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Origin of new beta cells during pregnancy using PCLT and TIS microscopy

Reference	BB/K018868/1
Principal Investigator / Supervisor	Dr Michael Khan
Co-Investigators / Co-Supervisors	Professor David Epstein, Professor Nasir Rajpoot
Institution	University of Warwick
Department	School of Life Sciences
Funding type	Research
Value (£)	872,535
Status	Completed
Type	Research Grant
Start date	01/11/2013
End date	31/10/2017
Duration	48 months

Abstract

Functional beta-cell mass (FBM) doubles in a few weeks of mouse or human pregnancy. The contribution of different sources to FBM expansion in pregnancy is still unclear. Our earlier published work [1] demonstrates that during pregnancy about half of new beta-cells arise from non-beta-cell precursors, the remainder coming from replication of existing beta-cells. The aim of this project is to clarify the identity and role of precursor cells (of which there could be more than one cell type). We will employ Pulse-Chase Lineage Tracing (PCLT) in two different experiments, using mice with transgenes CAIICreERT and ElastaseCreERT to label duct and acinar cells respectively. Our collaborator, Pedro Herrera of Geneva, will be doing PCLT for delta-cells and sending us tissue sections for analysis. We will see whether ductal, acinar or delta precursors are major contributors to FBM expansion during pregnancy. Analytical power is enhanced by coupling PCLT with a new microscopy platform, Toponome Image System (TIS), that can co-locate 40 or more different proteins in the same pixel of the same intact tissue section. The progeny of labelled cells are marked, and tracked from time point to time point, with the percentage contribution to beta-cell neogenesis unambiguously attributed. Imaging of the lineage label alongside detailed cell morphology, plus molecular co-expression patterns, formed using an additional 30 differentiation markers, will reveal the differentiation route for formation of new beta-cells. We will develop novel analytical tools based on mutual information, maximal information coefficient (MIC) and nonlinear manifold learning. Tracking of signatures from molecular co-expression of protein markers will reveal ancestors of beta-cells. This new knowledge will advance understanding of beta-cell formation and demonstrate the role of non-beta-cells in FBM expansion during pregnancy. Bibliography: [1] S. Abouna, et al., Organogenesis, vol. 6, pp. 125-33, 2010.

Summary

During normal human pregnancy, the mother's need for insulin is suddenly doubled. To fulfil this need, her body creates additional insulin-producing cells--these are called beta cells. This project's question is: Where do the additional cells come from in normal pregnancy? Understanding the body's production of new beta cell is an important biological problem in its own right. This understanding is also important for the treatment of diabetes, because a deep understanding of diseased physiology rests on understanding normal physiology. Experiments cannot be done on human subjects. We use mice, whose physiological response to pregnancy is fortunately similar to that of humans. For example, during pregnancy, the mass of beta cells more than doubles in mice, and the same is true for humans. This remarkable increase takes place over only a few days in a mouse, which is convenient for our study. This project's question can be recast: During pregnancy, which types of cells transform themselves into beta cells? Until recently, the accepted wisdom on this question was provided by a wonderful experiment carried out by Melton and Dor. They start with transgenic mice, that is, mice whose DNA has been altered in a certain way. This alteration has little effect on the physiology of the mouse until the mouse is injected with a certain hormone (tamoxifen), at which point the altered DNA starts to produce, but only in one particular type of cell--in the Dor-Melton case, only in beta cells--a certain protein (HPAP) visible under the microscope after staining. The cell is said to be "labelled by HPAP". What makes the experiment so informative is that, when an HPAP-labelled cell divides (replicates), then the cells resulting from division are also labelled. A labelled cell must have had labelled ancestors, and an unlabelled cell must have had unlabelled ancestors. This gives a mechanism for tracking the ancestry of a cell, which works whether or not the cell changes to acell of different type. Melton and Dor showed that, under circumstances that do not occur in nature (surgical removal of part of the pancreas), new beta cells come only from existing beta cells, via the usual process of cell division. Our group recently repeated the Melton-Dor experiment using normal pregnancy to create a large but normal demand for additional insulin. Under these conditions, the same labelling technique leads to a very different conclusion--about half of the new beta cells do NOT arise through replication of existing beta cells, but come from somewhere else. Our experiment was not designed to show where the new beta cells did come from, even though we could be sure that some came from division of existing beta cells. Our objective now is to determine with precision the various types of ancestors of new beta cells in pregnancy. This would be a major step towards understanding insulin-deficiency in diabetes, and may lead to effective treatment. It may become possible to cultivate outside the body, in large numbers, ancestor cells discovered during our project and beta cells that the ancestor cells generate. It is already standard treatment in both type 1 and type 2 diabetes to transplant into the body of a diabetic beta cells that are harvested from road traffic accident victims. However, a supply of beta cells from such a source can supply only a tiny fraction of the need. The University of Warwick's robotically controlled TIS microscope, the only one of its kind outside Germany, allows us to deduce, from the expression of different proteins at different time points, how a cell changes while becoming a beta cell. TIS detects 40 proteins at the same place in the same tissue section, instead of the 2 or 3 detectable through conventional microscopy. This great power of TIS will enable us to identify which of many suggestions are correct as to the identity of ancestors of beta cells.

