Background Ex vivo expansion of hematopoietic stem cells (HSCs) remains an unmet need in hematopoietic stem cell transplantation (HSCT), especially in settings with limited donor cell availability. Recent research has focused on mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) for optimal HSC expansion. MSCs can be sourced from various tissues, including bone marrow (BM) and placenta. Our study aims to investigate and compare the potential of EVs derived from BM and preterm and full-term placental MSCs to enhance ex vivo expansion of HSCs. Also we aimed to investigate the RNA cargo of these MSC-derived EVs and evaluate their transcriptomic impact on expanded BM CD34⁺ HSCs.

Methods EVs were isolated from MSCs derived from human preterm placenta, full-term placenta, and BM using size-exclusion chromatography. Human BM CD34⁺ HSCs were co-cultured with each EV type under cytokine-supplemented conditions for 10 days. Proliferation was assessed by total nucleated cell (TNC) count, stemness was evaluated by flow cytometry, and clonogenic potential was determined through colony-forming unit (CFU) assays.

Small RNA sequencing was performed on EVs to profile the abundance and differential expression of microRNAs (miRNAs). Bulk RNA sequencing of HSCs after co-culture was conducted to assess transcriptional changes induced by EV exposure. A network analysis was performed to explore interactions between differentially expressed mRNAs in HSCs and miRNAs from EVs. Raw read counts for miRNAs were normalized to reads per million mapped reads (RPM) across all samples, and log₂ fold changes between groups were calculated. miRNAs with significant differential expression were selected, and their predicted target genes were identified using miRNet 2.0, referencing experimentally validated interactions from miRTarBase v9.0. The resulting interaction networks were visualized and refined using Cytoscape.

Results Co-culture of human BM CD34⁺ HSCs with MSC-derived EVs resulted in a significant increase in TNC count in all EV-treated groups compared with the control. The largest effect was observed with BM MSC–derived EVs, which produced nearly a ninefold increase in TNCs, followed by full-term placental MSC-derived EVs with sixfold increase. Flow cytometric analysis demonstrated that the proportion of CD34⁺ cells was maintained across all treatment groups and remained comparable to the control. CFU assays revealed that EVs from full-term placental MSCs supported superior multilineage differentiation potential compared with other groups.

Small RNA sequencing of EVs identified six significantly upregulated miRNAs common to all MSC-derived EVs—hsa-let-7f-5p, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7b-5p, hsa-miR-21-5p, and hsa-miR-23a-3p. Network analysis integrating these miRNAs with differentially expressed mRNAs from expanded HSCs revealed convergence on genes that are associated to proliferation such as KREMEN1, IGF2BP1, PEG10, MYCL, EGFR, ERBB2, PDGFRA, and MTUS1. Genes related to differentiation and development such as SOX6, TGFB2, SMAD3, and EGR3 were also identified.

To explore differences between full-term placental and BM MSC–derived EVs, we identified differentially expressed miRNAs in the former compared with the latter. Hsa-miR-1-3p was identified and network analysis linked it to targets including ANK1, TUBB2B, CTTN, and DNAAF5, genes associated with cytoskeletal remodeling, membrane specialization, and differentiation-related processes in various cell types.

Conclusions MSC-derived EVs from BM and placental sources significantly enhance ex vivo expansion of human BM CD34⁺ HSCs while preserving stemness, with BM MSC–derived EVs producing the greatest overall proliferation and full-term placental MSC–derived EVs promoting superior multilineage differentiation. Integrated miRNA–mRNA network analysis revealed that common EV miRNAs target key regulator genes that control proliferation and differentiation. These findings suggest that MSC-derived EVs exert coordinated effects on HSC proliferation via distinct miRNA cargo, offering a promising strategy for improving HSC expansion.

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2025
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