Table 1.
Propagation and differentiation of HES3 to cardiomyocytes using different culture methods
| Culture conditions | MNL hESC cultures | hESC/MC aggregate spinner cultures | |||
|---|---|---|---|---|---|
| Propagation (7 days) | |||||
| Expansion fold (propagation) | 10.3 ± 0.4 | 15.4 ± 0.4 | |||
| % Pluripotent cells | |||||
| Tra-1-60-positive | 93.1 ± 1.4 | 96.2 ± 1.7 | |||
| mAb84-positive | 99.4 ± 0.5 | 98.6 ± 1.7 | |||
| Aggregate sizes (μm) | NA | 316 ± 11 | |||
| Differentiation to cardiomyocytes (20 days) | |||||
| MNL | MC-Rp | MC-SS | MC-AS | MC-Sp | |
| Expansion fold (differentiation) | 2.0 ± 0.2 | 2.2 ± 0.1 | 2.1 ± 0.2 | 2.0 ± 0.2 | 2.3 ± 0.1 |
| % beating aggregates | Beating sheet | 65.7 ± 1.8 (beating foci on plate) | 66.5 ± 2.6 | 53.4 ± 1.5 | 73.6 ± 2.8 |
| Beating aggregate size (μm) | NA | NA | 1063 ± 53 | 581 ± 54 | 655 ± 13 |
| % Cardiomyocytes | |||||
| MHC-positive | 45.7 ± 4.6 | 43.1 ± 1.2 | 42.6 ± 1.9 | 42.8 ± 1.6 | 47.7 ± 1.9 |
| cTnT-positive | 51.1 ± 0.5 | 53.1 ± 0.9 | 45.7 ± 2.9 | 42.4 ± 0.2 | 56.1 ± 1.4 |
| CM/hESC | 3.8 ± 0.2 | 1.2 ± 0.2 | 1.0 ± 0.2 | 0.8 ± 0.1 | 9.6 ± 0.3a |
| CM/ml (×x106) | 0.5 ± 0.02 | 0.2 ± 0.02 | 0.1 ± 0.02 | 0.1 ± 0.01 | 1.9 ± 0.05b |
| Total expansion fold (propagation + differentiation) | 20.0 ± 0.8 | 32.8 ± 1.1 | 31.8 ± 2.1 | 29.3 ± 2.5 | 34.3 ± 0.9c |
HES3 cells were propagated as either monolayer cultures (MNL) or human embryonic stem cell (hESC)/microcarrier (MC) aggregates for 7 days. Five differentiation culture regimes were tested. Data presented are mean ± standard deviation (n = 3 to 4). In HES3 cultures: asignificant difference between MC-Sp and MNL (P < 0.05), and between MC-Sp and the other three regimes (P < 0.01), in terms of cardiomyocytes produced per initial hESC seeded (CM/hESC); bsignificant differences (P < 0.01) between MC-Sp and the other four regimes in terms of cardiomyocyte density (CM/ml); csignificant difference between MC-Sp and MNL (P < 0.05) in terms of total expansion fold achieved. CM, number of cardiomyocytes (cTnT-positive cells) in the final cell population; Total expansion fold, cell expansion from seeding the hESC culture in the propagation phase, to final CM harvesting (day 20) in the differentiation phase. cTnT, Cardiac troponin T; MC-AS, hESC/MC aggregates in agitated suspension cultures; MC-Rp, monolayer replated cultures; MC-Sp, hESC/MC aggregates in spinner cultures; MC-SS, hESC/MC aggregates in static suspension cultures; MHC, myosin heavy chain; NA, not available.