Although we have discovered significant differences between human and mouse preimplantation development, many of the key mechanisms are conserved, including the expression of pluripotency transcription factors Nanog and Oct4.
To complement our human research and to mechanistically understand how stem cells differentiate, we are using mouse embryonic stem cells (mESCs), which are highly amenable to genetic manipulation.
For example, we have generated mESCs that overexpress transcription factors in a controlled (doxycycline inducible) manner, allowing for inducible differentiation (Niakan et al., 2010). We hypothesise that the unique ability of specific transcription factors to induce ESC differentiation results from a dual function in activation of endoderm genes and simultaneous direct repression of pluripotency genes.
To further understand the network of gene regulatory changes during ESC differentiation, we are determining the hierarchy of gene and protein changes during early differentiation. Our preliminary analysis has revealed known as well as novel candidate regulators of stem cell self-renewal and endoderm differentiation. In the future we will analyse the function of these candidates by performing gain- and loss-of-function analyses in the context of inducible differentiation. Combination of these data with gene expression analysis will generate hypotheses concerning the genetic hierarchy of pluripotency disengagement. These experiments will directly inform derivation of human extra-embryonic stem cells, as the core network involved in embryonic stem cell self-renewal is conserved in humans and mice.
How do transcription factors control pluripotent stem cell differentiation? We observed that Nanog and the SRY-related HMG-box transcription factor Sox17 were bound reciprocally to shared Nanog/Sox17 genomic sites, represented here by the region upstream of the Lama1 start site (arrow).
This suggests a mechanism for initiation of differentiation in which Sox17 displaces repressive Nanog complexes from shared binding sites and, in turn, activates gene expression.