(89a) Transcriptional Regulatory Network In Pluripotent Stem Cells

Stem cells maintain their pluripotent state through a robust gene network consisting of complex feedback and feedforward interactions between key transcription factors. In self-renewing stem cells, the gene network maintains expression of pluripotency-associated genes such as Oct4 and Nanog, while suppressing the expression of genes associated with differentiation to somatic lineages. In the context of embryonic development, this network is actively maintained in pluripotent cells of the inner cell mass while the trophoblast and primitive endoderm lineages lose expression of key genes like Oct4 and Nanog, respectively. Primitive endoderm-precursor cells, derived from E4.5 blastocyst, are separated from the pluripotent stem cells of the inner cell mass through a single differentiation event. These cells express many key pluripotency-associated genes including Oct4 and Sall4, but also express genes representative of the primitive endoderm lineage, such as Gata6 and Sox17. This unique gene expression profile, developmental proximity to pluripotent stem cells and, genetic tractability make these cells ideal candidates for probing the dynamics of pluripotency gene network. With these precursor cells as our model system, we have used a combinatorial approach involving experimental gene over-expression/knockdown studies and, a theoretical boolen model, to explore interactions within this network. Using this approach, we have been able to predict outcomes of experimental perturbations within the network. This has provided us with novel insights into how pluripotency is established and maintained in stem cells.