Oxygen Availability in Embryonic Development and Stem Cell Maintenance in Three-Dimensional Cell Culture

Abstract

Conventional cell culture techniques fail to capture the three-dimensional context of embryonic development. This physical space, as well as providing structural support, consists of a cytokine-rich environment with significantly lower oxygen tensions compared to the atmospheric levels used routinely. Together, chemical and physical cues establish the signalling network governing the hallmarks of stem cells, such as pluripotency and self-renewal. Oxygen levels are also known to affect cell-fate commitment since pluripotent stem cells in early embryogenesis respond to the oxygen-depleted environment by expressing hypoxia inducible factors, such as HIF-1α. In this project, I examined the dual role of oxygen, firstly exploring the link between stemness and the hypoxic stem cell niche, and secondly investigating how oxygen affects differentiation, focusing on HIF-1α activation in a model of neurite development.
Hypoxic culture induced slower proliferation without enhancing pluripotency marker expression in stem cell line TERA2.cl.SP12. Strikingly, HIF-1α activation in an in-house neural differentiation model resulted in a marked increase in neurite outgrowth. When this effect was coupled with inhibition of Rho-associated protein kinase (ROCK), known to promote neuritogenesis, I observed a synergistic effect that enhanced neurite outgrowth further. Collectively, these findings underline the role of oxygen as an active component shaping stem cell activity. Additionally, enabling the crosstalk between the HIF and ROCK pathways by incorporating this key environmental factor to the in vitro study of neurogenesis could be exploited as a novel approach to induce neural regeneration.