Oil red o adiopogenesis kit

Differentiation of WT and ATF6-deficient hMSCs from hESCs was carried out as previously described^{27 (link), 29 (link), 32 (link), 34 (link)}. Briefly, embryoid bodies were plated on Matrigel in differentiation medium (αMEM (Invitrogen) medium supplemented with 10% FBS (Gemcell), 5 ng/ml TGFβ (Human Zyme), 10 ng/ml bFGF (JPC), and 1% penicillin/streptomycin (Gibco)). About 10 days later, differentiated cells were passaged once. To further purify hMSCs, the differentiated cells were then subjected to fluorescence activated cell sorting (FACS) by evaluating the hMSC-specific surface markers (CD73, CD90, and CD105). The antibodies of hMSC-specific markers used in FACS were as follows: anti-CD90-FITC (BD Bioscience, 555595), anti-CD73-PE (BD Bioscience, 550257), and anti-CD105-APC (BD Bioscience, 17-1057-42). Anti-IgG-FITC (BD Biosciences, 555748), anti-IgG-PE (BD Biosciences, 555749), and anti-IgG-APC (BD Biosciences, 555751) antibodies were also used as isotype controls. The differentiation potentials of hMSC towards osteoblasts, chondrocytes and adipocytes were verified by histochemical staining with von Kossa (osteogenesis), Alcian blue (chondrogenesis), and Oil red O (adiopogenesis) Kit (IHC World), respectively.

hMSCs were differentiated from hESCs based on a published protocol^{31 (link)}. Briefly, embryoid bodies were left to differentiate in αMEM (Invitrogen) medium supplemented with 10% FBS (AusGeneX), 10 ng/ml bFGF (JPC), 5 ng/ml TGFβ (HumanZyme) and 1% penicillin/streptomycin (Gibco) until fibroblast-like cells appeared. The hMSCs were purified with different antibodies corresponding to hMSC-specific markers (CD73, CD90, and CD105) by FACS. Antibodies used for hMSC characterization were as follows: anti-CD105-APC (17-1057-42) antibody was purchased from eBioscience; anti-CD90-FITC (555595), anti-CD73-PE (550257), anti-CD34-PE (555822), anti-CD43-APC (580198), and anti-CD45-FITC (555482) antibodies were purchased from BD Biosciences. Anti-IgG-FITC (555748), anti-IgG-PE (555749), and anti-IgG-APC (555751) antibodies from BD Biosciences were used as isotype controls. The functionality of hMSC was further verified by differentiation towards cartilage, bone, and adipocytes^{31 (link)}. The tri-lineage differentiation abilities of hMSC lines were evaluated by histochemical staining with von Kossa (osteogenesis), Alcian blue (chondrogenesis), and Oil red O (adiopogenesis) Kit (IHC World), respectively.