Epithelial polarity as a fate determinant of multipotent endocrine progenitors during human pancreas development
Final Report Abstract
A fundamental unanswered question in developmental biology is how different cell fates are induced from the same multipotent progenitors during the formation of complex organs that are composed of many different cell types. We use the developing pancreas as a model system to study this central general question and hypothesize that apical‐basal polarization of epithelial cells plays a critical role for steering pancreatic cell fate specification, namely the commitment of multipotent endocrine progenitors to either glucagon‐producing alpha cells or insulin‐producing beta cells. We have successfully generated a hESC double reporter cell line for monitoring cells that express the endocrine progenitor marker NGN3 and their hormone‐producing progeny while simultaneously visualizing the apical polarity marker EZR. These double‐reporter hESCs can be directed to differentiate in vitro toward pancreatic endocrine cells. We have established a novel lineage‐tracing approach based on confocal live‐imaging and could demonstrate that this tool can be used to determine the correlation between progenitor cell polarization and eventual alpha or beta cell fate. Due to unexpected difficulties with the originally proposed NGN3 reporter design, we had to modify our strategy. As a result, the progress of the project was delayed and the collection of a comprehensive dataset that is required to obtain statistically significant results is still ongoing. A virus‐mediated RNA interference system was proposed to dissect the molecular mechanisms that link the polarity complex to Notch signaling and cell fate decisions in hESC‐derived multipotent progenitors. The observed results were not satisfying and forced us to develop an alternative strategy for perturbing apico‐basal polarity in differentiating CRISPR/Cas9‐modified hESCs by inducible genetic recombination of the polarity regulator CDC42. Preliminary data strongly indicate that this inducible knockout system will finally allow us to answer the mechanistic questions raised in the original proposal. The expected results will not only shed light on unknown details of basic biological processes and of human pancreas development in particular, but will also help design more refined protocols for the in vitro generation of beta cells from hESCs, thus contributing to the development of cutting‐edge cellular therapies for diabetes.
