Figure 1. Suspension culture of progenitor cardiac spheres and simulated microgravity increase cell viability and CM yield.

(A) Experimental design. hPSCs were induced to differentiate into CMs at day 0 using growth factors or small molecules. At day 4 or 5, cells were dissociated and forced to aggregate into progenitor cardiac spheres. The progenitor cardiac spheres were cultured under simulated microgravity at days 5–8 or 6–9 and maintained under standard gravity until day 20 (designated as 3D-MG). Parallel 2D and 3D cultures were maintained under standard gravity throughout, designated as 2D-SG and 3D-SG, respectively. At day 20, cells were analyzed for cell viability, CM purity, yield and density. (B) Representative flow cytometry analysis. Cell viability was analyzed by EMA staining and EMA negative cells were identified as live cells. Purity of CMs was analyzed by intracellular staining of α-actinin, a CM-associated marker. (C) Summary of IMR90-iPSC differentiated cell viability, CM purity, cell yield and density. (D) Summary of H7 hESC differentiated cell viability, CM purity, cell yield and density. (E) Summary of H9 hESC differentiated cell viability, CM purity, CM yield and density. The CM yield was defined as the number of viable CMs generated from one undifferentiated stem cell. Data are presented as mean ± SD of 3–6 biological samples for each culture condition. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.