New strategies in cell replacement therapies for diabetes: role of USP7 in iPSC and adult organoids beta cell differentiation

Type 1 diabetes is a disease in which patients have very few or no insulin-producing beta cells, which results in high blood glucose levels. Since the discovery of insulin almost a century ago, there has been an alarming increase in new diabetes cases, but the only treatment for Type 1 diabetes is still based on delivering insulin via injections or pumps. While insulin administration can successfully control diabetic symptoms, the risk of complications is very high, and the continual blood sugar management required can be a real burden in patients' lives. Regenerative medicine offers new hope of a curative treatment for diabetes by replacing lost beta cells. However, the source of replacement cells and the efficiency with which they can be produced and how well they work in the body are all still under investigation.
In our lab, we have exciting evidence of a novel mechanism by which the key protein involved in beta cell differentiation, Neurogenin 3 can be made more stable and therefore increase its potential to make beta cells from different cell sources. We believe that identifying the conditions that allow for the enhanced differentiation of iPSC or Adult pancreas cells could greatly improve the yield and functionality of beta cells to be translated for human therapy. We hope that our findings pave the way for the development of new, more efficient strategies to replenish lost beta cells in diabetes patients, in order to achieve the ultimate therapy goal for diabetes: a "diabetes free" life.

We hypothesize that exploiting the USP7-NGN3 axis will allow for the improved generation of insulin-producing beta cells. Using a multidisciplinary approach integrating molecular and cellular studies with advanced transcriptomics and imaging techniques we will leverage basic biology to improve beta cell generation. In Aim 1, We will explore, at the molecular level, the functional interaction between USP7 and Ngn3 using doxycycline inducible iPSCs overexpressing Ngn3 or USP7 or both, and by applying 10x scRNAseq techniques we will identify additional USP7 regulated pathways that are crucial for the generation of beta cells from human iPSCs. In Aim 2, We will focus on exploring the USP7 interactome at different stages of beta cell differentiation and IP-MS identify interactors that promote USP7 deubiquitinating activity. In Aim3, we will modulate the USP7-NGN3 axis and differentiate the iPSC-derived pancreas progenitors into beta cells. A comprehensive characterization of the generated cells will be performed to assess efficiency of differentiation. Specifically, we will test glucose challenge-induced insulin release, assess calcium dynamics, and carry out qPCR and immunofluorescence analyses of beta cell markers (glut2, pck1/3, insulin, gck). In Aim 4, we will assess the in vivo functionality of the generated cells using transplantation strategies in normoglycaemic or diabetes mice. We will assess transplanted cells' efficiency in insulin production. We will also carry out studies to assess the resultant immunological reaction to the transplanted cells using ELISA, immunostaining and qPCR analyses.