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Integrated signalling networks in muscle stem cells: cell fate regulation by heparan sulfates

Final Report Summary - SATCELLOMICS (Integrated signalling networks in muscle stem cells: cell fate regulation by heparan sulfates)

The overall aim of this project was to test the hypothesis that the extracellular environment experienced by muscle stem cells affects cell fate decisions and to explore how this can impact disease and ageing. We used two different approaches to test this hypothesis.

In the first case we studied how the composition of the muscle extracellular environment changes when a muscle degenerative disease, such as muscular dystrophy, progresses. It is known that as muscular dystrophy progresses, muscle stem cells lose their regenerative capacity and this is thought by many to be due to adverse effects of the dystrophic extracellular environment rather than an intrinsic defect in the stem cells themselves. To understand how the extracellular dystrophic environment affects muscle stem cells, we first sought to understand how the composition of the extracellular dystrophic environment changes with progression of the disease. We collected muscles from dystrophic mice and their non dystrophic siblings at the age of 3 months - when the disease has already shown typical features of muscular dystrophy but muscle function is still moderately preserved - and at the age of 7.5 months when muscle function is already compromised. We developed a protocol to obtain protein fractions enriched in extracellular matrix and used highly sensitive mass spectrometry to identify changes in the composition of the extracellular environment that are associated with muscle dystrophy at both ages analysed. With this approach we identified a class of proteins that were not previously known to play a role in the pathogenesis of muscular dystrophy, which accumulate in dystrophic muscle as the disease progresses and that have the potential to dramatically affect muscle stem cells. We tested this possibility by treating muscle stem cell cultures with synthetic compounds that mimic the action of the proteins that we found accumulate in dystrophic muscle over time and indeed we observed that muscle stem cell homeostasis and fate decisions are dramatically affected by these compounds. These data strongly support our original hypothesis that muscle stem cell function is compromised in muscular dystrophy due to the accumulation in the extracellular environment of proteins that negatively regulate muscle stem cell function.

In the second case we have studied how global gene expression changes in muscle stem cells exposed to different environments. We knew from previous studies that when muscle stem cells are isolated from their environment and cultured in a plastic Petri dish, they retain the capacity to differentiate into muscle cells and fuse to form muscle fibres. However, cells grown isolated in a Petri dish appear slightly different compared to cells grown adjacent to the muscle fibres that is their native niche of residence in the musculature. To understand how the muscle fibre niche affects muscle stem cell identity and properties, we cultured muscle stem cells for the same amount of time either as isolated cells in petri dishes or attached to their native muscle fibres. We then collected these cells at two subsequent time points carefully selected to be around the moment when we know muscle stem cells make important decisions regarding their destiny: either turn on genes that make up typical muscle proteins and generate muscle fibres by cell fusion (this process is called terminal differentiation and fusion) or turn down the muscle genes and return to be quiescent progenitors (this process is called self-renewal). We collected cells from isolated cultures and from fibre cultures at these two time points, extracted the total RNA from these cells and quantified the expression levels of all their genes, since knowing how much each gene is expressed is a good tool to understand what cells are doing, namely: their identity, their activity and the fate they are choosing to undergo. We used bioinformatics to compare gene expression in muscle stem cells that were grown isolated in a Petri dish with muscle stem cells that where grown attached to their native niche and identified many groups of genes belonging to the systems that control cell-environment communication (called signalling pathways) and, to some extent, genes belonging to cellular functions involved in cell proliferation and differentiation, that are differentially expressed in the two gene sets analysed.

Overall, our results demonstrate that the extracellular environment controls muscle stem cell homeostasis and fate decisions and thus, muscle stem cell regenerative capacity can be manipulated (e.g. boosted in muscle degenerative disorders or in ageing) by simply altering the composition of the extracellular environment, potentially with a targeted pharmacological approach. For example, in our muscular dystrophy case study, we have identified protein targets that accumulate in dystrophic muscle with disease progression and prevent muscle stem cell differentiation. Synthetic compounds that target the molecular processes regulated by these proteins already exist and some of them have already been proven safe in clinical trial settings. Our study provides the foundation for development of a wholly new approach to the treatment of muscular dystrophy and, potentially, other muscle disorders and muscle trauma, with the potential to dramatically improve the quality of life of many patients worldwide.

In addition to identifying and describing the molecular basis of a novel therapeutic strategy for muscle disorders, this project has a wider socio-economical impact involving several stakeholders. Firstly, researchers (in Academia and industry) involved in stem cell research and in muscle research will benefit from the proteomics and transcriptomics data that we have generated and that will be deposited in open source databases and shared with the scientific community alongside the two publications that will be produced from the work carried out in this project (see next section for dissemination details). These “big data” resources, which already represent an essential asset of the research group I have built while carrying out the research project of my fellowship, will provide many other researchers in related fields with extremely useful data that can be reanalysed using different bioinformatics approaches, correlated to other datasets, to clinical data, etc. to generate novel testable hypotheses on the biology of muscle stem cells in health and disease. Indeed, the proteomics data have been already used in collaboration with the group of Prof Andy Jones in the context of the development of a bioinformatic suite to analyse and plot proteomics data (mzqLibrary, Qi et al., submitted). Another pathway to impact that Dr Pisconti is currently pursuing is the exploitation of what we have learned about the way the environment affects muscle stem cell biology to improve the current muscle stem cell culture systems. Muscle stem cells from either rodents or humans cannot be maintained in culture for more than 3-5 passages due to occurrence of either spontaneous differentiation or cell senescence. Addition to the culture medium of and excess of growth factors – e.g. purified FGF2, chick embryo extract or high concentrations of serum – is the strategy often followed to promote proliferation and inhibit differentiation. However, an excess of growth factors often leads to premature senescence. If we could develop cell culture systems that reduce or even eliminate the amount of serum and growth factors in the culture medium by targeting specific signalling pathways in a reproducible way using synthetic compounds, we could achieve at least two important goals with highly valuable socio-economic impact: (a) the use of animals for muscle stem cell research would be dramatically reduced, (b) muscle stem cell cultures could be successfully used as a tool for preclinical tests and for basic research. Our comparison of global gene expression analysis between cells cultured isolated in Petri dishes, which quickly differentiate and or/senesce, and cells cultured in association with their native niche, which maintain their proliferative potential for a longer time, has led us to identify novel regulation mechanisms of key gene networks involved in muscle stem cell differentiation and senescence.