Mesencult acf chondrogenic differentiation medium

To confirm the multipotentiality of MSCs used in our research, experiments were performed in accordance with the minimal criteria for defining multipotent MSCs proposed by the International Society for Cellular Therapy (ISCT) (17 (link)).
The cultured plastic-adherent cells expressing the markers CD73 (sic passim; eBioScience, San Diego, CA, USA), CD90 and CD105 but not expressing the markers CD14, CD19, CD34, CD45 and HLA-DR were able to differentiate into adipocytes, osteoblasts and chondrocyte induced by products of Stem Cell Technologies (Vancouver, BC, Canada), which are respectively MesenCult^™ Adipogenic Differentiation Medium (human) and MesenCult^™ Osteogenic Stimulatory kit (human) and MesenCult^™−ACF Chondrogenic Differentiation Medium. Manufacturer's manuals were referred.
To determine whether the expanded MSC cultures maintained multipotency differentiation characteristics, we tested both HD-MSCs and AD-MSC for differentiation into adipogenic, osteogenic and chondrogenic cell lines. MSCs cultured in adipogenic differentiation medium showing lipid droplets were stained by Oil Red O staining. Osteogenic differentiation was demonstrated by calcium deposition, which was stained by Alizarin Red S. Histological sections of chondrogenic pellet were stained with Alcian Blue and Nuclear Fast Red. Undifferentiated AD-MSCs and HD-MSCs were used as controls.

For chondrogenesis, 1 × 10⁶ CSPCs at passage 2 were centrifuged in 15 mL polypropylene tubes to obtain cell pellets and cultured in MesenCult™-ACF chondrogenic differentiation medium (Catalogue #05455, Stem Cell) for 4 weeks. After differentiation, pellets were fixed and sectioned. Chondrogenesis was analysed by staining with toluidine blue (G3668, Solarbio). For osteogenesis, CSPCs at passage 2 were harvested and cultured in 24-well plates (20,000 cells/well). After 90% confluence, the osteogenic differentiation medium was replaced [low glucose DMEM (10567-014, Gibco) with 10% FBS, 10 mM β-glycerophosphate (50020, Sigma), 50 mM L-ascorbate-2-monophosphate (A7631, Sigma), and 1 μM dexamethasone (D1756, Sigma)] for 3 weeks. The osteogenic cells were fixed and stained with alizarin red (G1452, Solarbio). To induce adipogenesis, CSPCs at passage 2 were harvested and cultured in 24-well plates (20,000 cells/well). After 90% confluence, the osteogenic differentiation medium high glucose DMEM (11965-092, Gibco) was replaced with 10% FBS, 100 μM indomethacin (I7378, Sigma), 0.5 mM IBMX (I5879, Sigma), 10 μg/mL insulin (91077C, Sigma), and 1 μM dexamethasone (D1756, Sigma) for 2 weeks. Oil Red O (G1260, Solarbio) staining was performed to assess the adipogenicity potency.

Adipocytes, osteoblasts, and chondrocytes were generated from MSCs. For adipocyte differentiation, MSCs between passages 3 – 4 were plated on 6-well plates with MesenCult-ACF Basal Medium (StemCell Technologies, Cat. # 05449) at 4 – 5 3 10⁵ cells per well. Differentiation was performed using the MesenCult Adipogenic Differentiation Kit (StemCell Technologies, Cat. # 05412) according to the manufacturer’s protocol. Media changes were done every 3 – 4 d until 13 d. For osteogenic lineage differentiation, MSCs between passages 3 – 4 were plated on a 6-well plate with MesenCult-ACF Basal Medium (StemCell Technologies) at 3 – 4 × 10⁴ cells per well. Differentiation was performed using MesenCult Osteogenic Differentiation medium (StemCell Technologies, Cat. # 05465) according to the manufacturer’s protocol. Medium changes were done every 3 – 4 d until 13 d. For 3-D pellet chondrogenic differentiation, MSCs were first released from T25 flasks using ACF Enzymatic Dissociation/Inhibition Solution (StemCell Technologies, Cat. # 05426) and collected in polypropylene tubes at 2.5 – 3 × 10⁶ cells per tube with MesenCult-ACF Chondrogenic Differentiation Medium (StemCell Technologies, Cat. # 05455) according to the manufacturer’s protocol. Medium changes were done every 3 – 4 days until 13 d.

Bone marrow stem/stromal cells (BMSCs) were isolated from the hindlimbs of 6‐week‐old C57BL6J mice. A single‐cell suspension of BMSCs was cultured in DMEM with 10% FBS and 1% P/S, and only cells that adhered after 3 hours were maintained with periodic changes of fresh medium. Cultured BMSCs were then characterized by flow cytometry and evaluated for multipotency using adipocyte, osteocyte, and chondrocyte assays (Fig. S5). To generate cartilage pellets, BMSCs from WT and SIRT3 KO mice were transferred to MesenCult™‐ACF Chondrogenic Differentiation Medium (STEMCELL Technologies, #05455) with or without 1% BSA or 0.5 mM FA. Pellets were collected after 3–28 days of differentiation. Pellet images were captured with a stereomicroscope (Nikon SMZ800), and paraffin‐embedded sections were stained with safranin‐O (Fig. S6). Sections were also immunostained with antibodies for COL2 (abcam, ab185430), PRG4 (Millipore, AB2200), SIRT3 (Cell Signaling Technology, 5490), and perilipin (Cell Signaling Technology, 3470). Staining intensity and distribution throughout the pellet were quantified by Zeiss Zen software (Fig. S7). For each experiment, BMSCs were collected from separate animals to generate three biologically independent pellets (n = 3) tested in each treatment group per time point.