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Bulk RNA-seq of mouse bone marrow derived macrophages co-cultured with platelets

ABSTRACT: Bulk RNA-seq of mouse bone marrow derived macrophages co-cultured with platelets

PROVIDER: PRJNA1090441 | ENA |

REPOSITORIES: ENA

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Project description:Background: Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with 20% fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. Methods: A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. Results: Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways; cell proliferation and cell cycle pathways; and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. Supernatant analysis found that HPGF-C18 BMSCs displayed higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. Conclusions: Traditional measures; expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

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Bulk RNA-seq of mouse bone marrow derived macrophages co-cultured with platelets

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