Transforming growth factor beta 1 tgf β1

To determine the multi-lineage differentiation potential of cells from each tissue type, hDIAS cells from each donor were grown in established adipogenic, osteogenic, and chondrogenic culture conditions [10 (link)]. For adipogenic and osteogenic differentiation, 1.5x10⁴ cells from each donor were plated in each well of a 24-well plate. Adipogenic differentiation medium consisted of DMEM with high glucose/GlutaMAX™-I, 5% FBS, 1% P/S/F, 1% NEAA, 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine (Sigma-Aldrich), and 0.2 mM indomethacin (Sigma-Aldrich). Osteogenic differentiation medium consisted of DMEM with high glucose/GlutaMAX™-I, 10% FBS, 1% P/S/F, 1% NEAA, 100 nM dexamethasone, 10 mM β-glycerophosphate (Sigma-Aldrich), 250 mM ascorbate-2-phosphate, and 50 ng/mL bone morphogenetic protein-2 (BMP-2) (Peprotech). For chondrogenic differentiation, 2.5x10⁵ cells from each donor were added to a round bottom polystyrene 96-well plate (Costar 3799, Corning, NY) 0.2 mL of CHG, supplemented with 50 ng/mL BMP-2, and 10 ng/mL transforming growth factor beta-1 (TGF-β1) (Peprotech), and centrifuged at 500 xg for 5 minutes to form cell pellets [30 (link)]. All groups were then maintained at 37°C and 5% CO₂ for 4 weeks, with media exchanged every other day.

Two percent sodium alginate was prepared by dissolving sodium alginate (Sigma-Aldrich) in phosphate-buffered saline (PBS) and sterilizing by heating to 80°C in a water bath for 15 min (Leo et al., 1990 (link)). Anti-CD150-PE, anti-GRPC5C Alexa Fluor-405, and anti-Ki67-Alexa Fluor 405 antibodies for flow cytometry were purchased from R&D Systems. Anti-CD49f-PE and anti-CD90-VioBlue were purchased from Miltenyi Biotec. Transforming growth factor beta-1 (TGFβ-1) and fibroblast growth factor were obtained from PeproTech. Fms-like tyrosine kinase-3 (Flt-3), G-CSF, stem cell factor (SCF), and interferon alpha were purchased from ImmunoTools. All-trans retinoic acid (ATRA) was obtained from Sigma-Aldrich. Hoechst 33342, propidium iodide (PI), and pyronin Y (PY) were purchased from Sigma-Aldrich. The CountBright^TM Absolute Counting Beads was purchased from Thermo Fisher Scientific. MHY1485, AZD8055, rosiglitazone, KU-55933, LEE011, roscovitine (seliciclib, CYC202), glasdegib (PF-04449913), sodium butyrate, dasatinib, BIO, plerixafor (AMD3100), quizartinib, and sorafenib were purchased from Selleckchem. Adiponectin, triglitazone LE135, PD169316, harmine, nilotinib, ethylisopropyl amiloride, anisomycin, and curcumin were purchased from Sigma-Aldrich, while prostaglandin E2 was from BioVision/Cambridge Bioscience. Cytarabine and daunorubicin were purchased from Sigma-Aldrich.

The cyiPSCs were chondrogenically differentiated to produce cartilage using a previously described method for human iPSCs⁹ with one minor modification. In brief, after chondrogenic differentiation, the cells were transferred into suspension culture to induce ECM secretion and form cartilaginous particles 1–3 mm in diameter. Cells and particles were cultured in chondrogenic medium [Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis) with 1% ITS-X (Thermo Fisher Scientific, Waltham), 1% fetal bovine serum (FBS) (Thermo Fisher Scientific), 1 × 10⁻⁴ M nonessential amino acids (Thermo Fisher Scientific), 1 mM Na pyruvate (Thermo Fisher Scientific), 50 U penicillin, 50 mg/mL streptomycin (1% PC/SM, Thermo Fisher Scientific), 0.25 μg/mL amphotericin B (Thermo Fisher Scientific), 50 μg/mL ascorbic acid (Nacalai), 1 μM rosuvastatin (BioVision, Milpitas), 10 ng/mL BMP2 (Peprotech), 10 ng/mL transforming growth factor beta 1 (TGFβ1) (Peprotech), and 10 ng/mL GDF5 (BioVision)]. For human iPSCs, the chondrogenic medium is typically replaced with the conventional medium (DMEM supplemented with 10% FBS and 1% PC/SM) at 6 weeks after the chondrogenic differentiation.⁹ In contrast, cyiPSCs were kept in chondrogenic medium after the chondrogenic differentiation until the analysis.