Evaluation of adipogenesis of mesenchymal stem/ stromal cells from different parts of the synovium in patients referred to hip arthroscopy

Na področju biologije je dolgo veljalo, da je z embrionalnim razvojem v somatske plasti endoderma, mezoderma in ektoderma usoda celic v smislu njihove dokončne diferenciacije zapečatena. Odkritje matičnih celic je te dogme močno omajalo in povzročilo razvoj regenerativne medicine. Mezenhimske matične/stromalne celice (MSC) izvirajo iz zarodne plasti mezoderma, iz katere se med embrionalnim razvojem razvijejo kostno tkivo, hrustanec, mišice, maščoba in druga vezivna tkiva. MSC se nahajajo v številnih tkivih in lahko diferencirajo v osteoblaste, hondrocite, adipocite, hepatocite, astrocite, pnevmocite, nevrone, srčno-mišične celice in celo krvne celice. Med seboj se razlikujejo po diferenciacijskem potencialu, morfologiji, velikosti in izražanju posameznih označevalcev. Zaradi lahke dostopnosti se danes veliko uporabljajo v celičnih terapijah, , v relativno kratkem času jih lahko v celičnih kulturah namnožimo v velikem številu, ne povzročajo zavrnitvenih reakcij in niso etično sporne. Njihova zelo pomembna lastnost je imunosupresivno delovanje. Pri nas se v zadnjih letih uporabljajo za zdravljenje hudih oblik bolezni presadka proti gostitelju, pri zdravljenju nekaterih avtoimunskih bolezni, globokih hrustančnih poškodbah, prav tako pa se uporabljajo za zdravljenje kronične oblike srčnega popuščanja. Zaradi izredno redkih neželenih učinkov se je zdravljenje z MSC izkazalo za zelo varen in učinkovit način zdravljenja. MSC imajo zaradi številnih svojih lastnosti tudi velik potencial za zdravljenje degenerativnih bolezni in travmatskih poškodb. Med najpogostejše degenerativne bolezni mišično-skeletnega sistema spada tudi osteoartroza. Vloga MSC pri tej bolezni še ni povsem raziskana, zato smo se v okviru magistrske naloge usmerili v raziskovanje teh celic v pogojih in vitro pri osteoartroznih bolnikih. Namen raziskave, ki smo jo izvedli je boljše razumevanje značilnosti MSC pri osteoartrozi. Magistrska naloga je tudi del širših projektov, katerih rezultati bodo omogočili razvoj novih pristopov v regenerativni medicini za diagnostiko ali zdravljenje degenerativnih bolezni sklepov, predvsem osteoartroze v zgodnjih fazah. V raziskavi smo poskušali ovrednotiti sposobnost adipogeneze MSC pridobljenih iz različnih delov sinovije in trabekularne kosti kolčnega sklepa. Primarne MSC so bile izolirane iz tkiv odvzetih iz treh različnih anatomskih mest sinovije in trabekularne kosti preiskovancev napotenih na artroskopijo kolka na Ortopedsko kliniko v Ljubljani. Nato smo MSC v pogojih in vitro izpostavili adipogenemu diferenciacijskemu mediju. Vzporedno so bili vzorci izpostavljeni tudi bazalnemu gojitvenemu mediju. Ti so nam služili kot kontrola. Nastale adipocite smo obarvali z uporabo histoloških metod in posneli slike pod mikroskopom. Nato smo s pomočjo programa ImageJ iz teh slik kvantificirali adipocite. Cilj te naloge je na podlagi te kvantifikacije ovrednotiti sposobnost adipogeneze MSC v odvisnosti od vrste tkiva, starosti bolnikov in prisotnosti degenerativnih sprememb kolka. Preizkusili smo tri hipoteze, ki predpostavljajo, da je sposobnost adipogene diferenciacije vzorcev vseh sinovijskih MSC večja kot pri kostnih MSC, da je sposobnost adipogene diferenciacije MSC večja pri mlajših bolnikih, in da je sposobnost adipogene diferenciacije večja pri preiskovancih brez degenerativnih sprememb kolka. Pri skoraj vseh vzorcih lahko potrdimo uspešno adipogeno diferenciacijo, saj je nastalo več adipocitov pri vzorcih MSC, ki so bili izpostavljeni adipogenemu gojitvenemu mediju kot pri njihovih kontrolah. Nobene od preiskovanih hipotez pa nismo uspeli potrditi. Najverjetnejši razlog je majhno število vzorcev, zato bi za večjo moč testa morali vključiti več preiskovancev oz. večje število vzorcev MSC. Vseeno pa raziskava, kot je naša, prispeva dragoceno znanje o primernosti MSC, pridobljenih od bolnikovih v klinični praksi k boljšemu razumevanju nastanka in možnosti zdravljenja degenerativnih bolezni kolka.In the field