Genetic Approaches to Study Human Embryonic Stem Cell Self-Renewal and Survival

Abstract

Embryonic stem (ES) cells can be maintained indefinitely in culture while retaining the ability to give rise to cellular derivatives from the three germ layers. These unique characteristics hold great promise for regenerative medicine and underscore the importance of understanding the molecular mechanisms behind ES cell maintenance. The embryonic stem cell state is supported by a delicate equilibrium of mechanisms that maintain pluripotency, prevent differentiation, and promote proliferation and survival. We sought to find genes that could contribute to one or more of these processes in human ES cells by using a gain-of-function screen of over 8000 human open reading frames (ORFs). We identify Vestigial-like 4 (Vgll4), a co-transcriptional regulator with no previously known function in ES cells, as a positive regulator for survival of human ES cells. Specifically, Vgll4 protects human ES cells from dissociation stress, and enhances colony formation from single cells. These effects may be attributable in part to the ability of Vgll4 to decrease the activity of initiator and effector caspases. Based on global transcriptional analysis, we hypothesize that Vgll4 enhances survival of hES cells at clonal densities by regulating changes in the cytoskeleton, which may in turn regulate pathways known to result in hES cell death. This dissertation introduces a novel approach for studying hES cell survival in the context of cell dissociation and presents Vgll4 as a novel regulator of this process. We also propose that Vgll4 could have multiple functions in hES cells including possible roles in pluripotency, cell cycle dynamics, Hippo pathway regulation, and \(TGF\beta\) signaling. A direct regulator of survival in human embryonic stem cells could have important implications for facilitating the generation of transgenic cell lines and reporters, thus harnessing the therapeutic application of these cells.