Ky02111

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.

Undifferentiated hPSCs were seeded onto Matrigel-coated dishes at a density of 4 × 10⁴ cells/cm² and allowed to expand for 48 h (∼80% confluency). At this stage (d1 of differentiation), cultures were treated with medium comprising StemPro34 supplemented with (1:100 dilution) Matrigel and (1 ng/mL) BMP4 (R&D systems) and after 24 h (d2 of differentiation), medium comprising StemPro34 with (10 ng/mL) BMP4 and (8 ng/mL) Activin A (Life Technologies). Medium exchange was performed on d4 of differentiation using RPMI supplemented with 1xB27 (Life Technologies) and small molecule inhibitors, KY02111 (10 μM) and XAV939 (10 μM) (R&D systems). From d8 onward, cells were maintained in RPMI medium supplemented with B27 only, with medium changes every 3 days. Cardiac differentiation efficiency was accessed by using immunocytochemistry with primary mouse anti-human α-actinin antibody (No. A7811, 1:800; Sigma) dilution and secondary goat anti-rabbit Alexa633 (No. A21052, 1:400; Invitrogen), counterstaining with 0.5 μg/mL DAPI (No. D9542, 1:500; Sigma). Immunofluorescence images were captured using Operetta High-Content System (Perkin Elmer) and analyzed using Harmony High-Content Analysis Software.

In preparation for differentiation cells were seeded at 4–8 × 10⁵ cells/cm² and after 6 days of expansion, differentiation was induced (day 0) by washing the cells with PBS (Sigma-Aldrich, St. Louis, MO) and adding xeno-free differentiation medium (XF; Table 1) containing BMP4 and WNT3A (R&D Systems, Minneapolis, MN) at stated concentrations. The next day, this medium was replaced with XF medium without BMP4 and WNT3A. From day 2 to 8, cells were incubated in XF with 10 μM KY02111 (R&D Systems, Minneapolis, MN) replenished daily. From day 9 on cells were cultured in XF, which was replaced daily.
Differentiation in spinner flasks was carried out after 5 days of hPSC expansion as aggregates. Subsequently aggregates were transferred to centrifuge tubes and spun down at 100 × g for 5 min. The medium was aspirated, aggregates were washed with PBS, and XF was added containing 30 ng/ml BMP4 and 200 ng/ml WNT3A (alternatively, 6 μM CHIR99021 was added) before returning the aggregate suspension to the spinner flasks. Differentiation in the spinner flasks proceeded using the same media and timing as in dish cultures.