Transforming growth factor beta 1

RPMI 1640 medium, F-12 medium, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from Gibco-BRL (Life Technologies, Grand Island, NY, USA). AICAR, bovine serum albumin (BSA), phosphate-buffered saline (PBS), RIPA buffer, protease inhibitor cocktail, phosphatase inhibitor cocktail, stripping buffer, thioglycollate medium, and 3-(4,5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). AICAR was purchased from Cayman Chemical (Ann Arbor, MI, USA). Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). BCA protein assay reagent was purchased from Thermo Scientific. For western blotting, rabbit antibodies against human phospho-AMPK, AMPK, MYC, mTOR, PARP, phospho-p70S6K, p70S6K, TSC-1, TSC-2, β-actin and secondary antibodies were purchased from Cell Signaling (Farmingdale, NY, USA). Mouse antibodies against human N-cadherin and E-cadherin were purchased from BD Biosciences (San Jose, CA, USA). Caspase-Glo 3/7 assay kit was purchased from Promega (Madison, WI, USA). Transforming growth factor-beta 1 was purchased from PeproTech (Rocky Hill, NJ, USA). Docetaxel (Taxothere, 20 mg/mL) was obtained from Sanofi Aventis (Berlin, Germany).

The number of monoclonal colonies (defined as a cluster of more than 32 cells which represents a population of cells derived from more than five population doublings of a single cell) in each well was counted under light microscopy after 5 days of culture, which was considered as the initial number of progenitors that had adhered to the plate (Williams et al., 2010 (link)). Each type of progenitor population in each well was then trypsinised by 0.05% trypsin/EDTA (Gibco-Thermo Fisher Scientific, United States) and reseeded in a T25 tissue culture flask (Sarstedt, Germany) in culture medium supplemented with 1 ng/ml transforming growth factor beta 1 (TGF-β1) (PeproTech, United Kingdom), 5 ng/ml fibroblast growth factor 2 (FGF-2) (PeproTech, United Kingdom). Population doubling times (PDTs) were calculated using the formula: PDT = (t₂-t₁) x ln (2)/ln (n₂/n₁), where t₁ = the time of cell seeding, t₂ = the time of cell harvest and n = the cell population at the matching time points. PDTs at passage 0 to 2 were compared between mixed populations and progenitor cells, as progenitors could not be counted with sufficient accuracy at P0-1.

The undifferentiated hiPSC colonies were manually dissected into smaller pieces and cultured for 10 days in nonadherent petri dishes containing hiPSC medium without bFGF for EB formation. Approximately 25 % of the initial media was replaced with an equal amount of the differentiation media (DMEM, 20 % FBS, 1 × non-essential amino acids, 0.1 mM β-mercaptoethanol, 1 mM L-glutamine) every 2 days. EBs were then seeded onto 10 cm gelatin-coated dishes. Within 10 days of cellular confluence, the cells were incubated with 0.25 % trypsin/EDTA at 37 °C for 5 minutes, and reseeded on new gelatin-coated dishes. At 90–100 % confluence of cells, which is often observed in 5 to 7 days, the cells were harvested with 0.25 % trypsin/EDTA. The cells were sorted by CD73 and CD105 double positivity. Then they (3 × 10⁵) were placed in a 15-ml polypropylene tube, centrifuged at 1200 rpm for 3 minutes at room temperature, and resuspended in chondrogenic differentiation medium (DMEM-HG supplemented with 10 % ITS (Invitrogen), 10⁻⁷ M dexamethasone, 1 mM ascorbate-2-phosphate (Invitrogen), 1 % sodium pyruvate (Invitrogen), and 10 ng/ml transforming growth factor-beta 1 (Peprotech)). The cells were recentrifuged and maintained in small pellet form for 21 days. The culture medium was replaced every 3 days. The hMSCs were also collected and cultured in this chondrogenic differentiation medium for 21 days.