Proliferation and Clonal Analysis of Muscle Derived CD133+ Stem Cell Subpopulations Define a Depletion of Mesenchymal Population in DMD Patients

In the muscle exist different stem cell populations that are able to participate in muscle regeneration. In patients with Duchenne Muscular Dystrophies (DMD) there is a depletion of stem cell populations due to continuous regeneration cycles. Moreover, recent findings supported the notion that heterogeneity is a hallmark of stem cells. It is known that these cell populations express a variety of surface markers including CD29, CD56, CD44, CD90, CD105, STRO–1. Here we have focused our attention on the so-called MDSCs (Muscle-Derived Stem Cells) that can be rather considered as satellite precursors. In the past our group focused its attention on a stem cell population expressing CD133 antigens on its surface. The analysis of muscle-derived CD133+ cells showed that they have been characterized for their regenerative potential in vivo, as well as their ability to repopulate the satellite cell niche in healthy muscles. The cells obtained from muscle biopsies of DMD and orthopaedic patients, were sorted for specific subpopulations and then cloned. Original subpopulations and their clones were characterized for proliferation and differentiation behaviour, as well as for the expression of surface markers by flow cytometry. However, we found in the CD133+ MDSCs a phenotypic heterogeneity in the presence of some surface antigens: in the same cell population we found an oscillation of expression of endothelial, myogenic and mesenchymal markers. Based on these assumptions we sorted some cell subpopulations obtained from muscles of healthy patients and DMD children: mesenchymal-like cells (CD133+ CD73+ CD44+ CD29+ CD34- CD45-); endothelial-like cells (CD133+ CD90+ CD146+ CD31+ CD45-) and myogenic-like cells (CD133+ CD56+ CD45- CD34+/-). These subpopulations were cloned and analyzed for their capacity to proliferate and differentiate into the endothelial and myogenic lineages. In these experiments we found stem cell properties, particularly in the mesenchymal-like CD133+ clones. In fact these cells show clonogenic potential and well differentiate into endothelial and myogenic cells. Instead we found that this subpopulation is compromised in DMD patients both in terms of percentage of expression, proliferative and differentiative capacities. In conclusion, our study showed that muscle-derived CD133+ cells represent a heterogeneous population of mesenchymal, myogenic and endothelial progenitors and that the first population appears to be the most compromised in DMD patients. Further experiments are needed in order to understand whether CD133+ mesenchymal population could be a good candidate for clinical applications in muscular dystrophies.