Mesencult acf medium

Control, ODDD patient, and Cx43-/- human iPSC-derived MSCs were cultured on gelatin-treated dishes with glass cover slips in MesenCult-ACF medium (StemCell, Technologies, Vancouver, Canada). Once cells reached confluency, media was replaced with StemPro Adipogenesis Differentiation Kit (ThermoFisher #A1007001) per the manufacturer’s instructions. Media was changed every 2–3 days during the differentiation period of up to 28 days. At select intervals, cells were processed for immunocytochemistry and Western blotting.

CRL-2097 human normal skin fibroblasts (HFs) were purchased from the American Type Culture Collection (ATCC: Manassas, VA, USA). Human UCB-MSCs were purchased from the Japanese Collection of Research Bioresources cell bank (JCRB1109, Osaka, Japan) and ATCC (PCS-500-010, Manassas, VA, USA). The HFs were cultured in α-MEM (GIBCO, Carlsbad, CA, USA) fibroblast medium containing 10% fetal bovine serum, GIBCO), 1% minimum essential medium non-essential amino acid (GIBCO), 1% penicillin/streptomycin (GIBCO), and 1% sodium pyruvate (GIBCO). UCB-MSCs were cultured in MesenCult™-ACF Medium (STEMCELL technologies, Cambridge, MA, USA). The hiPSCs reprogrammed from HFs by using Sendai-virus vectors encoding Oct4, Sox2, Klf4, and c-Myc [33 (link)], or episomal vectors (Episomal iPSC reprogramming vectors, Thermo Fisher Scientific, Waltham, MA, USA) [34 (link)], were cultured with mTeSR1 (STEMCELL Technologies) medium.

Human AMSCs from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China) were soaked in MesenCult™-ACF medium (STEMCELL Technologies, CA) supplemented with 2 mM L-glutamine (Thermo Fisher Scientific, Wilmington, DE, USA) and 1% antibiotic antibacterial agent (Thermo Fisher Scientific). The phenotypes of the 3rd to 6th generation of AMSCs were evaluated by flow cytometry analysis (BD Accuri®C6 flow cytometry) after incubation with antibodies against CD29, CD31, CD44, CD45, CD73, CD90, CD105, and HLA-DR (Biolegend, CA, USA). IgG1 served as a negative control. Adipogenic differentiation medium (Invitrogen, Carlsbad, CA, USA), osteogenic differentiation medium (Invitrogen), and chondrogenic differentiation medium (Invitrogen) were used to induce the differentiation of AMSCs. After 14 or 21 days of induction, the cells were stained with Oil Red O dye (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), Alizarin Red S (Sigma-Aldrich), and Alcian Blue (Sigma-Aldrich).