Figure 4 - ''Harnessing developmental cues for cardiomyocyte production''
Figure 4: Overview of Wnt modulation during hiPSC-CM differentiation and expansion. (A) Schematic overview of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. (B-C) Brightfield images of hiPSC-CMs over multiple passages including expansion. Scale bars indicate 200 µM.(C) Brightfield images of hiPSC-CMs in expansion from days 15 through 20. Scale bars indicate 200 µM. (D) Immunofluorescence images for α-Actinin (green), proliferation marker Ki67 (red) and nuclei (blue) as indicated. Scale bars indicate 100 µM. (E) Quantification of Ki67+ cells indicates that increased proliferation (37%) of hiPSC-CMs can be promoted by administering 2 μM CHIR99021. (F) Quantification of hiPSC-CM number from P1 to P5 by sequentially expanding the hiPSC-CMs using CHIR99021. Abbreviation: P, passage; D, differentiation day; RPMI; Roswell Park Memorial Institute medium, CHIR; CHIR99021. Figure adapted from (Maas et al., 2021).
Materials and Methods:
(B-C) To obtain hiPSC-derived CMs, hiPSCs were grown to ~90% confluence in a 6-well format. hiPSC cells were maintained in E8 complete medium for at least three passages before starting cardiac lineage differentiations. The cells were efficiently differentiated as previously described, using the RPMI/B27 minus insulin medium (Gibco, Waltham, MA, USA) supplemented with 7–8 µM CHIR99021 (cell line specific) (SelleckChem, Houston, TX, USA) for the first 2 days and 2 µM Wnt-C59 (SelleckChem, Houston, TX, USA) for another 48 h. The proliferation of hiPSC-CMs can be conducted by the removal of cell–cell contacts and small-molecule inhibition with CHIR99021 (Buikema et al. 2020). Subsequential multiple low-density passaging and reintroduction of 2–3 µM CHIR99021 (cell line specific) to the RPMI/B27 media results in an expansion of hiPSC-CMs (Maas et al. 2021).
(D) hiPSC-CMs after two days of expansion were seeded into a µ-Plate 96 well (Ibidi) with a density of ~5 × 104 cells/well for continuously culturing with RPMI/B27 and RPMI/B27 supplemented with CHIR99021 (3 µM) for six days. Media was refreshed every other day. At day 6, the cells were washed and fixed with 4% formaldehyde for 10 min at room temperature, permeabilized with 0.25% PBS-Triton for 5 min and then blocked with 10% donkey serum for 30 min. The cells were then incubated overnight at 4 °C with primary antibodies for alpha-actinin (Merck, A7811), dilution 1:250, and Ki67 (D3B5, dilution 1:400, Cell Signaling Technology, Danvers, MA, USA). Next, the cells were incubated with the corresponding secondary Alexa fluor-555 and −647 antibodies (1:400 dilution, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Nuclear DNA was labeled with Hoechst. Images were acquired by an confocal microscope system (Leica).
