Effects of low oxygen culture on pluripotent stem cell differentiation and teratoma formation

Abstract

Pluripotent stem cells (PSC) hold promise for the study of embryonic development and the treatment of many diseases. Most pluripotent cell research is performed in incubators with a gas-phase oxygen partial pressure (p02) of 142 mmHg. However, embryonic cells in early development are exposed to a local P02 of 0-30 mmHg, and the effects of such conditions on differentiating PSC are poorly understood. Residual PSC within differentiated populations are problematic because of their potential to form tumors in vivo. This is a major safety issue that must be overcome before PSC-based therapies can be used in the clinic. In this study, we differentiated mouse and human embryonic stem cells and mouse induced pluripotent stem cells at different defined P02 on highly oxygen-permeable silicone rubber culture dishes and assessed differentiation to the three germ layers, endoderm, ectoderm, and mesoderm and to cardiomyocytes and assessed residual PSC within differentiated populations. Low P02 drastically affects differentiation of PSC to the three germ layers and cardiomyocytes. Overall, differentiation was higher to endoderm, lower to ectoderm, and higher or the same to mesoderm. Differentiation to cardiomyocytes was greatly enhanced without the need for purification, possibly by lineage selection via increased Mesp1 and Mesp2 expression. Understanding the effects of P02 during differentiation is an important step towards the development of protocols for regenerative medicine. Control of P02 to physiological levels typical of the developing embryo reduced the fraction of PSC within, and the tumorigenic potential of, differentiated populations. Culture under differentiating conditions at low PO2 reduced measured pluripotency markers by up to four orders of magnitude. Upon implantation into immunocompromised mice, low PO2-differentiated PSC either did not form tumors or formed tumors at a slower rate than high PO2 PSC. Low PO2 differentiation could be combined with cell sorting for improved benefits. Low PO2 culture alone or in combination with other methods is a potentially straightforward method that could be applied to future cell therapy protocols to minimize the possibility of tumor formation.