Molecular mechanisms controlling differentiation of pluripotent cells into endoderm.

During the last decade various stem cells have been derived from adult or embryonic human tissues. These cells offer new prospects to treat degenerative diseases, since they can specialise into many mature cell types that could be useful for cell based therapies. However, robust protocols allowing the production of fully functional specialised cells still need to be established. The generation of such cell types may ultimately only be achievable by recapitulating a normal path of development in vitro. But the sequential events leading to the formation of the primary germ layers, the building block of the body‘s organs, remain obscure. Thus, the understanding of the mechanisms controlling the specification of the primary germ layers has a major importance for regenerative medicine. Here, I propose to use human Embryonic Stem cells (hESCs) to study the mechanisms controlling differentiation of the endoderm germ layer, from which originate the pancreas, liver, lungs, and gut. While the objective of this project is to control differentiation of hESCs into endoderm lineages, the insight that will be gained could also be used to convert adult cells to endodermal cells by recapitulating the developmental pathway that forms this tissue during normal development.

Generation of all the adult tissues occurs by progressive cell fate decisions, starting with the specification of the three primary germ layers (ectoderm, mesoderm and endoderm). Molecular mechanisms controlling specification of the germ layers have been extensively studied in amphibia and fish. However, the understanding of these mechanisms in mammals (especially humans) is more limited, and this represents a major drawback for regenerative medicine. Indeed the generation of fully functional cell types from stem cells may only be achievable by recapitulating a normal species specific succession of cell fate decisions.
The studies of these mechanisms at the molecular level in vivo are restricted in mammals and particularly in humans, by the difficulty of obtaining sufficient biological material. The recent availability of human Embryonic Stem cells (hESCs) offers new possibilities to resolve this major limitation. Their embryonic origin confers upon them the ability to proliferate indefinitely in vitro while maintaining the capacity to differentiate into extra-embryonic tissues and into derivatives of the three primary germ layers. Here, I propose to define the network of transcription factors controlling differentiation of pluripotent cells into endoderm using first hESCs as an in vitro model of human development and then using mouse embryos to validate these results in vivo.
This project will be based on functional studies and on high throughput approaches including chromatin immunoprecipitation (ChIP) assays and gene expression profiling. Importantly, differentiation of hESCs into definitive endoderm cells will be achieved using fully chemically defined culture conditions which are devoid of animal products, thereby eliminating factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. This method of differentiation has been developed during my current fellowship and the endoderm progenitors generated have been extensively characterised by both molecular analytical and cellular functional assays.
In summary, the objectives of this proposal are (i) to identify the key transcription factors controlling endoderm differentiation and to define precisely their function, (ii) to reveal the organisation of the core transcriptional network controlling the expression of these transcription factors during the transition between the pluripotent state and the endoderm fate and (iii) to define the downstream network of genes controlled by these transcription factors. The overall outcome of this project will gain the knowledge needed to control differentiation of hESCs into specific lineages and also potentially to enable trans-differentiation of other cell types, including adult stem cells and adult somatic cells.