Lineage differentiation of embryonic stem cells.

Abstract

Understanding events that regulate the generation of multiple cell and tissue types during mammalian development is complicated by the relative inaccessibility of the early embryo. Embryonic stem cells (ESC) are derived from and are thought to share a similar gene expression profile as the pluripotent inner cell mass (ICM) of blastocyst-staged embryos. ESC can be maintained as a self-renewing population indefinitely in culture, and yet, like the ICM, retain the potential to differentiate into all cell types of the body. These properties, along with the ability to manipulate these cells in vitro, constitute a powerful model system with which to study the roles of genes involved in maintaining pluripotency as well as those involved in lineage segregation and differentiation. Based on the premise that manipulating the expression of genes in ESC can provide insight into the regulation of cell fate choice during early development, a loss-of-function approach utilizing RNA interference (RNAi) was employed to knock down expression of the pluripotency factor, <italic> Oct4</italic>, in ESC resulting in trophectodermal differentiation even in culture conditions that inhibit differentiation. These results demonstrate the critical role of <italic>Oct4</italic> in maintaining pluripotency and establish RNAi as a viable loss-of-function approach to study gene function in ESC. In subsequent studies, an inducible gain-of-function approach was taken to evaluate whether forced expression of a pro-neural bHLH gene, <italic> Neurogenin1</italic>, is sufficient to promote neuronal differentiation in ESC. Transient expression of <italic>Ngn1</italic> in ESC resulted in widespread neuronal differentiation, even in conditions that inhibit differentiation. However, the induced cells were demonstrably sensitive to patterning factors including retinoic acid, BMP4, noggin, Shh, and FGFs, indicating that neurogenesis induced by <italic>Ngn1</italic> expression likely proceeds through intermediate progenitor cell stages. Induced cells were also implanted into the neural tubes of chick host embryos, and preliminary results suggest that they integrated into domains of the peripheral and central nervous system where <italic>Ngn1 </italic> is expressed in vivo. Together, these studies show that RNAi, inducible transgene expression, and transplantation of ESC are powerful techniques to study factors regulating cell fate choice. These studies form the basis for future work to better understand lineage segregation during embryogenesis.