Effects of oxygen on embryonic stem proliferation, energetics, and differentiation into cardiomyocytes

Abstract

Most embryonic stem (ES) cell research has been performed using a gas-phase oxygen partial pressure (pO2gas) of 142 mmHg, whereas embryonic cells in early development are exposed to cellular pO2 (pO2cell) values of about 0-30 mmHg. Murine ES (mES) cells were used as a model system to study the effects of oxygen on ES cell proliferation, phenotype maintenance, cellular energetics, and differentiation into cardiomyocytes. It was found that undifferentiated mES cells are capable of surviving and proliferating at pO2 conditions in the range of 0-285 mmHg, with only moderately decreased growth at the extremes in pO2 over this range. Oxygen levels had no effect on the maintenance of the undifferentiated phenotype during culture with the differentiation-suppressing cytokine leukemia inhibitory factor (LIF) in the culture medium, and low oxygen had, at most, a small differentiating-promoting effect during culture without LIF. Aerobic metabolism was used to generate approximately 60% of the energy required by undifferentiated mES cells at high pO2, but substantially smaller fractions when cells were oxygen starved. This shift from aerobic to anaerobic respiration occurred within 48 hr with minimal cell death.

(cont.) Oxygen was found to substantially affect the differentiation of mES cells into cardiomyocytes. Reduced pO2cell conditions strongly promoted cardiomyocyte development during the first 6 days of differentiation, after which oxygen primarily influenced cell proliferation. Using silicone rubber membrane-based dishes to improve oxygenation and an optimized cardiomyocyte differentiation protocol, it was possible to reproducibly obtain 60 cardiomyocytes per input ES cells and a cell population that was 30% cardiomyocytes following 11 days of differentiation. These results, obtained using a pO2gas of 7 mmHg during the first 6 days of differentiation, represent a 3-fold increase relative to those obtained with a pO2gas of 142 mmHg throughout differentiation. This work has shown that undifferentiated ES cells are able to adapt to their environmental pO2 and are relatively insensitive to its variations, whereas during differentiation oxygen affects cell fate decisions. Oxygen control can be used to improve directed ES cell differentiation into cardiomyocytes and oxygen may play a more important role in early embryonic development than heretofore appreciated.