Mesenchymal progenitor kit

Human iMSCs were generated using the Stemcell Technologies Mesenchymal Progenitor Kit following the manufacturers’ instructions with small modifications. Following three passages using the recommended Mesencult ACF plus medium, cells were grown in DMEM high glucose (Gibco) supplemented with 10% fetal calf serum (FCS; Biowest), basic FGF (bFGF; 5 ng/ml; Life Technologies), and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Gibco) until elongated and with fibroblast-like morphology. At passage 5, MSC surface markers were analyzed by flow cytometry, and the trilineage potential of the hiMSCs was determined. Generation of hiCPCs was performed as described previously (Adkar et al. 2019 (link)). At day 14, analysis for cell surface markers was performed, and hiCPC aggregates were collected for chondrogenesis (Supplementary Fig. S1).

Mesenchymal stromal cells were generated from hiPSC (hiMSCs) of the OPG-XL hiPSCs (LUMC0103iOPG) and of the two thereof derived CRISPR/Cas9 isogenic control hiPSCs (B89 and C81) using Stemcell Technologies’ Mesenchymal Progenitor Kit following the manufacturer’s instructions. Chondrogenesis and osteogenesis was performed in organoids following our established protocol employing 750 000 cells per organoid as described before [25 (link)].