The comparison of human skeletal stem cell markers expression between primary mesenchymal stem/ stromal cells from different tissues of the knee joint of post mortem donors

Mezenhimske matične/stromalne celice (MSC) so heterogena skupina celic, ki so sposobne diferenciacije v adipocite, hondrocite in osteoblaste v pogojih in vitro, v določenih primerih tudi transdiferenciacije v celice ektoderma in endoderma. Za identifikacijo pomembne lastnosti MSC so še adherenca na plastiko, izražanje pozitivnih celičnih označevalcev CD105, CD73 in CD90 in odsotnost negativnih označevalcev CD45, CD34, CD14 ali CD11b, CD79α ali CD19 in HLA-DR. Glavni vir izolacije MSC je kostni mozeg, najdemo pa jih lahko tudi v ostalih tkivih. Zaradi sposobnosti diferenciacije in imunomodulatornega učinka so zanimivo področje raziskav. Podskupino MSC predstavljajo skeletne matične celice (SSC), ki jih najdemo le v skeletnem tkivu. Odkrita subpopulacija humanih SSC (hSSC) s celičnimi označevalci PDPN+ CD146- CD73+ CD164+ se lahko diferencira le v celice kosti, hrustanca ter strome in zato predstavlja ciljno skupino celic za namene zdravljenja degenerativnih bolezni skeleta. Namen magistrske naloge je bil določiti izražanje izbranih celičnih označevalcev PDPN, CD146, CD73 in CD164, ki so bili identificirani kot označevalci hSSC, v primarnih MSC, izoliranih iz 50 vzorcev donorjev post mortem. Celice so bile izolirane iz treh tkiv v območju kolenskega sklepakostnega mozga, sinovijske ovojnice in pokostnice. Po gojenju smo iz celic izolirali RNA, vzorce z zadostno koncentracijo in kvaliteto RNA pa v koraku reverzne transkripcije prepisali v komplementarno DNA (cDNA). Te vzorce smo uporabili za merjenje izražanja genov preiskovanih celičnih označevalcev z metodo kvantitativne verižne reakcije s polimerazo (qPCR). Rezultate smo normalizirali na referenčni gen GAPDH. Preverili smo tri hipoteze: izražanje genov za označevalce hSSC se razlikuje med MSC, pridobljenimi iz različnih kolenskih in obkolenskih tkivizražanje genov za označevalce hSSC je odvisno od časa post mortemizražanje genov za označevalce hSSC je odvisno od starosti donorja post mortem. Po statistični analizi rezultatov nismo dokazali statistično značilnih razlik med izražanjem preiskovanih označevalcev med tremi različnimi tkivi. Vzorce smo razdelili v tri skupine glede na čas post mortemI. skupina ⡤ 24 h, II. skupina 24–48 h in III. skupina ⡥ 48 h od smrti do izolacije primarnih celic. Za CD146 je bilo izražanje v II. skupini statistično značilno nižje kot v III. skupini, za ostale gene pa nismo dokazali statističnih razlik. Za gen CD164 smo dokazali tudi statistično značilno negativno korelacijo med starostjo donorjev in izražanjem tega označevalca. Na podlagi teh rezultatov lahko zaključimo, da med MSC, izoliranimi iz različnih tkiv v območju kolenskega sklepa, ne obstajajo razlike v izražanju novih označevalcev hSSC. Ugotovitvi, da je izražanje CD146 manjše pri vzorcih, izoliranih v obdobju od 24 do 48 ur post mortem, ter da je izražanje CD164 večje pri mlajših donorjih, pa sta osnova za nadaljnje raziskave teh označevalcev na MSC.Mesenchymal stem/stromal cells (MSCs) are a heterogeneous group of cells capable of differentiation into adipocytes, chondrocytes and osteoblasts in vitro. In some cases, researches report of transdifferentiation of these cells into ectoderm and endoderm cells. Important properties for identifications important properties are also plastic adherence and the expression of positive cell markers CD105, CD73 and CD90, with the absence of the negative markers CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR. The main source of MSC isolation is bone marrow, but they can be found in other tissues too. Due to their differentiating properties and immunomodulatory effects, the MSCs are an interesting area of research. Skeletal stem cells (SSCs) represent a subgroup of MSCs which are found only in skeletal tissues. The discovered subpopulation of human SSCs (hSSCs) with markers PDPN+ CD146- CD73+ CD164+ can differentiate only into bone, cartilage and stroma cells and therefore represents a target group of cells for the treatment of degenerative skeletal diseases. The purpose of this master\u27s thesis was to determine the expression of the selected markers PDPN, CD146, CD73 and CD164, identified as hSSC markers, in primary cells isolated from 50 post mortem samples. Cells were isolated from three tissues in the knee joint areabone marrow, synovial membrane and periosteum. After culture-expansion, RNA was isolated from the cells, and samples with sufficient RNA concentration and purity were reversely transcribed into complementary DNA (cDNA). These samples were used to mesure gene expression of the studied markers using quantitative polymerase chain reaction (qPCR) method. Results were normalized to the GAPDH reference gene. We tested three hypothesesgene expression for hSSC markers differs between MSC isolated from different tissues of the knee joint of post mortem donors, gene expression for hSSC markers depends on the post mortem time and gene expression for hSSC markers depends on the age of post mortem donors. After analyzing the results, we did not find any statistically significant differences between the expression of the examined markers and three different knee joint tissues. The samples were divided into three groups, according to the post mortem timeI. group ⡤ 24h, II. group 24-48 h, III. group ⡥ 48h from death of donors to isolation of primary cells. For CD146, expression in II. group was significantly lower than in III. group. No statistically significant differences were found for other genes. For the CD164 gene, we discovered a statistically significant negative correlation between the age and the expression of this marker. Based on these results, we can conclude that there is no difference in the expression of the new hSSC markers between MSCs isolated from different tissues of the knee joint area. However, the findings that CD146 expression is lower in samples isolated 24 to 48 hours post mortem and that CD164 expression is higher in younger donors are the basis for further studies of these markers