Primerjava osteogenega potenciala in kinetike rasti človeških mišičnih in kostnih mezenhimskih matičnih/stromalnih celic

Levstek, Tina (author), Marc, Janja (mentor), Zupan, Janja (comentor)

Mezenhimske matične/stromalne celice (MSC) so redka, heterogena skupina multipotentnih celic, ki izvirajo iz mezodermalne zarodne plasti. Nahajajo se v številnih tkivih fetusa in odraslega organizma, njihova izolacija pa je možna tudi post mortem. Izolacija MSC je dokaj enostavna zaradi njihove adherence na plastično površino, kljub temu pa vedno dobimo heterogeno kulturo celic. MSC so tkivno specifične, na njihovo rast v pogojih in vitro vplivajo številni dejavniki, kot so spol, starost, prisotnost sistemskih bolezni in poškodb pri donorju, uporabljeno gojišče in pasaža. V kulturi imajo omejen proliferacijski potencial, saj po določenem številu populacijskih delitev vstopijo v stanje senescence. Namen naše naloge je bil ugotoviti prisotnost razlik v kinetiki rasti in osteogenem potencialu med različnimi donorji ter celicami, izoliranimi iz različnih tkiv. V magistrski nalogi smo analizirali MSC, ki so bile izolirane iz skeletne mišice gluteus medius in trabekularne kosti, odvzete bolnikom z osteoporozo (OP), osteoartrozo (OA) ter donorjev post mortem brez mišično-skeletnih bolezni. Kinetiko rasti smo spremljali s kumulativnim številom populacijskih delitev, testom sposobnosti tvorbe kolonij (CFU) in časom populacijskih delitev (PDT). Osteogeni potencial smo določali s histokemijskim barvanjem z barvilom Alizarin Red S (ARS) in z merjenjem izražanja osteogenih genov s kvantitativno verižno reakcijo s polimerazo v realnem času (qPCR). Kinetika rasti je različna med vzorcema, izoliranima iz skeletne mišice in trabekularne kosti. Vzorci, izolirani iz skeletne mišice bolnikov z OP in OA, so bili sposobni tvoriti več kolonij kot vzorci iz trabekularne kosti. Sposobnost tvorbe kolonij se tekom naraščanja pasaž znižuje in tik preden večina celic vstopi v senescentno stanje, celice niso več sposobne tvoriti kolonij. Največji začetni CFU sta imela vzorca, izolirana iz bolnikov z OA, sledita vzorca iz bolnika z OP, najnižji CFU pa sta imela vzorca, izolirana iz donorja post mortem. Čas populacijskih delitev se je na začetku počasi podaljševal, preden so celice postale senescentne, pa se je zelo podaljšal. PDT vzorcev, izoliranih iz skeletne mišice, je bil krajši kot PDT vzorcev iz trabekularne kosti. Osteogeni potencial se z naraščajo pasažo znižuje in je bil pri bolnikih z OA v primerjavi z bolniki z OA in kontrolnimi vzorci višji, kljub temu pa je bil tudi pri bolnikih z OA nizek. To je verjetno tudi vzrok, da na molekularnem nivoju poteka osteogeneze nismo uspeli potrditi.

• Language: Slovenian
• Keywords: mezenhimske matične/stromalne celice (MSC), gojenje in vitro, kinetika rasti, osteogeni potencial
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107979
• Publication date in RUL: 11.06.2019

Secondary language

• Language: English
• Title: Comparison of osteogenic potential and growth kinetics of human muscle and bone-derived mesenchymal stem/stromal cells

Abstract

Mesenchymal stem/stromal cells (MSCs) are rare heterogeneous group of multipotent progenitors originating from the mesodermal germline. They have been isolated from many fetal and adult tissues as well as post mortem tissues. Isolation of MSCs is quite simple due to their adherence to the plastic surface; nevertheless, we always obtain a heterogeneous cell culture. MSCs are tissue specific. Their growth in vitro is influenced by many factors such as gender, age, the presence of systemic diseases and injuries, the medium used and passage number. In culture, they have a limited proliferation potential, since after a certain number of population doublings they enter the senescence. The aim of our research was to determine whether the growth kinetics and the osteogenic potential of the MSCs differ between cells isolated from different tissues and different donors. MSCs samples were isolated from skeletal muscle gluteus medius and trabecular bone harvested from patients with osteoporosis (OP), osteoarthritis (OA) and post mortem donors without musculoskeletal disorders. The growth kinetics were monitored by a cumulative number of population doublings, a colony forming units test (CFU) and a population doubling time (PDT). The osteogenic potential was determined with Alizarin Red S (ARS) staining and measurement of osteogenic gene expression using real-time quantitative polymerase chain reaction (qPCR). The results have shown that the ability of MSCs to form colonies decreases with passage number and that they are no longer capable to form colonies just before most of the cells enter the senescence. Samples isolated from skeletal muscle of donors with OA and OP formed more colonies than samples isolated from trabecular bone. The highest CFU had samples isolated from the patients with OA, followed by samples from the patients with OP. Samples isolated post mortem had the lowest CFU. Population doubling time slowly increased at the beginning however, it was considerably prolonged before cells underwent senescence. PDT of the samples isolated from skeletal muscle was shorter than those from trabecular bone. With passaging the osteogenic potential is decreasing and is higher in patients with OA than from patients with OP and cadavers; yet the osteogenic potential of OA samples was low. Consequently, the gene expression analysis did not confirm the osteogenesis.

• Keywords: mesenchymal stem/stromal cells (MSCs), in vitro culture, growth kinetics, osteogenic potential