A functional genomics and candidate gene validation approach to identify genes regulating pancreatic development and/or stem cell fitness

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Pluripotent stem cells are characterized by continuous self-renewal while maintaining the potential to differentiate into cells of all three germ layers. Despite extensive knowledge in this regard, a gap remains in the understanding of the precise signaling clues governing commitment to the respective cellular lineages particularly endodermal derivate. Differentiation strategies based on the use of cytokines and small molecules already allow the generation of virtually every cell type in the brain but also the heart while e.g. endocrine and exocrine pancreatic differentiation strategies remain rather inefficient. Forward genetic screening using RNA interference techniques and large-scale compound screens may provide novel and unorthodox avenues to reach this goal also during endodermal differentiation of human pluripotent stem cells. The first part of the current proposal aims to validate the results of an already performed shRNA screen while differentiating human embryonic stem cells towards definitive endoderm. Disease- and age-associated decreases in regenerative capacity and organ maintenance could represent major factors limiting the quality of life and life expectancy. In the second part of the proposal, we aim to identify such factors limiting stem cell fitness and therefore regenerative capacity in shRNA-based reprogramming screens. To do so, we have identified Dkk3 as a gene, which limits cellular reprogramming, but also liver and hematopoietic stem cell fitness. Herein, we aim to extend these findings towards pancreatic regeneration and try to define the molecular events governing this process. Taken together, the current proposal will provide novel clues to improve pancreatic differentiation of human pluripotent stem cells. This is the perquisite of applying these techniques to cell replacement therapies in e.g. diabetic patients. On the other, we will shed light on Dkk3 a stem cell fitness limiting gene to develop potential strategies for improved organ maintenance and regeneration.