Enhancement of pancreatic beta cell generation in vitro and in vivo by post-translational regulation of Neurogenin3

Many illnesses of middle and old age result from a progressive loss of tissue function including Alzheimers, Parkinsons Disease, heart failure and notably diabetes. These chronic conditions generally require long-term medication, but recent spectacular advances mostly grounded in basic biology have raised the possibility that defective cells could be directly replaced. An increase in the mass of beta islets cells, the type of cells that are missing or malfunctioning in diabetes, has been proposed to ameliorate type 1 diabetes, and is also likely to be an effective treatment for many sufferers of Type 2 diabetes. Indeed, successful islet transplantation has been shown to result in relief from a requirement for insulin therapy and relief from diabetes-related symptoms.
So called stem cells, cells that can both proliferate indefinitely and can be induced to form more specialised cell types in a culture dish, could act as a source for beta cell replacement. However, a major obstacle remains in that stem cells must be induced to adopt beta cell fate and maintain that fate indefinitely when transplanted back into the patient, retaining functionality and avoiding the risk of cancerous tumour formation. It is widely acknowledged that for this to occur, stem cells must be taken down the developmental pathway that beta cells usually take when undergoing formation in the developing embryo. Based on these developmental pathways, protocols have been published where human embryonic stem cells can be induced to form relatively immature beta cells, while their in vivo implantation back into animals results in further maturation and beta cell functionality. However, a caveat to this approach is that cancer formation was also occasionally observed, indicating the need for tranplantation of beta cells that are differentiated to a more mature state to minimise this risk. This is currently a road-block to generating more mature beta cells in vitro that we may have uncovered a way of moving through.
The protein Neurogenin 3 (Ngn3) is needed for formation of all beta cells during development and many experiments indicate that manipulating its activity could enhance beta cell formation from stem cells. However, normal Ngn3 only causes rather immature beta cells to be generated and these cannot respond to blood glucose levels in the same way as normal beta cells. We see that changing the chemical modifications of Neurogenin protein by preventing the addition of phosphates can significantly enhance the ability of Neurogenins to drive cell maturation and we hypothesise that this may make a modified form of Ngn3 much better at generating mature beta cells than the normal protein. This project is designed to test this hypothesis and, if correct, to understand the mechanism of this enhanced cellular maturation. To help us move this project towards clinical application, we are working with Prof Douglas Melton at the Harvard Stem Cell institute who is focussed on generating beta cells of sufficient quality for human trails.

Diabetes, currently one of the UK's most pressing public health problems, has the potential to be treated successfully by pancreatic islet transplantation, but islet supply is a critical issue that must first be addressed. Stem cells (either ES or iPS) could act as a source for beta cell replacement. However, major obstacles remain in that stem cells must be induced to adopt functional beta cell fate and maintain that fate indefinitely when transplanted back into the patient. This may be achieved by recapitulating developmental events that occur during pancreas formation. Endocrine pancreas and beta cell formation is ultimately controlled by the activity of the key proneural bHLH transcription factor Ngn3. We have identified multi-site phosphorylation as critical post-translational modifications of Ngn3 and we hypothesise that rational manipulation of Ngn3 phospho-status will enhance Ngn3 activity, promoting stable, more mature beta cell formation.
Specifically we will:
1) Characterise the contribution of distinct SP sites to phosphoregulation of Ngn3 in the rapidly analysed in vivo model, Xenopus.
2) Use RNA-mediated transfection to over-express Ngn3 and phospho-mutant Ngn3 in human ES cells induced to differentiate into pancreatic progenitor cells in vitro, comparing the efficiency of beta cell generation and maturation. To assay engraftment and functionality of human islet cells so generated. This work will be conducted in collaboration with Prof. Douglas Melton, Harvard Stem Cell Institute.
3) Differentiate ES cell lines derived from our Ngn3eYFP and 6S-ANgn3eYFP mice towards an endocrine pancreas fate and to compare both efficiency of endocrine cell differentiation and the endocrine subtypes so generated.
4) Generate inducible mES cell lines over-expressing wild-type and phosphomutant Ngn3 and use genome-wide ChIP seq and RNA seq to qualitatively and quantitatively compare transcriptional profiles to identify differentially regulated targets.

Who will benefit?

This proposal is centred on improving the efficiency of beta cell generation and so will benefit patients, health-care providers and biomedical science and allied industries. Diabetes is perhaps the most pressing public health concern in developed nations in the 21st Century with numbers of affected individuals rising fast. While insulin therapy gives some control, it is far from a cure. However, increasing islet cell mass (with immunosuppression as necessary) is a very real prospect for allowing long-term blood glucose stabilisation in both Type 1 and Type II diabetes. Currently one major block to such therapy is an acute shortage of pancreatic islets or beta cells for transplantation. Hence, huge resources world-wide are being applied to developing methods for generating and expanding islets/beta cells in vitro from ES and iPS sources. Our work in basic biology may have uncovered a way to promote islet cell generation and maturation from ES cells in vitro, benefiting all those groups with an interest in improving and enhancing this process.

How will they benefit?
This work, grounded in the basic developmental biology of proneural protein regulation, has indicated a way to enhance differentiation of endocrine and beta cells from ES cells in vitro. Patients will benefit from potentially enhanced cell based therapies, while health-care providers and allied industries will benefit by developing these therapies. Moreover, the scientific community will benefit from an enhanced understanding of the basic developmental biology of islet cell differentiation and proneural protein regulation.

This proposal involves close collaboration with Prof Doug Melton's laboratory at the Harvard Stem Cell Institute who are focussed both on promoting human ES cell differentiation in vitro and testing the functionality of generated cells in sophisticated animal models of diabetes. The UK (patients, biomedical science and allied inductries and the scientific community) will additionally benefit by the close ties with the Harvard Stem Cell Institute proposed. This collaboration is ideal as DM's lab is focused on moving ES cell-derived beta cells from the lab to the clinic, and as such is also developing methods to maximise expansion of progenitor cells on a scale large enough to be suitable for human trials. This project focuses on determining whether cell cycle-mediated phosphorylation of Ngn3 controls endocrine pancreas cell differentiation and whether supplying a non phosphorylatable form of Ngn3 by RNA-mediated transfection will drive ES cell differentiation into beta cells in vitro. A next step in using our findings to enhance beta cell generation for therapeutic application will be to determine whether exogenously added chemical inhibitors of cdk kinase activity (potentially in combination with inhibitors of other proline-directed kinases such as GSK3beta) will have a similar effect as introduction of phosphomutant forms of Ngn3; development of such small molecules that have already been trialed as anti-cancer therapeutics will accelerate the take-up of our findings as a way to potentiate beta cell production.

If phospho-mutant Ngn3 (or manipulating endogenous Ngn3 protein function with small molecules) does promote endocrine cell differentiation from ES cells as hypothesised, we will determine whether the cells so generated are glucose sensititive and can reverse experimentally-induced diabetes in mouse models (within the 3 years of this grant). If functional beta cells can be generated in vitro in sufficient numbers to be transplanted into patients using this in conjunction with other methods under development by DM, we may be be in a position to begin human trails in 5-10 years.

In summary, the clear and ultimate aim of this proposal is to benefit diabetes patients, the healthcare/biotech industry, the NHS and the tax-payer by enhancing beta cell generation for diabetes therapy