Characterisation of adult skeletal muscle-derived stem cell subpopulations in osteoporosis patients

Mišične matične celice (MuSC) predstavljajo tkivno specifično in heterogeno populacijo matičnih celic v skeletno-mišičnem sistemu. Osrednjo vlogo pri regeneraciji poškodovanih mišičnih vlaken pripisujemo satelitnim mišičnim matičnim celicam (SC), obstajajo pa tudi druge podskupine MuSC, ki so v regeneracijski proces vključene posredno preko izločanja različnih sekretornih molekul, ki preoblikujejo mikrookolje na mestu poškodbe. Poleg tega pa naj bi preko izločanja različnih miokinov in adipokinov MuSC vplivale tudi na kostno tkivo, pri čemer naj bi večja adipogena sposobnost MuSC prispevala k razvoju in/ali poslabšanju osteoporotičnega procesa pri bolnikih z osteoporozo. Natančna vzročno-posledična povezava med sarkopenijo, osteoporozo ter vlogo MuSC še ni poznana. Namen magistrskega dela je bil ugotoviti, ali se lastnosti MuSC razlikujejo med skupino zdravih starejših preiskovancev in skupino bolnikov z osteoporozo. V ta namen smo MuSC iz mišic osteoporoznih in zdravih starostnikov primerjali po naslednjih parametrih: sposobnost tvorbe kolonij (CFU), čas populacijskih delitev (PDT), prisotnost površinskih označevalcev mezenhimskih matičnih celic (MSC) (CD73, CD90 in CD105), sposobnost adipogene, osteogene in miogene diferenciacije ter v izražanju nekaterih genov, povezanih z različnimi podskupinami MuSC (PDGFRα, PDGFRβ, PW1, CD56, Pax7), gena, povezanega s klonogeno sposobnostjo MuSC (Gremlin1) in nekaterih genov, vključenih v preoblikovanje zunajceličnega matriksa (ECM) (MMP-1, MMP-3, MMP-9, TIMP-3). CFU smo določili kot razmerje med številom nastalih kolonij in številom vseh nasajenih celic posameznega vzorca, za določitev PDT smo določili število celic v dveh časovnih intervalih v času logaritemske faze rasti celic. Adipogeni potencial smo določili s histokemijskim barvanjem nastalih adipocitov z barvilom Oil Red O po 21-dnevnem gojenju celic v adipogenem diferenciacijskem gojišču. Osteogeni potencial smo določili s histokemijskim barvanjem nastalih mineraliziranih nodulov z barvilom Alizarin Red S po 21-dnevnem gojenju v osteogenem diferenciacijskem gojišču. Miogeni potencial pa smo določili z imunohistokemijskim barvanjem nastalih desmin+ diferenciaranih mišičnih celic po 7-dnevnem gojenju na 0,2-% želatini v miogenem diferenciacijskem gojišču. Analizo izražanja genov smo izvedli s kvantitativno verižno reakcijo s polimerazo v realnem času (qPCR). Pridobljeni rezultati kažejo, da ni razlik v CFU in PDT med vzorci izoliranimi iz pacientov z osteoporozo in zdravimi starejšimi preiskovanci. So pa razlike prisotne na nivoju adipogene in osteogene diferenciacije. Višjo stopnjo tako adipogene kot tudi osteogene diferenciacije izkazujejo kolonije, izolirane iz vzorcev pacientov z osteoporozo, kar nakazuje na potencialno dvojno vlogo MuSC pri osteoporotičnem procesu. Prav tako na razlike med skupinama nakazuje analiza izražanja genov. V skupini oseb zdravih starejših preiskovancev prevladuje podskupina PW1+/Pax7- intersticijskih mišičnih matičnih celic (PIC), ki je v skupini oseb z osteoporozo odsotna. Prav tako so razlike prisotne v izražanju genov, vključenih v preoblikovanje ECM. Rezultati nakazujejo na nižje izražanje gena TIMP3 in MMP1 v skupini pacientov z osteoporozo v primerjavi z zdravimi starejšimi preiskovanci, na drugi strani pa je pri pacientih z osteoporozo nekoliko višje izražanje genov MMP3 in MMP9. Na osnovi rezultatov lahko zaključimo, da se izolirani vzorci MuSC med osebami z diagnosticirano osteoporozo in zdravimi starejšimi preiskovanci v CFU in PDT statistično ne razlikujejo. Pride pa do razlik na nivoju adipogenega in osteogenega diferenciacijskega potenciala MuSC, ki se pri osteoporozi poveča, kar nakazuje na dvojno vlogo MuSC pri tej bolezni. Razlik v sposobnosti miogene diferenciacije nismo uspeli niti dokazati niti ovreči. Dokazali pa smo razlike v deležu podskupin MuSC in v izražanju genov, povezanih s preoblikovanjem ECM med obema skupinama preiskovancev. V skupini bolnikov z osteoporozo je odsotna podskupina PIC, ki v skupini zdravih preiskovancev predstavlja 50 % vseh izoliranih kolonij. Razlike v preoblikovanju ECM pa nakazujejo na višje izražanje gena MMP3 in MMP9 pri pacientih z osteoporozo.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