RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype

Corritore, Elisa;Dugnani, Erica;Pasquale, Valentina;Piemonti, Lorenzo;Lysy, Philippe;et.al.
(2016) 49th Annual Meeting of ESPGHAN (European Society for Paediatric Gastroenterology Hepatology and Nutrition) — Location: Athènes, Grèce (25.May.2016)

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  • Corritore, ElisaUCLouvain
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  • Dugnani, Erica
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  • Pasquale, Valentina
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  • Piemonti, Lorenzo
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  • Sokal, EtienneUCLouvain
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  • Lysy, PhilippeUCLouvain
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Abstract
RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype Elisa Corritore1, Erica Dugnani2, Valentina Pasquale2, Lorenzo Piemonti2, A. Vetere3,Susan Bonner-Weir4, Etienne M. Sokal1, Philippe A. Lysy1,5 1Institut de Recherche Expérimentale et Clinique, Pediatric Research Laboratory,Université Catholique de Louvain, Brussels, Belgium; 2San Raffaele Research Institute, Milan, Italy; 3Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, USA ; 4Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, Boston, USA ; 5Pediatric Endocrinology Unit, Cliniques Universitaires Saint Luc, Université Catholique de Louvain, Brussels, Belgium Pancreatic epithelial cells represent an attractive cell source for replacement therapy of type 1 diabetes. Previously, we designed a protocol for expansion of human pancreatic duct-derived cells (HDDCs) and showed their β-cell engineering potential. In this study, we reprogrammed HDDCs into β-cell-like lineage by over-expressing mRNAs of key pancreatic transcription factors (TFs). Pancreatic duct cells (n=6) were purified and propagated into endothelial growth-promoting media. Synthetic modified (sm) RNAs were manufactured by unidirectional subcloning of PDX1, NGN3 and MAFA into a plasmid containing 5’ and 3’ UTR regions. The UTR-flanked inserts were excised and poly(A)-tailed. The final smRNAs were synthesized through in vitro transcription followed by phosphatase and DNase treatments, before being daily transfected in HDDCs. In all donors, transfection of PDX1, NGN3 and MAFA led to upregulation of endogenous target (ex: NGN3) and β-cell marker (ex: INS, synaptophysin, SLC2A2, GCK) genes with the highest expression levels being reached after MAFA transfection. Co-transfection protocols failed to show significant improvement of β-cell differentiation. Acceptable impact on innate immune response and cell viability was noticed after 7 consecutive daily smRNA transfections, based respectively on minimal IFNA and RIG-1 gene expression and on annexin-V/PI staining. After MAFA transfection, HDDCs stained positive for MAFA and insulin (19.3 ± 3.3 %) proteins, while ELISA assays showed detectable amounts of C-peptide content and release (21.45 ± 2.42 pg/mL/106 cells) under basal conditions. In conclusion, we showed that MAFA RNA over-expression is sufficient to efficiently reprogram HDDCs toward β-cell-like phenotype in a timely manner. Further research is mandatory to demonstrate a controlled insulin secretion capacity after differentiation.
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Corritore, E., Dugnani, E., Pasquale, V., Piemonti, L., Vetere, A., Bonner-Weir, S., Sokal, E., & Lysy, P. (2016). RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype. Journal of Pediatric Gastroenterology and Nutrition. Published. 49th Annual Meeting of ESPGHAN (European Society for Paediatric Gastroenterology Hepatology and Nutrition), Athènes, Grèce. https://hdl.handle.net/2078.5/184034 (Original work published 2016)