Primerjava izražanja označevalcev diferenciranih in nediferenciranih mezenhimskih matičnih/stromalnih celic, izoliranih iz različnih delov sinovije kolka

Sluga, Nastja (author), Zupan, Janja (mentor), Stražar, Klemen (comentor)

Mezenhimske matične/stromalne celice (MSC) so redka, neraziskana in izjemno raznolika skupina postnatalnih celic, iz katerih se razvije mišično-skeletni sistem. Nahajajo se v številnih tkivih, pri čemer se med seboj razlikujejo po diferenciacijskemu potencialu, morfologiji, velikosti in izražanju posameznih označevalcev. Imajo imunosupresivne lastnosti in veliko sposobnost regeneracije tkiv ter velik potencial za zdravljenje številnih avtoimunskih, vnetnih in degenerativnih bolezni ter poškodb tkiv. Kljub velikemu potencialu za njihovo uporabo v klinični praksi, še zmeraj veliko težavo predstavlja ohlapna definicija in pomanjkanje specifičnih označevalcev za identifikacijo MSC. Te problematike smo se v naši raziskavi dotaknili tudi mi. Namen raziskave je bil ugotoviti razlike v izražanju genov za označevalce diferenciranih in nediferenciranih MSC iz treh različnih delov sinovije kolka in trabekularne kosti ob kolku. Meritve smo izvedli na 113 vzorcih MSC, 11 različnih pacientov. Tkivni vzorci so bili pridobljeni med rutinsko artroskopijo kolka na Ortopedski kliniki Univerzitetnega kliničnega centra v Ljubljani. Med posegom so bili odvzeti vzorci sinovije na treh predelih kolka ter vzorec trabekularne kosti. Iz tkivnih vzorcev so izolirali primarne MSC, ki so jih v pogojih in vitro izpostavili adipogenemu in osteogenemu diferenciacijskemu mediju.Vzporedno so bili vzorci izpostavljeni tudi bazalnemu gojitvenemu mediju. Ti so nam služili kot kontrola. Po gojenju so iz celic izolirali RNA in jo v procesu reverzne transkripcije prepisali v komplementarno DNA. Ta vzorec smo uporabili za določanje izražanja genov za tri adipogene označevalce ADIPOQ, PPARG in FABP4 ter štiri osteogene označevalce OC, ALP, COL1A1 in RUNX2. Magistrska naloga pa vključuje tudi določitev izražanja štirih novih označevalcev MSC, katerih metodo kvantitativne verižne reakcije s polimerazo v realnem času (qPCR), smo predhodno zoptimizirali. To so označevalci, ki jih kodirajo geni PDPN, CD73, CD146 in CD16 ter predstavljajo imunofenotip humanih skeletnih matičnih celic. Izražanje genov za označevalce smo merili z metodo qPCR. Preizkusili smo tri hipoteze, ki predpostavljajo, da je izražanje genov za adipogene, osteogene in nove označevalce v MSC, glede na mesto odvzema tkiv v kolku, za izolacijo teh celic enako. Pri adipogenih in osteogenih označevalcih, smo preverjali tudi razlike v njihovem izražanju med diferenciranimi MSC in njihovimi kontrolami, da bi ugotovili, če nam ti geni služijo kot učinkoviti označevalci adipo- in osteo-geneze. Pri vseh genih za adipogene označevalce lahko potrdimo uspešno adipogeno diferenciacijo, saj so se bolje izražali pri vzorcih MSC, ki so bili izpostavljeni adipogenemu gojitvenemu mediju kot pri njihovih kontrolah. Prvo hipotezo smo sprejeli, saj statistično značilne razlike v izražanju genov adipogenih označevalcev, glede na mesto odvzema tkiv, nismo uspeli dokazati. Drugo hipotezo delno sprejeli, saj smo dokazali statistično značilno razliko v izražanju osteogenih genov glede na mesto odvzema samo za dva gena, OC in COL1A1. Uspešno osteogeno diferenciacijo lahko potrdimo pri COL1A1, zato smo izražanje tega gena predlagali za označevalca osteogene subpopulacije MSC, izoliranih iz trabekularne kosti ob kolku. Ugotovili smo, da je izražanje genov za nove označevalce glede na mesto odvzema tkiv v kolku enako, zato smo tretjo hipotezo sprejeli. V MSC iz vseh tkiv se najbolje izraža CD73, najmanj pa CD146, kar je bilo tudi pričakovano, saj je CD146 negativni označevalec humanih skeletnih matičnih celic. Na podlagi rezultatov naše raziskave, predlagamo nadaljne raziskave tega področja na proteinskem nivoju.

