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In Vitro Derivation of Beta Cells by Ectopic Expression of Pancreatic Transcription Factors

Final Report Summary - TRANSBETA (In Vitro Derivation of Beta Cells by Ectopic Expression of Pancreatic Transcription Factors)

The TRANSBETA Project aimed to use human cells to produce insulin producing cells by transdifferentiation of a non beta cell population to beta cells by expression of pancreatic specific transcription factors. In the last 15 years, modest success has been achieved by using up to 4 exogenous transcription factors to produce cells capable of producing insulin, focusing mainly on acinar cells, alpha cells, duct cells or gut cells as starting populations for transdifferentation. We proposed to significantly expand the number of transcription factors and initial cell types tested in order to find novel and more efficient combinations of transcription factors capable of generating insulin producing cells that could eventually be used as a source of cells for transplant.
Results: we attempted to develop an improved reporter system designed to track the beta cell state consisting of a lentiviral vector with 3 expression cassettes: a) a constitutive promoter driving expression of the puromycin resistance gene, allowing for selection of human cells transduced with the vector; a pdx1promoter driving expression of RFP and an insulin promoter driving expression of GFP, allowing unequivocal identification of pancreatic beta cells. We cloned 17 genes into doxycicline regulatable lentiviral vectors. We have co-transduced pools of candidates into human fibroblasts, selected for puromycin resistance and activated expression of transgenes by addition of doxycicline. Within 5 days, 4% of cells expressed GFP. However, upon withdrawal of doxycycline GFP fluorescence was quickly lost. We are currently defining what subset of factors is responsible for reporter activation and characterizing the phenotype of the fluorescent cells. However, a major development in the field of beta cell production during the course of this project has been achieved by the development of an effective protocol to differentiate beta cells from human embryonic stem cells. This achievement somewhat dampens the translational potential of transdifferentiation for beta cell production. The PI funded by this CIG has successfully integrated himself as a researcher and educator at the University of Algarve. His group is currently composed of a postdoc, a PhD student and a research assistant, he has published several papers and book-chapters and presented his work at several scientific meetings. He has served in several institutional positions, including Director of the Master’s Program in Biomedical Sciences and as Vice-Director of the Center for Biological Research. He was given the 2012 Young Investigator Award by the pharma company Genzyme-Sanofi, who has funded the candidate's research on Gaucher's Disease. The fellow has also obtained a national portuguese research grant and developed two additional research lines at his laboratory. Furthermore, he teaches 50% of his time in several courses at the Department of Biomedical Sciences and Medicine. The CIG grant has been a fundamental source of support for the PI, who has successfully passed his tenure examination in March 2016 and has therefore been confirmed for a permanent Professorship at the University of Algarve.