Acf enzyme inhibition solution

Induced mesenchymal progenitor cells (iMPCs) were generated from iPSCs using a spontaneous differentiation protocol as described before [26 (link),32 (link)]. When iPSCs reached 70~80% confluency, the iPSCs’ maintenance medium was switched to STEMdiffTM-ACF Mesenchymal Induction Medium (Stemcell Technologies, Vancouver, BC, Canada) for three days, and the medium was changed every day afterwards. The medium was subsequently replaced by MesenCultTM-ACF Plus Medium (Stemcell Technologies) for four days of differentiation. The cells were detached and plated onto pre-coated plates within the MesenCultTM-ACF Plus Medium. When the cultures reached 70~80% confluency, ACF Enzymatic Dissociation Solution and ACF Enzyme Inhibition Solution (Stemcell Technologies) were used for detachment; the differentiated cells were then grown in tissue culture flasks (Corning, NY, USA) for future experiments. The iMPCs were cultured in the fibroblast differentiation medium for four weeks as described above for MSCs before use.

iMPCs were generated from iPSCs using a protocol established in our lab (Diederichs and Tuan, 2014 (link)). When iPSCs reached 80% confluency, expansion medium was replaced with STEMdiff™-ACF Mesenchymal Induction Medium (Stemcell Technologies) for 3 days. Afterwards, MesenCult™-ACF Plus Medium (Stemcell Technologies) was used. On day 6, differentiated cells were detached and re-plated onto flasks that were pre-coated with Animal Component-Free Cell Attachment Substrate (Stemcell Technologies). The MesenCult™-ACF Plus Medium was used to expand these cells to 80% confluency. After being dissociated with ACF Enzymatic Dissociation Solution and ACF Enzyme Inhibition Solution (Stemcell Technologies), the iMPCs were grown on regular tissue culture flasks in expansion medium [DMEM/F-12 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), and 1% antibiotics-antimycotics (Gibco)]. iMPCs at passage 4 were used for all experiments.