• Language: Slovenian
• Keywords: Mezenhimske matične/stromalne celice, metoda qPCR, adipogeni označevalci, osteogeni označevalci, imunofenotip humanih skeletnih matičnih celic.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-117135
• Publication date in RUL: 25.06.2020

Secondary language

• Language: English
• Title: Comparison of differentiated and undifferentiated mesenchymal stem/ stromal cell markers expressed on cells isolated from various parts of hip synovium

Abstract

Mesenchymal stem/stromal cells (MSCs) are rare, unexplored and heterogeneous postnatal cell population. They are capable of differentiating into mature cells of muscle and skeletal system. MSCs are located in various tissues and organs, but they do not all have the same differentiation capacity toward certain linages. The difference between them is also in their size, morphology and in the expression of surface markers. MSCs have immunosuppressive properties and the ability to regenerate injured tissues. Based on these properties they have the potential for the treatment of diverse diseases. This field faces several challenges and the biggest one is the absence of specific MSCs markers. Therefore the purpose of this master thesis is comparison of differentiated and undifferentiated MSC markers expressed on cells isolated from three different parts of the hip synovium and trabecular bone. We have included113 samples of MSCs from 11 different patients in our study. Tissue samples were obtained during routine arthroscopy procedure at the Department of orthopedic surgery of University Medical center in Ljubljana. MSCs were isolated from tissue samples and exposed to adipogenic and osteogenic differentiation media. At the same time we exposed cell replicates to basal culture media. These samples served as controls. In the next step RNA was isolated from the cells reversely transcribed into complementary DNA (cDNA). We used cDNA for measuring the expression of three adipogenic markers, ADIPOQ, PPARG and FABP4, and four osteogenic markers, OC, ALP, COL1A1, and RUNX2. We also measured the expression of four new surface markers. Initially, we optimised the method of quantitative polymerase chain reaction (qPCR) for measuring their expression. These are the markers, that are encoded by PDPN, CD73, CD146, and CD164. These markers have recently been discovered as markers for self-renewing, multipotent human skeletal stem cells. Gene expression of all listed markers was measured using qPCR. We tested three hypothesis that presume the expression of genes for adipogenic, osteogenic and new MSC markers is similar in MSCs isolated from various parts of hip synovium. We also tested the differences in expression of adipogenic and osteogenic markers between samples that were treated with adipogenic or osteogenic media and controls. Since all adipogenic markers showed higher expression in treated samples we confirmed successful adipogenesis. We did not prove that the expression of adipogenic markers differs between MSCs obtained from various parts of hip, so we accepted the first hypothesis. We proved statistical difference for the expression of OC and COL1A1 in MSCs isolated from different parts of hip synovium. For osteogenic marker COL1A1 we also confirmed successful osteogenesis, therefore this gene could serve as an identification marker for osteogenic subpopulation of MSC isolated from trabecular bone. We found out that the expression of the new markers is similar betweeh MSCs isolated from different tissues. Therefore we accepted the third hypothesis. CD73 showed the highest expression in all synovial tissues. CD146 is a negative surface marker for human skeletal stem cells, and as expected the expression of CD146 was the lowest. Based on our results and findings we suggest further research on protein level.

• Keywords: Mesenchymal stem/stromal cells, qPCR method, adipogenic markers, osteogenic markers, human skeletal stem cells immunophenotype.