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Mezenhimske matične/stromalne celice so heterogena populacija matičnih celic, ki se nahajajo v tkivih odraslih in imajo edinstveno sposobnost multilinijske diferenciacije oz. pretvorbe znotraj mezodermalne zarodne plasti. Iz slednje se med embrionalnim razvojem razvijejo kostno tkivo, hrustanec, mišice, maščobno tkivo in druga vezivna tkiva. Mezenhimske/stromalne matične celice (MSC, angl. mesenchymal stem/stromal cells) so že nekaj časa najpogostejša tarča raziskav na področju regenerativne medicine. S poznavanjem njihove fiziološke in patofiziološke vloge pri degenerativnih boleznih kolka se kaže njihova potencialna uporaba v diagnostiki teh bolezni. Najpogostejše degenerativne bolezni mišično-skeletnega sistema so osteoporoza, osteoartoza (OA) in sarkopenija. OA je raznolika in večfaktorska bolezen sinovijskih sklepov, pri kateri sodelujejo tudi vnetni dejavniki. Osnova bolezni je napredujoča izguba strukture in popravljalne funkcije sklepa z endogenim regenerativnim procesom, ki naj bi bil pri omenjeni bolezni oslabljen. Namreč, pri mlajših zdravih posameznikih regenerativni proces po poškodbi tkiv izvedejo prav MSC. Namen magistrske naloge je bil ugotoviti razlike v izražanju genov za mediatorje vnetja v MSC, pridobljenih iz različnih tkiv kolka preiskovancev, z in brez degenerativnih sprememb. Tekom artoskopije kolka so bila preiskovancem odvzeta tri tkiva sinovije na različnih mestih kolka (S1-S3) in stebriček trabekularne kosti femurja (KO). Primarne MSC so nato namnožili v pogojih in vitro, iz njih izolirali ribonukleinsko kislino, mi pa smo nato optimizirali metodo kvantitativne verižne reakcije s polimerazo (qPCR) za merjenje izražanja genov za mediatorje vnetja. Na podlagi rezultatov optimizacije smo za nadaljnjo analizo izbrali gene TGFB1, IL6, COMP, MMP13 in VEGFA. Rezultate pridobljene s qPCR smo normalizirali glede na izbrani referenčni gen GAPDH. Rezultate smo nato primerjali med bolniki z zgodnjo OA in tistimi brez nje ter med različnimi tkivi. Preverili smo 3 hipoteze, in sicer: izražanje genov za vnetne molekule se razlikuje med MSC bolnikov z zgodnjo OA v primerjavi s tistimi brez OA, izražanje genov za vnetne molekule se razlikuje med MSC iz različnih tkiv bolnikov z zgodnjo OA in izražanje genov za vnetne molekule se razlikuje med MSC iz različnih tkiv bolnikov brez OA. Statistična analiza rezultatov ni pokazala statistično značilnih razlik v izražanju preiskovanih genov, zato smo vse 3 hipoteze zavrnili. Iz tega lahko zaključimo, da se MSC iz različnih tkiv kolka pri bolnikih z OA in pri tistih brez degenerativnih sprememb kolka ne razlikujejo v izražanju genov za vnetne molekule in da je verjetno razlog za razlike med MSC v drugih molekulah. Pomanjkljivost te študije je predvsem majhna skupina vzorcev, zato predlagamo raziskavo še na večji populaciji, da bi bila moč statističnega testa večja. Mesenchymal stem/stromal cells are a heterogeneous population of stem cells that are found in adult tissues and have the unique ability of multilineage differentiation, in particular the differentiation within the mesoderm. During embryonic development connective tissues such as cartilage, muscles, fat and others develop from the mesoderm. Mesenchymal stem/stromal cells (MSCs) have been the most common research target in the field of regenerative medicine for some time. Their physiological and pathophysiological role in degenerative disorders of the hip shows their potential use in the diagnosis of these disorders. The most common degenerative disorders of the musculoskeletal system are osteoporosis, osteoarthritis (OA) and sarcopenia. OA is a diverse and multifactorial disorder of the synovial joints, where inflammatory factors are also involved. The basis of the disease is the progressive loss of the structure and repair function of the joint through the endogenous regenerative process, which is suggested to be weakened in the aforementioned disease. Namely, in younger healthy individuals, the regenerative process after tissue damage is carried out by MSCs. The aim of the master's thesis was to determine differences in the expression of genes encoding mediators of inflammation in MSCs obtained from different hip tissues of subjects with and without degenerative changes. During arthroscopy of the hip, synovium at 3 different locations of the hip (S1-S3) and a pillar of the trabecular bone of the femur (KO) were taken from the subjects. Primary MSCs were then isolated from these tissues and cultured in vitro and RNA was isolated from culture expanded cells. We then optimized a quantitative polymerase chain reaction (qPCR) method to measure the expression of genes for inflammatory mediators. Based on the optimization results, we selected TGFB1, IL6, COMP, MMP13 and VEGFA genes for further analysis. The results obtained by qPCR were normalized to reference gene GAPDH. The results were then compared between patients with early OA and those without, and between different tissues. We tested 3 hypotheses, namely: gene expression of inflammatory molecules differs between MSCs from patients with early OA compared to those without OA gene expression of inflammatory molecules differs between MSCs from different tissues of patients with early OA and gene expression of inflammatory molecules differs between MSCs from different tissues of patients without OA. Statistical analysis of the results did not show statistically significant differences in the expression of the investigated genes, so we rejected all 3 hypotheses. Based on these results we can conclude that MSCs from different tissues of the hip and patients with OA and those without hip degenerative changes do not differ in the expression of genes for inflammatory molecules. The differences between these MSCs are probably result of other molecules. The limitation of this study is also small group of samples, so we suggest further research in larger population in order to increase the power of the statistical test.