of biology, it has long been held that with embryonic development into the somatic layers of the endoderm, mesoderm, and ectoderm, the fate of cells in terms of their definitive differentiation is sealed. The discovery of stem cells severely shaken these dogmas and led to the development of regenerative medicine. Mesenchymal stem/stromal cells (MSCs) originate from the germ layer of the mesoderm, from which bone tissue, cartilage, muscle, fat, and other connective tissues develop during embryonic development. MSCs are found in many tissues and can differentiate into osteoblasts, chondrocytes, adipocytes, hepatocytes, astrocytes, pneumocytes, neurons, cardiovascular cells, and even blood cells. They differ from each other in terms of differentiation potential, morphology, size and expression of individual markers. Today, they are widely used in cell therapies, due to their availability, in a relatively short time they can be culture expanded in large numbers, MSCs do not cause rejections and are not ethically controversial. Their very important property is immunosuppressive action. In recent years, they have been used in our country to treat severe forms of graft-versus-host disease, certain autoimmune diseases, deep cartilage injuries, and are also used to treat chronic forms of heart failure. Due to the extremely rare side effects, MSC treatment has proven to be a very safe and effective treatment. MSCs also have great potential for the treatment of degenerative diseases and traumatic injuries due to their many properties. Osteoarthritis is the most common degenerative diseases of the musculoskeletal system. As the role of MSCs in this disease has not yet been fully investigated, our master\u27s thesis has focused on the research of these cells in vitro in patients with osteoarthritis to better understand the characteristics of MSCs in this particular disorder. This master\u27s thesis is also part of larger research project, the results of which will enable the development of new approaches in regenerative medicine for the diagnosis or treatment of joint degeneration and osteoarthritis in the early stages. The aim of our thesis is to evaluate the adipogenesis of MSCs derived from different parts of the synovium and trabecular bone of the hip joint. Primary MSCs were isolated from tissues harvested from three different anatomical sites of synovium and trabecular bone of subjects referred to hip arthroscopy at Department of Orthopaedic Surgery, University Medical Centre Ljubljana. The MSCs were exposed to adipogenic differentiation medium under in vitro conditions. In parallel, the samples were also exposed to basal culture medium. These served as controls. The adipocytes formed were stained using histological methods and images were taken under a microscope. Adipocytes were then quantified from these images using the ImageJ program. Based on this quantification, we aimed to evaluate the ability of the adipogenesis of MSCs depending on the type of tissue, the age of the patients and the presence of the degenerative changes in the hip. We tested three hypotheses, first, we assume that the ability of adipogenic differentiation of all synovial MSCs is higher than that of bone-derived MSCs, second, we assume that the ability of adipogenic differentiation of MSC is higher in younger patients, and third, we assume, that the ability of adipogenic differentiation is higher in samples without degenerative changes of the hip. Successful adipogenic differentiation can be confirmed in almost all samples, as more adipocytes were formed in MSC samples exposed to adipogenic culture medium than in their controls. We were not able to confirm any of the three hypotheses. This is most certainly due to the small number of samples. To achieve higher power of the study, we should include more subjects or analyse more MSC samples. Nevertheless, our research provides valuable knowledge on the suitability of MSCs derived from patients in the clinical practice and contributes to better understanding of the onset and treatment options for degenerative hip diseases