Final Activity Report Summary - SPAM (Stem cell Potency and the Apoptotic Machinery: Do apoptotic genes affect stem cell differentiation?) This project re-located a European Union citizen back to the European research area after a period of ten years training and working in the United States (US). The researcher was an expert in the field of programmed cell death, called apoptosis, which is a mechanism of cellular suicide found in all multicellular organisms. This form of cell death occurs when proteins, often called the 'death machinery', are activated within a cell. There are many signals that trigger this activation; some occur during normal development and act to remove cells which are no longer needed, some are produced by stresses and remove damaged cells that are unable to perform their function and others are generated by cancer-causing genes and lead to the death of cells that could otherwise cause cancer. Many of the proteins that make up the 'death machinery' were identified and it appeared that many of them, while vital for apoptosis, led double lives and were also keys for other cellular processes. Interestingly, some of the 'death proteins' appeared to be important for the differentiation of stem cells, although the way they acted and why they did not kill the stem cells remained unknown. Understanding the regulation of the apoptotic 'death proteins' and how they could induce a very different cell fate, namely differentiation, without killing the cell was a fundamentally important question. But answering this question might also have an important social impact. The median age of the population of the first world is high and rising. Consequently, the burden of degenerative diseases is becoming a significant problem in health care, with important implications for mortality and morbidity and imposes an economic price as health care costs rise. In recent years regenerative medicine, which includes stem cell therapies, has emerged as potential new strategy for combating degenerative conditions. However, translating stem cell-based approaches into widely available therapy requires an understanding of basic stem cell biology that allows manipulation of stem cells in order to maximise their therapeutic potential while minimising the risks associated with their transplantation, which is a level of understanding that we currently lack. Initially, we investigated a number of different stem cells including mouse embryonic stem cells, human adult mesenchymal stem cells and a series of stem cell-like cell lines. While several different experimental models gave promising data, the most robust data was obtained using a mouse cell line that behaved like a muscle progenitor cell and differentiated into multinucleated cells called myotubes. Within a tissue myotubes progressed to form the mature muscle fibre. The normal fusion of progenitor cells into myotubes during skeletal muscle regeneration and differentiation was a complex process, while the mechanisms for this remain poorly understood. Nevertheless, caspase-3, a cysteine protease that usually induces apoptosis, appeared to be important for this differentiation. One key question was how caspase-3 was activated during differentiation; was there a novel pathway that activated caspase-3 or did its activation rely on other 'death proteins' of the apoptotic process? We took several different approaches to answer this question, using gene overexpression, small intereferring ribonucleic acids (RNAs) and pharmacological inhibitors; all the approaches implicated a known apoptotic pathway in the differentiation process. This was a remarkable finding, since a large body of evidence supported the idea that once this apoptotic pathway was activated the cell was doomed to die. Here, we showed that, despite the activation of this pathway, the cell not only survived, but also needed this pathway to undergo differentiation. By the time of the project completion we were investigating the mechanism that allowed for the muscle cells to tolerate activation of a normally lethal pathway.