A recessive genetic screens to identify regulators of embryonic stem cell self-renewal and lineage commitment

Embryonic stem (ES) cells exhibit indefinite growth in culture while retaining the potential to differentiate into all fetal or adult cell types. The application of ES cells in regenerative medicine requires that ES cell differentiation in culture be directed to become a specific cell type, such as dopaminergic nerve cells, which could be used to replace the damaged nerves in Parkinson‘s disease patients. However, at present we are unable to reliably direct cell differentiation in this way, so even when we successfully generate nerve cells they tend to be mixed together with other unwanted types. Better understanding of the genes and molecular signaling involved in ES cell growth and differentiation in culture will help develop new culture systems for directed ES cell differentiation. Application of new techniques, such as the proposed recessive genetic screen, to the study of ES cell biology and in vitro differentiation will allow us to find novel components as well as bring new views to the established ES cell regulatory pathways.

Embryonic stem (ES) cells are pluripotent cells. They can self-renewal in culture for an indefinite time while retaining the capability of differentiating into all adult cell types. ES cells can be induced by growth factors and hormones to differentiate to many cell types in culture, opening the potential of using in vitro differentiated cells in regenerative medicine and pharmaceutical screening. However, the fundamental molecular signaling that controls ES cell self-renewal and in vitro differentiation has not been well defined. I aim to address this question using a phenotype-driven genetic screening strategy. Unbiased genetic screens can identify novel molecular components of a biological process. Recessive genetic screens have been performed in many model systems such as bacteria, yeast, fly and worm. However performing a recessive genetic screen in cultured mouse cells is limited by the diploid nature in the genome. A new tool, Blm-deficient ES cells have recently been developed in which homozygous mutations arise via highly efficient loss of heterozygosity. In my previous work, I have demonstrated the use of Blm-deficient ES cells in a genetic screen to isolate components of the DNA mismatch machinery. Here, I propose to apply this tool to identify novel molecules and pathways involved in stem cell self-renewal and in vitro differentiation.