Stemmacs ips brew xf culture medium

Xeno-free differentiation of hPSCs toward cardiomyocytes was carried out as we described recently [18 (link)]. Briefly, 5 × 10⁶ cells were seeded as single cells in spinner flasks (Corning Inc., Corning, NY, USA) with 50 mL StemMACS iPS-Brew XF culture medium (Miltenyi Biotech, Auburn, CA, USA). After 5 days of hPSC expansion as aggregates, the medium was replaced with xeno-free differentiation medium (DM) supplemented with 200 ng/mL WNT3A and 30 ng/mL BMP4. Medium was changed after 24 h. Forty-eight hours later, DM with 10 µM KY02111 (R&D Systems, Minneapolis, MN, USA) Wnt inhibitor was added and replaced daily for 6 days. Cells were then cultured in DM until day 13 with daily medium exchange.

To establish iPSC lines, BM-MNCs or MSCs from three GS-2 patients and one healthy donor were transduced for 2 consecutive days with bicystronic (pSIN4-CMV-K2M, Addgene, 21164; pSIN4-EF1α-O2S, Addgene, 21162) or polycystronic lentiviral vectors (pRRL-PPT-SF-OSKM-IRES-idTomPRE), kindly provided by Prof. Dr. Axel Schambach, Hannover Medical School, Germany [17 (link)], carrying the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc. After transduction, MSCs were cultured in DMF10 and MNCs in StemMACS HSC expansion medium (Miltenyi, 130-100-463) supplemented with StemMACS HSC cytokine cocktail (STF, Stem Cell Factor/Thrombopoietin/Flt3-ligand, Miltenyi, 130-100-843) for 7 days. Transduced cells were plated on Matrigel-coated dishes (Corning, 354277), and the culture medium was replaced by StemMACS iPS-Brew XF culture medium (iPS-Brew, Miltenyi, 130-107-086) with 20 ng/mL basic fibroblast growth factor (bFGF, Immunotools, 11343627). Colonies were picked around day 18 after reprogramming, disrupted by microdissection, and re-plated in iPS-Brew/bFGF supplemented with 10 μM Rock inhibitor Y27632 (Miltenyi, 130-103-922).