Mezenhimske matične celice so nediferencirane celice z veliko sposobnostjo diferenciacije v tkiva mezodermalnega izvora in visokim potencialom za uporabo v imunomodulatorni in regenerativni terapiji, na primer pri osteoartrozi. Tu gre za degenerativno bolezen sklepov, ki se začne z mikroskopskimi spremembami v hrustancu, ki vodijo v njegovo prekomerno obrabo in sčasoma tudi v poškodbo kosti. V tej magistrski nalogi smo nameravali oceniti sposobnost matičnih celic, da tvorijo hrustančno tkivo v pogojih in vitro. Matične celice smo pridobili iz vzorcev tkiv, ki so se med seboj razlikovali glede na patologijo darovalca (z ali brez degenerativnih sprememb kolka) in mesto odvzema (vzorci sinovije na treh predelih kolka ter vzorec trabekularne kosti). Tkivne vzorce smo pridobili od 15 bolnikov, napotenih na Ortopedsko kliniko UKC Ljubljana. Primarne celice smo gojili v pogojih in vitro bodisi v hondrogenem ali kontrolnem mediju, iz tako nastalih hondrogenih pelet pa pripravili obarvane preparate ter jih nato pregledali pod mikroskopom. Morfologijo pelet smo ocenili z uporabo metodologije po Bernu, izmerili pa smo tudi njihov premer. S statistično analizo smo dokazali vpliv sestave gojitvenega medija na morfologijo pelet ter vpliv mesta odvzema tkiv na premer pelet, medtem ko patologija kolka ni vplivala na nobenega izmed preiskovanih parametrov. Domnevamo, da so ključen vzrok za dobljene rezultate specifične lastnosti mezenhimskih matičnih celic in njihovega mikrookolja ter relativno nizko število vzorcev. Zato verjamemo, da bo za kakovostno oceno sposobnosti hondrogeneze mezenhimskih matičnih celic potrebna izvedba nadaljnjih sorodnih študij z večjim številom vzorcev ter v pogojih z večjo podobnostjo stanju in vivo. Mesenchymal stem cells are non-differentiated cells with a high capacity to differentiate into tissues of mesodermal origin and a high potential for use in immunomodulatory and regenerative therapy, e.g. osteoarthritis. This is a degenerative disease of the joints that begins with microscopic changes in cartilage that lead to its excessive wear and eventually to bone damage. In this work, we aimed to investigate the ability of mesenchymal stem cells to form cartilage under in vitro conditions. We obtained stem cells from samples that differed by the underlying pathology of the donor (with or without degenerative changes of the hip joint) and by the site of collection (sinovium samples from three sites in the hip joint and samples from trabecular bone tissue). Samples were collected from 15 patients at the Orthopaedic Clinic of the University Medical Centre Ljubljana. Primary cells were cultured under in vitro conditions in either hondrogenic or control medium, and the hondrogenic pellets formed were used to prepare stained slides, which were then examined under the microscope. The morphology of the pellets was evaluated using the Bern score, while their diameter was also measured. Using statistical analysis, we demonstrated the influence of growth medium on pellet morphology and the influence of collection site on pellet diameter, while pathology had no effect on either parameter. We hypothesise that the key to these results lies in the specific characteristics of the mesenchymal stem cells and their microenvironment, and in the relatively small number of samples. Therefore, we believe that a larger number of samples, as well as conditions more similar to those in vivo, are needed for a better evaluation of the cartilage-forming ability of mesenchymal stem cells.

