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

BACKGROUND Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10{\%} DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry,as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel,each sample was flow sorted into fibroblast,T-cell,B-cell,and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS Upon dissociation,cryopreserved synovial tissue fragments yielded a high frequency of viable cells,comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with {\~{}} 30 arthroplasty and {\~{}} 20 biopsy samples yielded a consensus digestion protocol using 100 mu$g/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes,distinct populations of memory B cells and antibody-secreting cells,and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes,fibroblasts,and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell,including transcripts encoding characteristic lineage markers identified. CONCLUSIONS We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. View Publication

OBJECTIVE: Endothelial-like vascular progenitor cells (VPCs) can be collected in peripheral blood stem cell (PBSC) products that are used in hematopoietic stem cell transplantation (HSCT). The association between VPCs in PBSC products and transplant-related toxicity caused by high-dose chemo/radiotherapy was assessed to identify potential mediators of vascular repair. MATERIALS AND METHODS: PBSC grafts in 29 patients (mean age: 48 years; range,20-67 years) undergoing autologous HSCT were analyzed using a cell culture assay for VPC cluster formation in fibronectin-coated dishes in serum-rich angiogenic conditions. Transplant toxicity was estimated using total length of hospital stay (LOS) following HSCT and the Seattle criteria for transplant-related organ toxicity for 8 organ systems (grade 0-4). RESULTS: LOS following graft reinfusion was lower (14.7 vs 20.0 days,p = 0.002) and the mean number of organs with any toxicity (1.0 vs 2.4,p = 0.016) or with toxicity grade textgreater or = 2 was reduced (0.2 vs 1.6 organs,p = 0.007) in patients with high graft VPC content (n = 10,textgreater2.0 x 10(3) VPCs/kg) compared with reduced VPC content (n = 19,textless or = 2.0 x 10(3) VPCs/kg). An association between graft CD34(+) levels and LOS or organ toxicity was not observed. In addition,graft VPC levels were independent of graft CD34 counts,peripheral blood monocytes and hemoglobin levels,age,and disease (p = NS). CONCLUSION: PBSC products enriched for VPCs are associated with reduced toxicity following HSCT. Identifying specific factors that contribute to high graft VPC levels is needed. View Publication

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver,very few HSCs efflux Hoechst 33342 efficiently,and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death,but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis,or of its underlying transcriptome network. Therefore,the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes,whereas MeHg altered fewer transcripts. To attenuate batch effects,analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (backslashtextless20 % overlap). Moreover,within one test system,little overlap between the PS changed by the two compounds has been observed. However,using TFBS enrichment,a relatively large ‘common response' to VPA and MeHg could be distinguished from ‘compound-specific' responses. In conclusion,the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles. View Publication

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study,we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively,while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs,multiple uniquely shared amino acid configurations were identified,warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes,which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation. View Publication

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A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side population" (SP) of bone marrow. This SP from bone marrow View Publication

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens,or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here,to yield functional human haematopoietic stem cells,we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG,HOXA5,HOXA9,HOXA10,LCOR,RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid,B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. View Publication