I am reaching out for advice on some challenges we have faced with inducing pluripotent stem cells (iPSCs) to differentiate into mesenchymal stem cells (MSCs).

Plan 1: We choose P30 generation iPSC, plated with 1% Matrigel; after stable passage, the seeding density in the 6-well plate is 200,000-250,000 total cells. The first stage: mesoderm induction for 5 days; commercialization Medium. The second stage: αMEM medium + 15% FBS + 1% PS + 1% NEAA + 1% L-glutaMAX + 10ng/mL TGF-β1 and 20ng/mL bFGF, induction for 12-15 days. Experimental results: mesoderm marker, Brachyury: 90%; α-SMA: 98%. Final detection: CD90: 54.6%, CD73: 96.3%, CD105: 97.2%, the expression of other markers met the requirements, but the index of CD90 was low。 The MSC-like cells showed good adipogenic and osteogenic differentiation, but could not form spheres in chondrogenic medium.

Plan 2: The basic conditions are as in Plan 1, select the p38 generation cells in the bottle, culture at 37°C throughout, 8% CO2 incubator, 1% Matrigel plating; 16 days in the first stage: mTESR1 medium containing 10 μM TGF-β inhibitor; Section 2 Phase II, 30 days: knock out DMEM+10% knock out serum replacement+1% PS+1% NEAA+1% L-glutaMAX+1% β-mercaptoethanol+10 ng/mLbFGF+10uM SB431542. Experimental results: Phase I: CD90: 95.8%；CD73: 94.5%; CD105: 77%; Stage II: CD90: 80%; CD73: 62.7%; CD105: 78%. In this experiment, in the first stage, our three-dimensional differentiation experiment results found that the cells have better chondrogenic and adipogenic effects, but poor osteogenesis.

If anyone has experience overcoming issues with iPSC-MSC differentiation, I would greatly appreciate your insights and suggestions on improving CD90 expression and osteogenesis. Please let me know if I can provide any other details on our protocols and results so far.