Human second-trimester amniotic fluid as a source of induced mesenchymal stem cells for regenerative applications

Human fetal mesenchymal stem/stromal cells (hfMSCs) display several advantageous properties compared with their adult counterparts and human amniotic fluid stem cells (AFSCs) have been shown to improve bone extracellular matrix quality and structure. However, the clinical translation of primary hfMSCs remains limited by donor-to-donor variability, limited expansion capacity and replicative senescence, which are associated with progressive loss of tissue-repair potential. To address these limitations, we reprogrammed AFSCs to pluripotency and subsequently directed AFSC-derived hiPSCs towards the mesodermal lineage to generate induced mesenchymal stem/stromal-like cells (AF-iMSCs). AF-iMSCs retained key MSC features, including expression of canonical mesenchymal markers and trilineage differentiation potential. Comparative gene-expression analysis showed that AF-iMSCs expressed higher levels of genes associated with mesenchymal stromal identity, cell signalling, adhesion, migration, extracellular matrix remodelling and osteogenic differentiation, whereas AFSCs expressed higher levels of genes linked to proliferative and growth-factor signalling. In addition, AF-iMSCs secreted small extracellular vesicles (iEVs) that promoted fibroblast migration and supported type I collagen and elastin production in vitro, indicating functional pro-reparative activity. Together, these findings suggest that iPSC-mediated derivation of AF-iMSCs provides a scalable and reproducible strategy to overcome key limitations of primary AFSCs and support the development of cell-based and extracellular vesicle-based regenerative therapies.