Rejuvenated human amniotic fluid stem cells: a superior source of standardized induced mesenchymal stem cells for enhanced therapeutic applications

Human fetal mesenchymal stem cells (hfMSCs) present advantageous characteristics compared to their adult counterparts and have emerged as potent cells in the field of regenerative medicine. In the context of skeletal regeneration, human amniotic fluid stem cells (AFSCs) have been shown to improve the quality and structure of the bone extracellular matrix in an experimental model of severe osteogenesis imperfecta. However, primary hfMSCs undergo replicative senescence during in vitro expansion, along with a progressive decrease in plasticity and tissue repair potential. To overcome this challenge, we rejuvenated AFSC to pluripotency using non-integrative episomal reprogramming and subsequently re-derived the cells towards the mesoderm to obtain induced MSCs (iMSCs). We found that iMSCs have a slower proliferation rate compared to their parental cell line (40h±2h vs. 29h±5h) but retain the multipotency and differentiation potential characteristic of MSCs. Comparative genomic analysis revealed that iMSCs express higher levels of genes involved in maintaining stemness, cell signaling, adhesion and migration, as well as promoting osteoblast differentiation, whilst AFSC expressed higher levels of genes involved in cell proliferation. In addition, iMSCs secrete small extracellular vesicles (iEVs) that have the potential to stimulate fibroblast migration, a key process in tissue repair and wound healing. Together, these data suggest that resetting the epigenetic clock of primary hfMSCs may represent a promising strategy to address the limitations associated with primary cell use and enhance their therapeutic potential.