MMSR · The Molecular Mechanisms of Stem Cell Self Renewal

Over the past few years, a view has emerged that pluripotency in mouse embryonic stem cells (ESCs) is cell-autonomous and does not depend on external signals. This was based on work that showed that a combination of a small molecule ERK inhibitor with a GSK3 kinase inhibitor was sufficient to maintain self-renewal, in absence of any growth factors. I have now discovered that autocrine or paracrine signalling via the Wnt pathway plays an essential role in ESC self-renewal, and is required to inhibit their differentiation towards primitive ectoderm. Moreover, I have discovered that the combination of Wnt protein with the cytokine LIF is sufficient to support the maintenance and derivation of germ-line competent ES cells in fully defined conditions not containing any other growth factor. Thus, two different fully defined conditions both maintain self-renewal, but do so in different ways. However, the intracellular events downstream of those conditions must both converge on the self-renewal machinery. By analyzing and comparing these events in both systems, unmatched synergy in zooming in on the relevant entrances into the self-renewal machinery can be obtained. I propose to analyze the events downstream of these two sets of self-renewal stimuli, in an effort to identify novel self-renewal genes. The downstream effects of the self-renewal stimuli will be analyzed using gene expression profiling, ChIP-Seq, and phosphoproteomic approaches. Combining and comparing the different data sets will identify candidate genes that will be screened in a self-renewal assay designed for high sensitivity and throughput. Genes that pass this test will be further analyzed for their roles in proliferation, survival, and differentiation in an effort to compartmentalize the self-renewal machinery into functional blocks. This analysis will provide the first comprehensive resource documenting the molecular workings of self-renewal.