Stemdiff mesenchymal progenitor kit

MSCs were induced from hiPSCs using the STEMdiff Mesenchymal Progenitor kit (Stemcell Technologies) according to the manufacturer’s protocol. In brief, hiPSCs were first induced into early mesoderm progenitor cells with STEMdiff Mesenchymal Induction Medium. Four days later the medium was changed to MesenCult-ACF medium. The cells were then passaged into 6-well plates coated with MesenCult-ACF Attachment Substrate. At day 24, the induced cells were analyzed for cell surface markers, proliferative potential, and differentiation potential. In this study, iMSCs were used between passages 4 and 8.

We used STEMdiff™ Mesenchymal Progenitor Kit (STEMCELL, USA) to differentiate iPSCs and IL-24-iPSCs into iMSCs and IL-24-iMSCs, respectively, according to the manufacturer’s protocol. Briefly, after iPSCs were cultured with mTeSR1 medium to a confluence of 30%, they were cultured with Mesenchymal Induction Medium for 4 days, and the medium was changed daily, and then cultured with MesenCult™-ACF Medium for 3 days. When the cell confluence reached 90%, they were passaged into a 6-well plate pre-coated with the MesenCult™-ACF attachment substrate, and the ACF medium was changed every day. After 4 days of cultured, cells with 90% confluency were passaged into a gelatin-coated 10-cm dish and continue to culture with MSC medium.

MSCs were differentiated from a healthy control relative and ODDD patient iPSCs (harboring a Cx43 p.V216L mutant) that were originally derived from dermal fibroblasts [10 (link)], or iPSCs where Cx43 was ablated (referred to here as Cx43-/- iPSCs), using the STEMdiff mesenchymal progenitor kit (StemCell Technologies #05240, Vancouver, BC) according to the manufacturer’s instructions. MSCs were cultured on gelatin-coated dishes in MesenCult-ACF basal media (StemCell Technologies #05445) in a 37 °C humidified incubator under 5% CO₂. MSCs were passaged using the ACF-free cell dissociation kit (StemCell Technologies #05426). Cells at passages 3–5 were considered early passage, while cells at passages 9–12 were defined as late passage.

For DNA extraction, we used three cell lines and lentiviral vectors. hiPSCs (HPS0002: 253G1), mesenchymal stem cells (hMSC), and undifferentiated type of hepatoma cells (HLF) were provided by the RIKEN BioResource Center Cell Bank [21 (link)] and the Cell Resource Center for Biomedical Research, Institute of Development, Takara Bio (Kusatsu, Shiga, Japan), and Aging and Cancer Tohoku University, respectively. HLF, MSC, and iPSC (253G1) were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin, in Mesenchymal Stem Cell Growth Medium 2 (Takara Bio, Kusatsu, Shiga, Japan), and in ReproStem medium (ReproCell, Tokyo, Japan) with 10 ng/ml of bFGF-2, respectively. Additionally, for the transfection to HLF cells or hMSCs, the human mesangial cell line 293FT (Invitrogen Japan K.K., Tokyo, Japan) was used for producing 520d-5p expressing-lentiviral particles. 293FT cells were cultured in DMEM supplemented with 10% FBS, 0.1 mM MEM nonessential amino acid solution, 2 mM L-glutamine and 1% penicillin/streptomycin. Also, we induced hMSCs from iPSCs using STEMdiff Mesenchymal Progenitor kit (STEMCELL technologies, Seattle, WA, USA) and defined them as 520d/MSC after they were transfected by miR-520d-5p.