor edge in PC3 tumors. Our findings suggest that HCs are involved in tumor angiogenesis and regulate the angiogenic switch during tumorigenesis. View Publication

Hematopoietic stem cells are resistant to HIV-1 infection. Here,we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21(Waf1/Cip1/Sdi1) (p21),a known regulator of stem cell pool size,restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs,p27(Kip1) and p18(INK4C),had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase,SIVmac-251,and the other cell-intrinsic inhibitors of HIV-1,Trim5alpha,PML,Murr1,and IFN-alpha,were unaffected by p21. Therefore,p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected sanctuary" in HIV disease." View Publication

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However,there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein,we provide a detailed protocol for the production,enrichment,and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol,researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines,researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks,and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis,as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease,$\beta$-thalassemia,and primary immunodeficiencies. View Publication

Mesenteric lymph node (mLN) CD103 (alphaE integrin)(+) dendritic cells (DCs) induce regulatory T cells and gut tolerance. However,the function of intestinal CD103(-) DCs remains to be clarified. CD47 is the ligand of signal regulatory protein alpha (SIRPalpha) and promotes SIRPalpha(+) myeloid cell migration. We first show that mucosal CD103(-) DCs selectively express SIRPalpha and that their frequency was augmented in the lamina propria and mLNs of mice that developed Th17-biased colitis in response to trinitrobenzene sulfonic acid. In contrast,the percentage of SIRPalpha(+)CD103(-) DCs and Th17 responses were decreased in CD47-deficient (CD47 knockout [KO]) mice,which remained protected from colitis. We next demonstrate that transferring wild-type (WT),but not CD47 KO,SIRPalpha(+)CD103(-) DCs in CD47 KO mice elicited severe Th17-associated wasting disease. CD47 expression was required on the SIRPalpha(+)CD103(-) DCs for efficient trafficking to mLNs in vivo,whereas it was dispensable on both DCs and T cells for Th17 polarization in vitro. Finally,administration of a CD47-Fc molecule resulted in reduced SIRPalpha(+)CD103(-) DC-mediated Th17 responses and the protection of WT mice from colitis. We thus propose SIRPalpha(+)CD103(-) DCs as a pathogenic DC subset that drives Th17-biased responses and colitis,and the CD47-SIRPalpha axis as a potential therapeutic target for inflammatory bowel disease. View Publication

Although adipose tissue is an expandable and readily attainable source of proliferating,multipotent stem cells,its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly,both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF,TGFbeta,fibronectin,and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication