Mitochondrial function and transition from glycolysis to oxidative phosphorylation in differentiating induced pluripotent stem cells

Induced pluripotent stem cells (iPSC) can be derived from human tissues such as the skin and have two characteristic properties. Firstly, we can grow them indefinitely in the laboratory in either small or large scale and secondy, by use of appropriate methods, they seem to be able to differentiate into almost all of the cell types found in the adult body. At a first glance this makes them similar to human embryonic stem cells which have been hailed as an attractive resource for regenerative procedures that involve transplantation of specific differentiated cells into human patients. Embryonic stem cells are derived from individual Human embryos so any cells we produce form these cell lines are likely to be rejected by the body's immune system. iPSC should not suffer from this problem if the cells we make from them are transplanted back into the same individual from which they were made but there is a potentially greater advantage of iPSC namely the creation of 'disease in a dish' models. This relies upon making an iPSC line from a patient suffering from a genetic disease then differentiating that iPSC line to the type of cells affected by the disease so we can see what problems arise and how we might be able to fix them. All of this relies upon the hiPSC line being able to differentiate into cells that are functionally identical to those found in the body. Available evidence and our pilot data suggest that although they may produce cells that look similar to adult cells (such as those of the bone marrow or the nervous system), these may not work as effectively as the genuine article therefore in order to realize the promise of iPSC technology, we have to find out why they are defective and develop ways to fix these problems. Our pilot data suggest that problems with mitochondria, the energy suppliers of the cell, may be a significant reason why iPSC and their differentiated problem could be dysfunctional. Mitochondria exist as distinct structures within the cell and while some of their components are produced from their own indepdent genes, the majority of their structure and function is controlled by genes residing in the cells nucleus. The ways in which mitochondria function are quite different in various cell types; for example hESC don't use them to generate energy very much but differentiated cells derived from hESC do. The human cells (somatic cells) we reprogram to make hiPSC fall into the latter category but it seems that the reprogramming is incomplete and hiPSC may still try to control their mitochondria in similar ways to the original somatic cells. This is potentially bad for the survival of the hiPSC but even worse, they may not be able to regulate mitochondria properly on differentiation. In view of this our objectives are to compare the mitochondria of hiPSC and hESC and then differentiate them to three cell types with differing energy requirements (and therefore different mitochondrial regulation). We will compare the expression of genes involved in mitochondrial function, analyse their control mechanisms and attempt to explain why this might not work optimally. Once this understanding is gained, we will be able to develop means to solve these problems and produce hiPSC with fullly functional mitochondria although this in turn may allow us to improve other aspects of iPSC function that rely upon completion of reprogramming. The successful execution of this project will be enormously beneficial not only to ours and other groups investigating iPSC biology but it will also promote development of disease treatments using these cells which will provide significant social and economic impact.

Our pilot data suggest that the mitochondria of induced pluripotent stem cells (iPSC), may not function in an analogous manner to those of human embryonic stem cells (hESC). There are similarities in terms of mitochondrial genome copy number and mitochondrial mass in both hiPSC and hESC but mitochondrial membrane potential and production of superoxide are significantly lower in iPSC. In addition, several nuclear encoded genes central to mitochondrial function, are mis-expressed compared to hESC both in the pluripotent cells and their differentiated progeny. Crucially, the extent to which mis-expression occurs varies between isogneic iPSC lines suggesting that the causes of these differences may be epigenetic and implies that the epigenetic reprogramming originally required to generate the iPSC from dermal fibroblasts may have been incomplete. If this is a general phenomenon with iPSC, they could be a poor source of cells for regenerative medicine or disease modelling so we plan to analyse mitochondrial parameters such as morphology, membrane potential and expression levels of genes associated with mitochondrial function across a range of hiPSC and hESC lines and somatic cell types we can produce from them. This point is important since hiPSC and hESC do not use the oxidative phosphorylation ATP generation system intrinsic to the mitochondria to the same extent as many somatic cells. It is therefore important to determine that hiPSC are capable of generating the numbers of mitochondria required by the somatic cell types they differentiate into and that these mitochondria function in the same way as we would expect of the progeny of hESC. We will analyse mitochondrial numbers and morpholgy using established histochemical staining protocols but the key steps of this proposal will be analyses of gene expression using microarrays and epigenetic architecture of nuclear encoded mitchondrial associated genes using chromatin immunoprecipitation.

Since the first publication detailing the derivation of Human iPSC in 2007, nearly two thousand articles have appeared in the scientific literature describing new methods to generate these cells and the attempts to develop protocols for their differentiation into somatic cells with potential clinical uses. Many studies of this type were already in progress using embryonic stem cells but the enormous advantage of iPSC is that they are isogenic with the individual from whom they are derived. If however they do not behave in the same predictable manner as ESC, they may not be so useful either as a source of cells in regenerative medicine or as in vitro models of human diseases. This proposal aims to determine if mitochondria in iPSC behave in a similar fashion to those of ESC and to explain any differences in terms of incomplete epigenetic reprogramming or other factors that contribute to mitochondrial function. Moreover, if the dysfunction results from problems with epigenetic regulation of nuclear encoded genes associated with mitochondrial activity, we hope to be able to 'repair' this so the mitochondria behave in a more normal fashion. In view of this, the most immediate group to benefit from our research will be the large number of academic groups who are working with iPSC as we have described in the academic beneficiaries section however the potential benefits of this work extend beyond the boundaries of purely academic research. Clinical trials are beginning with embryonic stem cell derived somatic cells so it is highly probable that iPSC represent the next logical step in development of stem cell based therapies if current problems with their growth and differentiation parameters can be overcome. A recent article in Nature Biotechnology (Nat Biotech 2010, 28(6):535-536) further underlines considerable industrial interest in pluripotent stem cells as sources of clinically useful tissue thus the results of this project are highly likely to be beneficial to the future utility of iPSC in commercial regenerative medicine. Regenerative medicine is likely to have a highly significant economic impact upon nations with the scientific and financial capabilities needed to implement its development. To set this statement in context, the USA currently spends $175.8 billion annually for the treatment of diabetes and myocardial infarction, two conditions which are primary targets for stem cell based regenerative therapies. The United Kingdom is a leading contributor to Stem Cell related research and if the results of this project allow the development of clinically useful iPSC, and these can undergo successful translation into commercial cell therapy products, the economic benefits to the UK will be considerable. The social impact of such developments cannot be understated. If effective treatments for the two conditions mentioned above and others arise, even indirectly from the results of this project we will have contributed in an effective way to improving the health and quality of life of significant numbers of individuals both in the UK and in other countries.