Journal: Regenerative Therapy
Article Title: Generation of canine induced pluripotent stem cell-derived mesenchymal stem cells: Comparison of differentiation strategies and cell origins
doi: 10.1016/j.reth.2025.05.008
Figure Lengend Snippet: Schema to obtain induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs). (A) A method for iMSC generation via lateral plate mesoderm (LPM). To induce mesodermal lineage, canine iPSCs (ciPSCs) were cultured on Matrigel with 20 % knockout serum replacement (KSR) medium containing transforming growth factor β (TGFβ) signal inhibitor, SB431542 (SB). After passages, the cells were cultured on tissue-culture dishes with fetal bovine serum (FBS)-MSC medium containing basic fibroblast growth factor (bFGF). We described this iMSC induction protocol as the LPM protocol. (B) A method for iMSC generation via neural crest cells (NCCs). To induce NCCs, ciPSCs were cultured with laminine-511 (LN511) and StemFit without solution C (StemFit without C) containing glycogen synthetase kinase 3β (GSK3β) inhibitor, CHIR99031 (CHIR), SB, and bFGF. After neural specification, the cells were maintained in NCC maintenance medium, which was StemFit without C containing SB, bFGF, and epidermal growth factor (EGF) on fibronectin (FN). When NCCs differentiated into iMSCs, they were cultured with StemFit for mesenchymal stem cells and LN511. We described this iMSC induction protocol as NCC + StemFit. (C) iMSC induction method via NCCs using other iMSC culture conditions. NCCs generated as described in (B) were induced to iMSCs by culturing with PRIME-XV MSC expansion XSFM on FN. We described this iMSC induction protocol as NCC + PRIME.
Article Snippet: After 2–5 days, the medium was changed to KSR + SB medium, which consisted of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Nacalai Tesque, Kyoto, Japan) with 20 % knockout serum replacement (KSR; Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine (Nacalai Tesque), 100 IU/mL penicillin, 100 μg/mL streptomycin (Nacalai Tesque), 0.1 mM non-essential amino acid (Nacalai Tesque), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), and 10 μM transforming growth factor β (TGFβ) inhibitor (SB431542; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan).
Techniques: Derivative Assay, Cell Culture, Knock-Out, Generated
