Generation of a mouse model to study the Pancreatic Polypeptide-producing PP-cells

The pancreas is an organ composed of the exocrine and endocrine glands. The exocrine gland consists of acinar cells and duct cells responsible for digestion of protein, nucleic acids and lipids. The endocrine compartment is formed of islets of Langerhans, cell clusters, in which five cell types are present: glucagon producing alpha cells, insulin producing beta cells, somatostatin producing delta cells, pancreatic polypeptide producing PP cells and ghrelin producing epsilon cells (not present in adult mice). These hormone-producing endocrine cells play a major role in regulating the glucose metabolism. In diabetes mellitus type I and II, there is a dysregulation of the function and or loss of beta cells which result in hyperglycemia (high level of blood sugar). In the last decade, researchers developed an approach toward regeneration of beta cells. In our laboratory, we showed in vivo intra islet cell conversion (from alpha or delta cells) into beta cells with combination of lineage tracing of cells (doxycycline dependent lineage tracing system) and near total beta cell loss. However, no lineage tracing tool exist for PP cells and moreover the identity and function of these cells remains unclear. Furthermore, no known antibodies for PP seem reliable. Hence, we developed a lineage tracing tool for PP cells in mice with Crispr/ Cas 9 system which is a novel and revolutionary technology to produce genomic modification at targeting site. In this study, we demonstrated the generation of genome edited PP KO and KI PP-rTA mice with Crispr/ Cas 9 technology. We performed two injection sessions with PX330, a plasmid based delivery system. In the first session, we used the SCR7 drug, an inhibitor of ligase IV which is a key component of the NHEJ pathway. Previously, SCR7 was shown to negatively regulate NHEJ pathway and favors HDR-mediated genome editing frequency in vitro and in vivo. We assessed whether the presence of SCR7 improves the efficiency of generation of KI PP-rtTA mice. From the two injection sessions, 6 out of 21 mice harbored a large deletion for PP (Δ) and strikingly 2 out of 21 mice were KI. To our knowledge the generation of a mice with an insertion of a large sequence by using plasmid based delivery system was not previously reported. Our results showed that the 2 sgRNAs used are efficient. These important findings reveal that plasmid based delivery system is fast and easy compare to the use of RNA. Nevertheless, PX330 efficiency has to be further assess in future injection sessions. 6 Moreover, our observations suggest that SCR7 seems to have a positive effect on the efficiency of generation of KI mice. Indeed, we obtained 3 KO out of 21 mice and the two KI in the first injection session with the SCR7. One KI mice showed a correct integration of the transgene. This mice that carried also a large deletion for PP was used to establish colonies. We successfully generated colonies for homozygous KO Δ/ Δ mice and KI/KI mice to study the effect of a genetic inactivation of PP. Surprisingly, with these mouse models we demonstrated that the PP antibody available in the laboratory is specific. KO Δ/ Δ mice and KI/KI mice showed a lack of PP staining in PP cells compare to WT mice. More importantly, we showed that the KI mouse model is inducible. Administration of doxycycline to the mice induced labelling of putative PP expressing cells with GFP in the periphery of pancreatic islets. We are currently assessing the specificity of our mouse model to demonstrate that with doxycycline we observe PP+GFP+ cells. We obtained 2 KI mice which show that Crispr/ Cas 9 system needs to be improved for generation of KI. With the KI mouse model, we will be able to address questions regarding the identity of PP cells during development (when do PP embryonic cells emerge? Are PP embryonic cells giving rise only to PP adult cells?) , the role of PP cells in glucose metabolism and the implication in regeneration of beta cells. Taken together, this novel tool will allow to provide new insights on pancreatic cell plasticity which potentially opens up to new therapeutic strategies

Pancreatic Ppy-expressing γ-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by β-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon β-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential