The comparison of molecular marker expression and osteogenic capacity of skeletal stem cells in patients with early and end-stage osteoarthritis

Osteoartroza (OA) je kronična degenerativna bolezen, za katero je značilna obraba sklepnega hrustanca. Gre za heterogeno bolezen, s prevladujočimi vnetnimi in presnovnimi procesi, ki privedejo do razgradnje sklepnega hrustanca in spremenjene strukture kostnega tkiva. Napredovana bolezen najpogosteje prizadene kolčne in kolenske sklepe, hrbtenico ter sklepe dlani in stopal. Trenutno terapijo OA sestavljajo različni načini za blažitev bolečine in vnetja, v končni fazi bolezni pa kirurška zamenjava poškodovanega sklepa. Velike obete pri zdravljenju, in ne le zgolj lajšanju simptomov bolezni, zaradi svojih regenerativnih zmožnosti, ponujajo matične celice. Mezenhimske matične/stromalne celice (MSC, angl. mesenchymal stem/ stromal cells) so heterogena populacija multipotentnih celic, z veliko zmožnostjo samoobnove in diferenciacije v različne celične linije. Podvrsta MSC so nedavno odkrite človeške skeletne matične celice (hSSCs, angl. human skeletal stem cells), ki so ravno tako multipotentne, a za razliko od MSC tkivno specifične, saj imajo natančno določen imunofenotip (CD73+/CD146-/PDPN+/CD164+) in zmožnost preobrazbe v osteoblaste in hondrocite, tako v pogojih in vitro kot in vivo. Ker imajo pomembno vlogo pri razvoju, regeneraciji in homeostazi kostnega tkiva, veliko obetajo v zdravljenju degenerativnih sklepnih bolezni, med katere sodi tudi OA. Celične terapije z MSC, ki se trenutno uporabljajo v klinični praksi, namreč omejuje pomanjkanje specifičnih celičnih označevalcev, ki bi omogočili njihovo natančno identifikacijo. V okviru magistrske naloge smo proučevali, kako različna stopnja OA kolka vpliva na lastnosti kostnih MSC teh bolnikov, in sicer na nivojih njihove sposobnosti osteogeneze ter imunofenotipa novih označevalcev hSSC v pogojih in vitro. Najprej smo z metodo kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) izvedli preliminarno študijo na vzorcih cDNA, pridobljenih iz kostnih MSC 32 preiskovancev, s katero smo preverili, ali med bolniki z zgodnjo in pozno obliko OA ter skupino kontrolnih oseb brez OA morda obstajajo razlike v izražanju genov za nove označevalce hSSC. Na podlagi teh rezultatov smo nadaljevali s študijo na izbranih vzorcih kostnih MSC 12 preiskovancev, pri čemer smo ovrednotili njihovo sposobnost osteogeneze ter s pomočjo pretočne citometrije določili njihove imunske fenotipe za označevalce hSSC. Med skupinami bolnikov z različnima stopnjama OA kolka nismo dokazali statistično značilnih razlik v sposobnosti osteogeneze in imunofenotipu novih označevalcev hSSC njihovih kostnih MSC. Čeprav je naša raziskava prva, ki je določila lastnosti kostnih MSC na nivoju nedavno odkritih hSSC pri bolnikih z različnima stopnjama OA, pa ostajamo do rezultatov kritični in dopuščamo možnost, da bi lahko z vključitvijo večjega števila preiskovancev dobili drugačne rezultate.Osteoarthritis (OA) is a chronic degenerative disease with typical loss of joint cartilage. It is a heterogeneous disease accompanied by an inflammatory and catabolic processes that lead to decomposition of joint cartilage and changes in tissue structure. It most commonly affects hip and knee joints, spine and joints of palms and feet. The therapy of OA currently involves pain relieving and anti – inflammatory medications. When disease progresses the only treatment option is surgical replacement of the joint with the artificial prosthesis. Significant breakthrough in the treatment options are mesenchymal stem/ stromal cells (MSCs) since they possess regenerative abilities and would therefore be capable of disease-modifying not only of relieving symptoms of OA. MSCs are heterogeneous population of multipotent cells with regenerative properties and capability of multilineage differentiation. Recently discovered sub-population of MSCs are human skeletal stem cells (hSSCs), also multipotent but in a contrast to MSC, hSSC are tissue specific, with defined immunophenotype CD73+/CD146-/PDPN+/CD164-, capable of in vitro and in vivo differentiation into cartilage and bone. hSSC play a significant role in bone forming and bone remodelling process and are therefore promising treatment option for degenerative diseases such as OA. The clinical use of the current cell therapies is limited by the lack of reliable cell surface markers, which hinders their proper identification and isolation. In this thesis, we researched the influence that different stages of hip OA might have on bone-derived MSC features such as in vitro osteogenic differentiation and immunophenotype for new hSSC markers. First, we performed a gene expression analysis on cDNA samples of bone-derived MSCs from 32 donors, where we searched for potential differences in gene expression of new molecular markers for hSSC in patients with early and late OA, and patients without OA. We then chose 12 samples of bone-derived MSCs for further in vitro analyses of osteogenic differentiation using Alizarin red S staining. On the same set of samples, we then performed flow cytometry and determined the immunophenotype for new hSSC markers. Results showed that there are no significant differences in the osteogenic potential and immunophenotype for new hSSC markers in bone-derived MSCs between patient groups with different stages of hip OA. This is the first research study determining the recently identified hSSC-like features of bone-derived MSCs, in patients with different stages of hip OA. Due to small group of donors, further studies are suggested to confirm the results of our study