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Abstract Background Umbilical cord blood (UCB) is a valuable source of hematopoietic stem and progenitor cells (HSPC) for transplantation. However, limited cell numbers require ex vivo expansion for effective treatment of adult patients. Mesenchymal stromal cell (MSC)-based co-cultures offer a supportive environment for HSPC expansion and a model to study niche interactions. Human platelet lysate (hPL) provides a xeno-free alternative to fetal bovine serum (FBS) for MSC culture, but its effect on MSC hematopoietic support remains unclear. This study investigates transcriptomic and functional changes induced by hPL-based culture and their impact on HSPC regulation. Methods MSC from three bone marrow donors were expanded using hPL- or FBS-supplemented media under three regimens: continuously in cell isolation medium (Direct), adapted from the isolation medium to the other formulation (Adapted), or re-adapted back to the isolation medium (Re-adapted). Co-cultures with UCB-derived CD34 + HSPC assessed functional support over a 7-day period. Bulk transcriptomic profiling (RNA-seq) was performed on MSC under each condition. Differential gene expression and pathway enrichment (GO, KEGG) analysis characterized molecular differences and impacted signaling networks. Results MSC properties were reversibly affected by the culture medium. Comparison of hPL-MSC and FBS-MSC revealed 13% differentially expressed genes (DEG), predominantly involved in extracellular matrix organization, chemokine signaling, and cell-cell communication. Minimal transcriptomic variation (1–2% DEG) was observed between Direct and Adapted / Re-adapted MSC. Co-culture with hPL-MSC resulted in significantly lower CD34 + cell expansion (2.4-fold reduction vs. FBS-MSC), though both outperformed the no feeder layer control. While proliferation was reduced, hPL-MSC promoted greater enrichment of primitive subsets, with increased CD34 + CD45RA − and CD34 + CD45RA − CD90 + populations. Clonogenic potential remained comparable across all conditions. Network analysis identified dysregulation in TGF-β, PI3K-Akt, Notch, Wnt, and JAK/STAT pathways. hPL-MSC showed elevated expression of inhibitory and reduced expression of stimulatory hematopoietic regulatory factors. Conclusions MSC cultured in hPL- or FBS-supplemented media display significant and reversible transcriptomic differences impacting HSPC expansion. Medium adaptation rapidly reprograms MSC phenotype and gene expression, highlighting responsiveness to environmental cues. Differential expression of key hematopoietic regulatory genes supports the observed functional disparities. These results provide a mechanistic basis for MSC-mediated HSPC support and lay groundwork for optimizing xeno-free expansion systems for clinical application.
Carreira et al. (Sat,) studied this question.