Influence of Reduced Oxygen Tension on the Differentiation of Mouse Embryonic Stem Cells into Definitive Endoderm and Distal Lung Epithelial Cells
Authors:   , Pimton Pimchanok , Lelkes Peter I. 
Published: July 2021

Chronic lung diseases are the second leading cause of death in the world. It kills 4 million people every year, and causes approximately 7% of all deaths worldwide. It afflicts people of all ages in every country and every socioeconomic group. Current pharmacological approaches to treating lung diseases, such as pulmonary hypoplasia and emphysema, are largely ineffective, while organ transplants are hampered by limited donor availability. Cell-based therapies and tissue engineering might offer an alternative approach for pulmonary alveolar repair and regeneration. If embryonic stem (ES) cell-derived somatic cells are ever to be used for treating human diseases, including lung diseases, it will likely require the derivation of large quantities of cells with high purity under defined conditions. In this thesis work, we aimed to optimize an endodermal cell source for pulmonary tissue engineering and regenerative medicine. The study focused on directing the differentiation of mouse ES (mES) cells into definitive endoderm, as this first step is a prerequisite for efficient differentiation of more committed progenitor cells, and eventually of endoderm-derived lung epithelia. During normal embryonic development, differentiation of stem cells into definitive endoderm and early lung commitment occurs in low oxygen tension. In order to efficiently derive distal lung epithelial cell from ES cells, it is critical to investigate how reduced-oxygen tension might coordinate and modulate the in vitro differentiation of embryonic stem cells into definitive endoderm and then into epithelial cells of the distal lung. Previous studies have demonstrated that Activin A (AA), a cytokine of the transforming growth factor-beta (TGF-[beta]) superfamily, alone or in combination with other growth factors, such as Wnt-3a, can generate definitive endoderm cells from ES cells. Based on an established definitive endoderm differentiation protocol using AA, we used hypoxic conditions known to exist during early embryonic development as part of a novel differentiation protocol that improves expression and yield of definitive endoderm by about a factor of 5. We further demonstrated that the enhanced definitive endodermal differentiation of mES cells in hypoxia is mediated by HIF-1[alpha], a known mediator of hypoxia-sensitive gene modulation. Finally, we demonstrated that ES-derived definitive endoderm cells obtained in hypoxia, were competent to differentiate into the distal lung lineage, specifically into SPC-expressing alveolar type II (AEII) lineage cells. Furthermore, we present evidence that during this complex developmental process hypoxia is only effective during the initial phases of endoderm differentiation, but it is detrimental during the subsequent differentiation into the lung epithelial phenotypes. Overall, this thesis demonstrates, for the first time, the feasibility of systematically utilizing oxygen tension to enhance differentiation of ES cells into definitive endoderm and subsequently into distal lung epithelia. This knowledge will be relevant for effectively engineering not only the lung, but other endoderm derived organs, i.e., liver, pancreas, stomach, intestine, and thymus, as well as the lung.