Abstract
Despite numerous studies on the molecular switches governing the conversion of stemness to differentiation in embryonic stem cells (ESCs), little is known about the involvement of the retromer complex. Under neural differentiation conditions, Vps26a deficiency (Vps26a-/-) or knockdown suppressed the loss of stemness and subsequent neurogenesis from ESCs or embryonic carcinoma cells, respectively, as evidenced by the long-lasting expression of stemness markers and the slow appearance of neuronal differentiation markers. Interestingly, relatively low reactive oxygen species (ROS) levels were generated during differentiation of Vps26a-/- ESCs, and treatment with an antioxidant or inhibitor of NADPH oxidase (Nox), a family of ROS-generating enzymes, led to restoration of stemness in wild-type cells to the level of Vps26a-/- cells during neurogenesis. Importantly, a novel interaction between Vps26a and Nox4 linked to the activation of ERK1/2 depended highly on ROS levels during neurogenesis, which were strongly suppressed in differentiating Vps26a-/- ESCs. Moreover, inhibition of phosphorylated ERK1/2 (pERK1/2) resulted in decreased ROS and Nox4 levels, indicating the mutual dependency between pERK1/2 and Nox4-derived ROS during neurogenesis. These results suggest that Vps26a regulates stemness by actively cooperating with the Nox4/ROS/ERK1/2 cascade during neurogenesis. Our findings have important implications for understanding the regulation of stemness via crosstalk between the retromer molecule and redox signaling, and may contribute to the development of ESC-based therapeutic strategies for the mass production of target cells.
Highlights
Embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocyst stage embryos, are capable of unlimited proliferation and can differentiate into multiple cellEdited by R.A
To determine whether Vps26a is involved in the regulation of stemness and differentiation, ESCs were established from wild-type (WT) and Vps26a-/- blastocysts at 3.5 days post coitum by expanded culture on mitotically-inactivated mouse embryonic fibroblast feeder layers (Supplementary Fig. 1), and were committed into neural-lineage cells by cultivation in neurobasal medium (NBM)
The number of cells continuously increased in the Vps26a-/- group compared with WT during neural differentiation from ESCs (Fig. 1c)
Summary
Embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocyst stage embryos, are capable of unlimited proliferation and can differentiate into multiple cellEdited by R.A. Embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocyst stage embryos, are capable of unlimited proliferation and can differentiate into multiple cell. These three authors contributed : Seon-A Choi, Young-Hyun Kim, Young-Ho Park. Extended author information available on the last page of the article lineages [1] Their self-renewal capabilities and pluripotency are representative features of ESCs, making them a robust and suitable model to aid our understanding of developmental biology. They are a useful resource for the development of therapeutic strategies for incurable degenerative diseases. Further clarification of the molecular mechanisms governing the selfrenewal and differentiation processes of ESCs is needed to yield effective target cells for academic and industrial areas
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