Impact Summary

Tissue plasticity and adaptive growth are areas of profound relevance to mammalian physiology. Disorders of these processes lie at the heart of some of the most important and prevalent diseases of man and animal, eg diabetes, a serious and widespread disease. Our research is on mice--one cannot research on humans. Even though there are differences, rodent and human systems are close in normal physiology of pregnancy. Recent studies [1] show that in human, as in mouse, pregnancy [2], neogenesis contributes a significant proportion of new beta cells required by the mother's increased insulin requirements. Almost all types of diabetes are associated with inadequate beta-cell mass. To cure a diabetic requires adding to the patient's supply of functioning beta-cells. A standard modern approach is to implant islets from cadavers. However, this can meet only a tiny proportion of demand. If we understood the cellular origin of new beta-cells in adult life, the same processes could be encouraged in the diabetic patient with drugs, or cells could be manufactured outside the body and implanted. Currently islet transplants survive rejection for around 18 months, which is acceptable as an alternative to diabetes, and provide effective treatment, the only real limitation being lack of suitable transplant material. Survival times for implants are rapidly increasing under the influence of intensive research. Pharma would be a major beneficiary, through manufacturing and selling beta-cells. Novo Nordisk has a major facility near Copenhagen, devoted to the creation of beta cells and to beta-cell therapy. All big pharma is similarly interested. In fundamental research it is impossible to be specific about timelines. However, a mouse beta cell precursor identified in this project would be rapidly validated in man, in view of the worldwide interest and existing infrastructures. Trials to expand these precursors and generate beta cells on a large scale would also follow rapidly.We think the time-lag would be 5 to 10 years. A particular beneficiary in the UK of a successful outcome for our research would be the NHS, currently spending £14 billion per year on diabetes (http://www.diabetes.co.uk/cost-of-diabetes.html). Training: Both wet and dry PDRAs will make contact with industry at our workshops, and when pharma visit our labs. Their experience at our workshops and the training they receive while on visits to Bielefeld and Geneva will greatly enhance their employment prospects. Weekly attendance at our multidisciplinary meetings will draw their attention to the importance of interactions with other disciplines and they will be taught the relevant details of the other disciplines by face-to-face tuition from the Investigators. Presentations to our team, to University seminars and to national and international conferences will develop their skills, making them more employable. Our project will have a beneficial effect on academics from Warwick and elsewhere. Schubert, the inventor of TIS, is working on a benchtop TIS system, making it eminently suitable as a diagnostic and research tool. Our methods of analysis and our software will be provided with such machines. Our ideas will also be useful whenever similar multi-channel studies are undertaken, and are not confined to TIS. The impact would be felt in laboratories worldwide. For example, our computer programs automate segmentation of images of tissue sections into cells, saving hours of intensive and uninteresting labour, simultaneously greatly improving accuracy. Through WISDEM (see Google), we will organize two meetings in Year 1, one for regional medical professionals and one aimed at the interested public, to explain our plans. In Year 3 we will again have two similar meetings to report on progress. [1] A. E. Butler, et al., Diabetologia, vol. 53, pp. 2167-76, 2010. [2] S. Abouna, et al., Organogenesis, vol. 6, pp. 125-33, 2010.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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