Impact of differentiation protocols on the functionality of mesenchymal stem cells derived from induced pluripotent stem cells

BACKGROUND The discovery of induced pluripotent stem cells revolutionized regenerative medicine, providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). AIM To evaluate and compare five iMSC differentiation protocols, assessing their efficiency, phenotypic characteristics, and functional properties relative to primary mesenchymal stem cells (MSCs). METHODS Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1, iMSC3), an iMatrix-free method (iMSC2), growth factor supplementation (iMSC4), and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria, requiring expression of surface markers (CD105, CD73, CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic), proliferation, mitochondrial function, reactive oxygen species, senescence, and migration. RESULTS All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001, respectively), while adipogenic potential was reduced in iMSC2 (Adipsin ; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01, respectively). Proliferation was comparable or superior to bone marrow MSCs, except in iMSC1, with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001), reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001), and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours). CONCLUSION While all protocols generated functional iMSCs, variations in differentiation, proliferation, and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications.