A three-stage differentiation method for generating induced mesenchymal stem cells from human pluripotent stem cells via gelatin-based screening

Human pluripotent stem cell (hPSC)-derived induced mesenchymal stem cells (iMSCs) offer advantages over tissue-derived MSCs in their in unlimited expansion, minimal batch-to-batch variation, controllable pathogen status, and large-scale production potential. Yet traditional iMSC differentiation methods face limitations that necessitate a more efficient approach. Based on the classic TGF-β inhibitor (SB431542) induction system, this study established a three-stage iMSC differentiation method: 10-day Laminin-521/Gelatin induction, 11-day screening on 0.1 % gelatin-coated dishes (enriching precursors via differential cell-gelatin adhesion), and 3-5-day amplification. A xeno-free system was established. Flow cytometry, trilineage differentiation, RNA Sequencing, karyotype analysis, and nude mouse tumorigenicity tests validated iMSC quality. This straightforward, cost-effective method (≈￥2000-3000 for 1 ×10⁸ P0 iMSCs) yields high-purity iMSCs with strong proliferation, tissue-derived MSC-like transcriptomes, and enhanced bone repair capacity in a mouse femoral fracture model. It also further implicates the gelatin DGEA motif (which mimics gelatin's effect) as key for iMSC differentiation, addressing classical iMSC industrialization issues and informing clinical translation.