Muscle stem cells (MuSC) represent a tissue specific and heterogeneous population of stem cells present in skeletal muscle tissue. During skeletal muscle repair some populations play a pivotal role, among them the most important are muscle satellite stem cells (SC). Other MuSC subpopulations contribute to muscle repair in a more indirect manner. They contribute to microenvironmental changes through the production of specific secretory molecules (myokines and adipokines). There might also be the connection between myokines’ and adipokines’ secretion and bone tissue remodeling, where adipokines potentially contribute to a deterioration of the osteoporosis process in osteoporosis patients. The cause-and-effect relationship between sarcopenia, osteoporotic progress and the role of muscle stem cells is not clearly understood.
The aim of the study was to determine weather characteristics of muscle stem cells change in the osteoporosis patients. For this reason, we compared MuSC isolated from gluteus medius from healthy elderly people and osteoporosis patients in following parameters: a colony forming unit (CFU), a population doubling time (PDT), expression of mesenchymal stem cell surface markers (CD73, CD90 and CD105), in vitro multilineage differentiation ability (adipogenesis, osteogenesis and myogenesis), expression of genes representative for specific muscle stem cell subpopulation (PDGFRα, PDGFRβ, PW1, Pax7 in CD56), expression of gene potential involved in clonogenic potential of muscle stem cells (Gremlin1) and genes involved in extracellular matrix (ECM) remodelling (MMP1, MMP3, MMP9 in TIMP3).
CFU was determined as a ratio between the number of formed colonies and the number of all seeded cells. For a PDT assessment we determined the number of cells in 2 different time intervals in the exponential growth phase. Adipocytes were detected with Oil Red O staining after 21 days of culturing in adipogenic differentiation medium. Alizarin Red S staining was used to evaluate formation of mineralized nodules to assess osteogenic differentiation potential after 21 days of culturing in osteogenic differentiation medium. For assessment of myogenic differentiation potential, cells were cultured for 7 days on 0,2 % gelatine in myogenic differentiation medium. After 7 days desmin+ multinucleated cells were detected with the immunofluorescent staining. Gene expression analysis was conducted with quantitative polymerase chain reaction (qPCR).
Final results show no difference in CFU and PDT measurements between isolated cells from osteoporosis patients and healthy control group of elderly people. However, difference was noted at the level of adipogenic and osteogenic differentiation. Cells isolated from osteoporosis patients show higher degree of both adipogenic and osteogenic differentiation ability. These results indicate a possible dual role of MuSC in the osteoporosis process. Additionally, differences between groups were detected in the gene expression analysis. In the healthy control group, the majority of isolated colonies belong to subpopulation PW1+/Pax7- interstitial muscle stem cells (PIC), which is completely absent in the group of osteoporosis patients. Furthermore, a difference was noted in expression of genes involved in ECM remodelling. Lower expression of TIMP3 and MMP1 genes were found in the group of osteoporosis patients compared to the healthy control group. On the other hand, higher expression of MMP3 and MMP9 genes were detected in the group of osteoporosis patients compared to the healthy control group.
To conclude, there is no significant difference in MuSC CFU and PDT between a group of healthy elderly people and patients with osteoporosis. However, higher level of adipogenic and osteogenic differentiation of MuSC were present in osteoporosis patients, which may indicate a dual role of MuSC in this disease. Difference at the level of myogenic differentiation was neither proved nor rejected. Nevertheless, there was a difference in subpopulations of MuSC isolated from osteoporosis patients and in the expression of genes involved in ECM remodelling. PIC subpopulation, which represents 50 % of isolated colonies in a group of healthy subjects, is absent in the group of osteoporosis patients. Differences in expression of genes involved in ECM remodelling, indicate higher expression of MMP3 and MMP9 and lower expression of TIMP3 and MMP1 in osteoporosis patients compared to the healthy group.
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