Bone morphogenetic protein 2

Sca-1⁺CD31⁻ and Sca-1⁺CD31⁺ cells were initially seeded at 1 × 10⁴ cells/cm² in normal medium containing DMEM/F12 supplemented with 2% FBS, 2% B27 (Gibco), 20 ng/mL endothelial growth factor (Peprotech), 40 ng/mL fibroblast growth factor-basic (Peprotech), and 20 ng/mL leukemia inhibitory factor (Peprotech) on gelatin-coated dishes. For cardiomyocyte induction, cells were cultured with DMEM/F12 supplemented with 10% FBS, 100 ng/mL bone morphogenetic protein-2 (Peprotech), and 100 ng/mL fibroblast growth factor-4 (Peprotech) on gelatin-coated dishes for 14 days. For smooth muscle cell induction, cells were cultured in DMEM/F12 supplemented with 10% FBS with 50 ng/mL platelet-derived growth factor-BB (Peprotech) for 14 days. For endothelial cell induction, cells were cultured in DMEM/F12 supplemented with 10% FBS plus 20 ng/mL vascular endothelial growth factor-165 (VEGF₁₆₅, Peprotech) and grown on gelatin-coated dishes for 14 days. In control groups, cells were cultured in the same medium for differentiation without growth factors. Control and treatment media were replenished for two days to ensure cytokine and growth factor activity. When induction was complete, lineage-specific markers were analyzed by IF staining.

Chondrocytes were obtained from distal femoral and proximal tibial growth plates of CON or CKO mice at P1. Epiphyseal cartilage tissues were dissected under a stereo light microscope and digested with 0.25% trypsin for 10 min at 37 °C, followed by digestion with 0.1% collagenase type II (Life Technologies, USA) for 12 h at 37 °C. Chondrocytes were seeded in a 6-well plate and cultured at 37 °C with 5% CO₂. DMEM/F12 with l-glutamine (Gibco, USA) supplemented with 1% penicillin–streptomycin solution (Gibco, USA) and 10% fetal bovine serum (PAN, Germany) was used as the culture medium. Chondrocytes were used within three passages for the hypoxia studies. Once the chondrocytes had grown to approximately 75% confluence, cells were exposed to normoxic (21% O₂, 5% CO₂, and 74% N₂) or hypoxic (1% O₂, 5% CO₂, and 94% N₂) conditions. ITS culture medium was prepared by adding 1% ITS Liquid Media Supplement (100×) (I3146, Sigma, America) and 50 ng/ml bone morphogenetic protein-2 (120-02C, PeproTech, Amarica) into the culture medium.

The MPCs underwent multi-lineage differentiation analysis to determine their osteo/chondro/adipo-genic capacity.
Osteogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (final concentration (FC): 100. nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), β-Glycerolphosphate (FC: 10. mM) (Sigma).
Adipogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (FC: 1 μM) (Sigma), Insulin (FC: 10 μM) (Sigma), Indomethacin (FC: 200 μM) (Sigma), and Isobutylmethylxanthine (FC: 500 μM) (Sigma).
Chondrogenesis: For each replicate, 5 × 10⁵ cells were pelleted through centrifugation and placed into DMEM/F-12 media that contained Dexamethasone (FC: 10 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), MEM Non-Essential Amino Acids (FC: 1%) (MEM-NEAA Gibco), Transforming growth factor (TGF)-β3 (FC: 10 ng/mL) (Peprotech), Bone morphogenetic protein (BMP)-2 (FC: 500 ng/mL) (Peprotech), Insulin transferrin selenium (FC: 1%) (Lonza- BioWhittaker), and sodium pyruvate (FC: 1%) (ThermoFisher). Media was adjusted to neutral pH (7.0–7.6).
After 21 days, differentiation was assayed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).

The MPCs underwent multi-lineage differentiation analysis to determine their osteo/chondro/adipo-genic capacity^{30 (link),34 (link)}.
Osteogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (final concentration (FC): 100 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), β-Glycerolphosphate (FC: 10 mM) (Sigma).
Adipogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (FC: 1 μM) (Sigma), Insulin (FC: 10 μM) (Sigma), Indomethacin (FC: 200 μM) (Sigma), and Isobutylmethylxanthine (FC: 500 μM) (Sigma).
Chondrogenesis: For each replicate, 5 × 10⁵ cells were pelleted through centrifugation and placed into DMEM/F-12 media that contained Dexamethasone (FC: 10 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), MEM Non-Essential Amino Acids (FC: 1%) (MEM-NEAA Gibco), Transforming growth factor (TGF)-β3 (FC: 10 ng/mL) (Peprotech), Bone morphogenetic protein (BMP)-2 (FC: 500 ng/mL) (Peprotech), Insulin transferrin selenium (FC: 1%) (Lonza- BioWhittaker), and sodium pyruvate (FC: 1%) (ThermoFisher). Media was adjusted to neutral pH (7.0–7.6).
After 21 days, differentiation was assayed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and histological staining.

The murine multipotential stem cell line, C3H10T1/2 clone 8, was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Monolayer cultures were maintained in DMEM containing 10% fetal calf serum (FCS) (growth medium). To induce chondrogenesis, we used a miromass culture system, ITS  +  premix (insulin, transferrin, selenous acid, BSA, and linoleic acid; Corning, Corning, NY, USA) and bone morphogenetic protein (BMP)-2 (Peprotech, Rocky Hill, NJ, USA) using 10 µL drops of cells at 1 × 10⁷ cells/mL as described previously [56 (link)]. Micromass cultures were maintained in the growth medium with or without 100 ng/mL recombinant murine BMP-2 and P15-1 (17 µg/mL) for up to 6 days. The medium was changed every other day. Total RNA was isolated after 3 days of micromass cultures and analyzed for the mRNA levels of chondrocyte markers, Sox-9 and type II collagen, and the stem cell marker, type I collagen. In addition, Alcian blue staining (1% Alcian blue (Sigma-Aldrich, St. Louis, MO, USA) in 3% glacial acetic acid solution) of the micromasses was performed to determine the degree of chondrogenic differentiation.