Regulation of pancreas organoid plasticity: new strategies for diabetes

Since the discovery of insulin almost a century ago, there has been an alarming increase in new diabetes cases, but the only treatment for 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 hopes of a curative treatment for diabetes by replacing lost beta cells, which respond to blood glucose levels and produce insulin. However, the source of replacement cells, the efficiency with which they can be produced and how well they work in the body are all still under investigation. Recent evidence suggests that the ductal cells of the pancreas, which are not damaged in diabetes, can change into other cell types when given the right trigger. Ductal cells can be grown long term outside the body as "organoids" in 3D culture. We and others have shown that when grown in special conditions or genetically modified to produce three key proteins responsible for making beta cells in the embryo (Ngn3/Pdx1/MafA), ductal cells change to beta-like cells.
In our lab, we have exciting evidence that the reason the efficiency of conversion and functionality of the generated cells does not match "real" mature beta cells is firstly, because not all cells are equally responsive to the signals to become beta-cells, and secondly, because of the strong degradation mechanisms that operate on Ngn3, Pdx1 and MafA, inactivating them before they have a chance to work. We believe that by identifying the cells that can convert into beta-cells, and discovering how to stabilise Ngn3, Pdx1 and MafA we will be able to improve the chances of curing diabetes. In this proposal, we want to address these questions: What are the proteasomal mechanisms regulating proendocrine factors stability? How does the lysosome regulate the proendocrine factors? Can the variation of such mechanisms at a single cell level explain the heterogeneity of the ductal organoids?
Altogether this research proposal will substantially increase our knowledge of beta cell generation from ductal cells. 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 increasing the stability of Ngn3/Pdx1/MafA and targeting the right subpopulation of cells within pancreatic organoids will result in more efficient beta cell generation and more functional beta cells.
Using a multidisciplinary approach integrating molecular and cellular studies with advanced imaging techniques, NGS, FACS and proteomics, we will leverage basic biology to improve beta cell generation. In Aim 1, we will characterise the Fbw7:Pdx1/MafA and Ngn3-Huwe1 axes (coIP, ubiquitin assays, mapping critical residues, Fbw7 and Huwe1 loss of function experiments, identification of novel interactors). In addition, the functional role of both axes in organoid differentiation into beta cells will be evaluated. In Aim 2, a comprehensive characterization of the role of the lysosome on the regulation of the proendocrine factors and in the differentiation process will be assessed. We will inhibit lysosome function using pharmacological agents (Lysosome inhibitors, NH4Cl, Leupeptine) or loss of function strategies (lysosome components). We will test glucose challenge-induced insulin release, calcium measurements, and qPCR and IF analysis of beta cell markers (glut2, pck1/3, insulin, gck) to assess the efficiency of differentiation upon perturbation of the lysosome pathway. In Aim 3, we will perform Genomix 10X single cell RNAseq analysis (3000 cells/sample) in mouse adult organoids using differentiating and undifferentiating conditions. Comprehensive bioinformatics analysis will be focused on the identification of novel FACS-sortable markers for the subpopulation which is permissive to differentiate to beta cells and we will address whether the lysosome and huwe1 signature could explain the organoid heterogeneity. Functional validation of the identified populations will be assessed as in aims 1 and 2.

This proposal focuses on basic biosciences and will improve our understanding of the molecular regulation of beta cell differentiation and will therefore ultimately contribute to new strategies for cell replacement therapies for the treatment of diabetes.

Main beneficiaries will be:

The academic community. Within the beta cell regeneration community, there are a small number of groups focusing on the molecular regulation of the transcription factors essential for beta cell differentiation. Our approach of biochemical characterisation coupled with new culturing and differentiation techniques will greatly benefit their research by providing experimental tools and new knowledge in the field.

Clinicians. By identifying novel signaling pathways and regulators that enhance beta cell differentiation, we will be providing clinician scientists with research tools with which to potentially establish new cell replacement strategies for diabetes or increase the success of islet cell transplantation. This will greatly facilitate research into this area and could be of clinical benefit to diabetes patients.

Pharmaceutical Industry/Biotech companies/medicinal chemists. The development of relevant inhibitors /activators to enhance beta cell differentiation could in the long term stimulate economic growth. Furthermore, given the implication of the molecular pathways in different organs, the chemical inhibitors could be utilized for other therapeutic purposes.

Diabetes Patients. Despite being treatable with insulin, living with diabetes is a burden for patients. Diabetes is still a disease associated with dramatic complications with a real need to identify novel therapies aimed to achieve a "diabetes free life". Cell replacement therapies provide the groundwork for this achievement, but the lack of knowledge about the regulation of beta cell differentiation has made the process inefficient. My proposal will ultimately lead to better approaches for the generation of beta cells for diabetes therapy, which will benefit patient quality of life in the long run.

The NHS and Diabetes Charities. These are major stakeholders in the search for curative treatments for diabetes. Complications associated with diabetes are a major financial burden on the NHS, and a better understanding of the molecular regulation of beta cell differentiation will ultimately lead to the development of cell replacement therapeutic strategies, thus limiting complications associated with uncontrolled insulin therapy. Our research will also be used to educate charitable organisations and fundraisers on the science behind cell replacement research in diabetes and keep them informed of advancements in the field.

Transferable skills gained by staff working on the project. The post-doctoral scientist employed on this project and the PhD/Rotation/Master's students associated to this project will develop extensive project management skills required for research and acquire a skill set highly desirable for future employment prospects. With my help, they will also gain additional general project management skills that are obtained from running a research project, which are transferable to many other disciplines in addition to the scientific employment sector. The post-doc will gain communication and supervision/teaching skills that will be transferable to any management discipline.