Figure 3
Lactate purification of hiPSC-CMs prior to microtissue formation with 5% hCFs improves excitability and lengthens APD. (A) Representative Vm traces from microtissues formed from hiPSC-CMs with (red) and without lactate purification (black) plus 5% hCFs. HiPSC-CMs from the same differentiation batch but grown in 2D culture without the lactate selection protocol for 28 days (to match the age of the cells) were used as the most appropriate control for comparison. (B) Percent of hiPSC-CM microtissues showing APs during 0.5 Hz pacing increases with lactate purification of hiPSC-CMs (n = 4 batches, minimum 2 molds per batch). (C) APD80 distribution from 35 microtissues in a single mold with hiPSC-CMLP and 5% hCF. (D) The average APD80 was 259.0 ± 42.0 ms (n = 201). APD80 variation between batches is small with hiPSC-CMLP: APDs were measured at 0.5 Hz cycle length pacing. The standard deviation from beat-to-beat in the same microtissue (technical replicates) was 6.4 ± 6.5 ms (n > 35 microtissues per batch, n = 4 batches), from microtissue-to-microtissue in the same mold (biological replicates) was 36.1 ± 21.4 ms (n = 2–3 molds per batch, n = 4 batches), from mold-to-mold in the same batch (also biological variability) was 31.8 ± 3.8 ms (n = 2–3 molds per batch, n = 4 batches), and from batch-to-batch, the standard deviation (another level of biological variability) was 19.3 ms (n = 4 batches). (E) Cumulative probability plot for APD80 (n = 3–4 batches, at least 2 molds per batch) shows consistently longer APD80 with hiPSC-CMLP (red) vs. hiPSC-CM (black). (F) Comparison of APDs from microtissues from the same experimental batch (each color) with and without lactate purification (n = 4 batches, ≥ 2 molds per batch). Values are means ± SD. * P < 0.05). Note that in batch 4, the microtissues generated from hiPSC-CM without lactate purification were not excitable.
