Transcriptomic Insights into the effect of Mesenchymal Stromal Cells Culture Medium on the Hematopoietic Stem/Progenitor Cells Co-culture
To investigate the molecular mechanisms underlying the altered hematopoietic support of mesenchymal stromal cells (MSC) expanded in human platelet lysate (hPL)-supplemented medium, we performed a comprehensive transcriptomic analysis comparing hPL-MSC and fetal bovine serum (FBS)-expanded MSC. MSC from three bone marrow donors were cultured under different adaptation regimens and co-cultured with hematopoietic stem and progenitor cells (HSPC). Transcriptomic analysis revealed distinct gene expression profiles, with 13% of genes differentially expressed between hPL-MSC and FBS-MSC, impacting cell signaling, extracellular matrix, and chemotaxis. Key pathways, including TGF-beta, PI3K-Akt, and Wnt signaling, were altered, affecting MSC-mediated HSPC expansion. hPL-MSC exhibited increased expression of inhibitory factors and reduced expression of promoters of HSPC proliferation, influencing Notch, TGF-ß, JAK/STAT, and Wnt regulation. These findings highlight critical molecular interactions in MSC-HSPC co-culture and provide insights for optimizing xeno-free MSC expansion for hematopoietic applications. Overall design: This study analyzed mesenchymal stromal cells (MSC) from three bone marrow donors (50, 86, 99) expanded in fetal bovine serum (FBS)- or human platelet lysate (hPL)-supplemented medium under different adaptation regimens over four passages. "F" denotes one passage in FBS, and "H" one passage in hPL. The conditions included Direct (continuous expansion in the same medium: F, FF, FFF, FFFF or H, HH, HHH, HHHH), Adapted (medium switch: HF, HHF, HFF, HHFF, or FH, FFH, FHH, FFHH), and Re-adapted (returned to the original medium: FHHF or HFFH). Bulk RNA sequencing was performed after RNA extraction using the High Pure RNA Isolation Kit (Roche). Library preparation and paired-end 150 bp sequencing were conducted by Novogene on an Illumina NovaSeq 6000 platform.