Introduction: Cardiomyocytes derived from differentiated embryonic stem (ES) cells hold promise as a treatment for heart disease, but generation of sufficient quantities of differentiated cells remains a challenge. Most ES cell research is performed in incubators with a humidified 95% air/5% CO2 gas mixture, resulting in a gas-phase oxygen partial pressure (pO_2gas) of 142 mmHg. Embryonic cells in early development are exposed to pO_2cell values of 0-30 mmHg, and the effects of such conditions on differentiating ES cells are poorly understood. Here we show that control of the pO_2cell to levels experienced by the developing embryo enhances differentiation of ES cells into cardiomyocytes.

Methods: Embryoid bodies (EBs) were formed in hanging drops containing 500 ES cells in 20 µl of DMEM supplemented with 10% FBS and ascorbic acid. After 2 days, EBs were transferred to culture dishes fabricated with a highly oxygen permeable, fibronectin-coated silicone rubber membrane on the bottom, to which the cells attached and grew. Three days later, the medium was changed to a serum-free ITS medium with ascorbic acid and grown 5-6 more days with daily medium changes. pO_2gas was controlled by placing culture dishes in airtight containers purged with premixed gas containing 5% CO2 and either 142, 36, or 7 mmHg oxygen. By using silicone rubber membrane-based dishes for all conditions, precise control of pO_2cell at the cell-membrane interface was achieved. Cardiomyocytes were identified by flow cytometric analysis of dispersed cells immunostained with an antibody to sarcomeric myosin heavy chain (MF-20) and confirmed with MF-20 immunostaining of 5-µm tissue sections.

Results: After differentiation of ES cells at different, constant pO_2gas conditions for 11 days, the fraction of cells that were cardiomyocytes, assessed by flow cytometry, was 29%, 20%, and 9% at a pO_2gas of 7, 36 and 142 mmHg, respectively. The total number of cardiomyocytes was similar at 7 and 142 mmHg but significantly higher at 36 mmHg. The number fraction of MF-20 positive cells by immunostaining tissue sections was 45%, 20%, and 5% for samples taken from tissue cultured at 7, 36, and 142 mmHg, respectively. Cells cultured for 11 days at 7 mmHg preferentially formed thin cell sheets and smaller aggregates than were found at 36 or 142 mmHg.. Culture of differentiating cells at pO_2gas of 142 and 7 mmHg oxygen for different time periods in different orders for 10 days resulted in the fraction and number of cardiomyocytes always being greater when cells were initially at 7 compared to 142 mmHg oxygen; the fraction increased with time cultured at 7 mmHg oxygen up to 6 days, with little change thereafter. Differentiation for 6 days at 7 mmHg followed by 4 days at 142 mmHg gave the best results. In a single series of experiments, the maximum cardiomyocyte fraction, 33%, was 4.7 times larger, and the number of cardiomyocytes was 4 times larger, than for constant culture at 142 mmHg oxygen, and 45 cardiomyocytes were generated for each initial ES cell. Calculations with a model of oxygen consumption and diffusion in the aggregates suggest that enhanced differentiation occurs for pO_2cell in the range of 1 to several 10s of mmHg.

Conclusions: These results demonstrate that culture at low pO_2cell markedly increases differentiation of ES cells into cardiomyocytes. This finding may enhance prospects for their therapeutic use in heart disease.