(560c) Effects of Oxygen on Murine Embryonic Stem Cell Energetics and Growth

Introduction: Prior to vascularization in vivo, embryonic stem (ES) cells and many of their differentiated progeny exist in an environment with an oxygen partial pressure (pO₂) that is often far less than 40 mmHg. Most in vitro culture of these cells is done in a 5% CO₂ / 95% air incubator that produces a gas phase pO₂ of 142 mmHg. As an initial step to test whether culture pO₂ significantly affects ES cell growth and differentiation, we studied the proliferation and cellular energetics of undifferentiated CCE and D3 murine ES cells maintained in different known pO₂ conditions. Methods: ES cells were grown attached to gelatin coated culture dishes using DMEM containing 1000 units/ml leukemia inhibitory factor. Gas phase pO₂ was controlled by placing culture dishes in small airtight containers purged with premixed gas containing 5% CO₂ and either 0, 1, 5, or 40% oxygen, resulting in environments with pO₂ values of 0, 7, 36, or 286 mmHg, respectively. Culture in a standard incubator was used as a control. We measured cell growth rate, oxygen consumption rate (OCR) immediately following cell detachment and reoxygenation, lactate production rate (LPR), cell specific lactate dehydrogenase (LDH) activity, and ATP content. We estimated the cell surface pO₂ by equating oxygen diffusion across the culture medium to the measured OCR. ATP production rate (APR) by aerobic and anaerobic metabolism, respectively, was estimated by assuming 6 mol of ATP were produced per mol of O₂ consumed and 1 mol of ATP was produced per mol of lactate formed. Cells were verified to be undifferentiated by flow cytometric analysis of Oct-4 and SSEA-1 expression. Results: ES cell specific growth rates were about 0.6 day^-1 at a pO₂ of 0 mmHg, increased to about 1.15 day^-1 in the range of 40-142 mmHg, and decreased to 0.9 day^-1 at 286 mmHg. OCR decreased from about 2.9 nmol/10⁸ cell sec at 142 mm Hg to 2.4, 1.7, and 1.0 nmol/10⁸ cell sec at 36, 7, and 0 mm Hg, respectively. Over the same pO₂ range there were concomitant increases in LPR and LDH activity (about 2-fold). The estimated APR was constant down to 7 mmHg, followed by a 20% decrease under complete anoxia. There was no significant decrease in intracellular ATP content or cell viability over the entire range of pO₂ values studied if sufficient medium was used to prevent excessive waste accumulation. Anaerobic metabolism accounted for 40% of the total ATP produced at 286 and 142 mm Hg, and 50, 70, and 100% at 36, 7, and 0 mmHg, respectively. Conclusions: ES cells utilize anaerobic metabolism for a significant fraction of their total energy production, even after having been grown at high pO₂ conditions (142 mmHg) for extended periods of time. With decreasing pO₂, ES cells further down-regulate aerobic and up-regulate anaerobic metabolic pathways well before oxygen becomes limited. Our results imply that precise oxygen control is not critical in bioprocess development for the culture of undifferentiated stem cells. Maintaining a pO₂ of 40 mm Hg or above is probably preferable to minimize lactate production, but the cells are not sensitive to either higher or lower pO₂ conditions. As long as glucose and other nutrients and growth factors are present, and excessive amounts of wastes do not accumulate, ES cells are tolerant of any pO₂ less than about 300 mm Hg. Further work needs to be done to determine whether human ES cells behave in a similar manner.