Drug resistance selection of hESC-derived cardiomyocytes.

(A) Schematic diagram of the drug selection regimen for isolation of cardiomyocytes (see Methods). Puromycin selection is typically done for a 1.5–2 day window between day 12 and 120 days (latest attempted) after EB formation. (B,C) Phase contrast micrographs of hESC colonies at day −2 (b) and again at day −1, after 24 hours exposure to G418 (C). (D,E) Beating cardiomyocyte clusters at day 12 (D) and again following Puromycin treatment at day 13.5 (E). Dark color is due to opacity of the tissue. Supplemental Movie S4 shows brightfield and fluorescence images of CSs at day 16. (F) Expression of αMHC and βMHC mRNA during differentiation in EBs (day 0–4) and in Puror CSs (day 18–90), by quantitative RT-PCR (error bars represent standard deviation) showing that αMHC transcripts are evident as cardiomyocytes emerge and persist for at least 90 days, consistent with efficacy of Puror selection when performed from day 12 and up to 150 days, the latest attempted (see text). β-MHC mRNA, in contrast, was first detected at day 90. (G,H) Histological sections of a day 18 EB (G) and day 20 CS (H) showing immunostaining of Troponin-I (red) and DAPI (blue). Cardiomyocytes were enriched in CSs, comprising 96.0%±8.6, as quantified from multiple sections of 10 independent biological replicates. (I) 3D confocal reconstruction of a 16 µm section through a CS isolated from a day 100 EB by the Neor, Puror selection protocol from a three-vector hESC line (αMHC-Puror_Rex-1-Neor, αMHC-mCherry_Rex-Blar, and PGK-H2BeGFP) showing cytoplasmic mCherry (magenta) and nuclear H2BeGFP (green). Numerous nuclei are not apparent since they are inside the reconstructed tissue.