ESPE2015 Free Communications Diabetes (6 abstracts)
RNA-Based MAFA Over-Expression is Sufficient to Drive Human Pancreatic Duct-Derived Cells Toward a β-Cell-Like Phenotype
Elisa Corritore a , Erica Dugnani b , Valentina Pasquale b , Lorenzo Piemonti b , Amedeo Vetere c , Susan Bonner-Weir d , Etienne M Sokal a & Philippe A Lysy a,
aUniversité Catholique de Louvain, Bruxelles, Belgium; bSan Raffaele Research Institute, Milan, Italy; cBroad Institute of Harvard and MIT, Cambridge, Massachusetts, USA; dJoslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA; ePediatric Endocrinology Unit, Cliniques Universitaires Saint Luc, Bruxelles, Belgium.
Background: 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.
Objective and hypotheses: In this study, we reprogrammed HDDCs into β-cell-like lineage by over-expressing mRNAs of key pancreatic transcription factors (TFs).
Method: 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.
Results: 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 seven 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 per 106 cells) under basal conditions.
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.
Volume 84
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