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

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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 a cell 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.