Nenadna pandemija COVID-19 je zelo vplivala na naše življenje. Znanstveniki in zdravniki povsod po svetu se še vedno trudijo, da bi poleg učinkovitih cepiv poiskali tudi najustreznejše zdravilo za posledice, ki jih povzroča ta bolezen. Kot morebitna rešitev se je pojavila ideja o terapiji z mezenhimskimi matičnimi celicami. Te so zaradi svojih imunomodulatornih, antimikrobnih in regeneracijskih lastnosti postale potencialno zdravilo za bolnike s hujšimi posledicami te bolezni. Prav tako se mezenhimske matične celice že kar nekaj časa uspešno uporabljajo pri zdravljenju vnetij, za imunosupresijo, regeneracijo tkiv in zdravljenje nekaterih prej neozdravljivih bolezni. Ker so posledice bolezni COVID-19 zelo širokega spektra, bi lahko z uporabo mezenhimskih matičnih celic izvajali terapijo na več področjih. Terapijo so že uspešno preizkusili na laboratorijskih živalih, sedaj pa so pričeli tudi s kliničnimi testiranji na ljudeh. Prvi rezultati študij so obetavni, čeprav so znanstveniki naleteli na precej ovir in izzivov, ki jih bo še potrebno rešiti. The entire course of our lives was suddenly greatly affected by the COVID-19 pandemic. Besides trying to make the efficient vaccines, scientists and doctors all over the world, are still struggling to find the most affective cure for the consequences caused by this disease. As a possible solution, the idea of mesenchymal stem cell therapy emerged. Due to their immunomodulatory, antimicrobial and regenerative properties, they have become a potential drug for patients with more severe consequences of a COVID-19 disease. Also, for some time now, mesenchymal stem cells have been used successfully in the treatment of inflammation, for immunosuppression, tissue regeneration and for the treatment of previously incurable diseases. Since the consequences of a COVID-19 are on the wide spectrum, using mesenchymal stem cell therapy could cure many things at the same time. The therapy for COVID-19 consequences has been already tested on laboratory animals. The results were promising, so the researchers are now begining clinical trials on humans. The first results of the studies are promising, although scientists have encountered a number of obstacles and challenges that will need to be addressed.

Konjska astma je pogosta kronična bolezen dihal, ki pomembno vpliva na zdravje in dobrobit konj. Trenutne strategije zdravljenja konjske astme temeljijo na lajšanju simptomov in ne zdravijo bolezni. Lahko imajo tudi znatne stranske učinke. Cilj naše raziskave je bil oceniti lokalne in sistemske učinke intrabronhialne administracije avtolognih mezenhimskih matičnih celic, pridobljenih iz maščobnega tkiva (Adipose derived mesenchymal stem cells, AD-MSC) na potek konjske astme. Konje smo naključno razdelili v dve skupini in jih zdravili z AD-MSC ali deksametazonom. Za ugotavljanje terapevtskih učinkov smo natančno opredelili klinično izražanje bolezni pri posameznem konju in opravili meritve pljučnih funkcij in endoskopski pregled dihalnih poti. Ob endoskopiji smo opravili tudi bronhoalveolarno lavažo, izpirke pa citološko pregledali in izmerili vrednosti izražanja sporočilne RNK določenih vnetnih citokinov in koncentracijo njihovih beljakovin. Po končani obravnavi smo konje spremljali še eno leto in primerjali dolgoročno stabilnost kliničnih znakov med obema skupinama. Kljub temu, da zdravljenje z AD-MSC ni povzročilo takšnega izboljšanja kliničnih znakov astme kot zdravljenje kontrolne skupine, smo zaznali znatno izboljšanje pri izražanju in koncentraciji posameznih vnetnih spremenljivk. V primerjavi s kontrolnim zdravljenjem, pa je imelo zdravljenje z AD-MSC pozitiven dolgoročni učinek na stabilnost klinične slike. Na podlagi rezultatov te raziskave zaključujemo, da ima intrabronhialno zdravljenje z AD-MSC omejen kratkoročen učinek in pozitiven dolgoročen učinek na simptome kronične astme pri konjih. Equine asthma is a common chronic disease of the respiratory tract that significantly affects horses' health and well-being. Current therapeutic strategies are based on symptomatic treatment and do not alter the disease's long-term progression, and their side effects are worrying. Our study aimed to evaluate the local and systemic effects of intrabronchial administration of autologous adipose-derived mesenchymal stem cells (AD-MSC) on severe equine asthma. Horses were randomly divided into two groups and treated with AD-MSC or dexamethasone. To determine the therapeutic effects of intrabronchial treatment with AD-MSC, we precisely defined clinical signs, and performed lung function tests and endoscopic examination of the airways in horses included in the study. Bronchoalveolar lavage was performed, and the samples were cytologically examined, and the mRNA expression values of specific inflammatory cytokines and the concentration of their proteins were measured. After treatment, the horses were monitored for another year, and the long-term stability of clinical signs between the two groups was compared. Although non-inferiority of AD-MSC treatment was not confirmed, improvement in certain inflammatory variables was observed after cell therapy. However, intrabronchial treatment with AD-MSC had a positive long-term effect on clinical stability of horses with severe asthma. Results of the study indicate that intrabronchial treatment with AD-MSC has a limited short-term anti-inflammatory effect and a positive long-term clinical effect in asthmatic horses.

Glioblastom je najpogostejši in najagresivnejši možganski tumor. Za visoko odpornost na standardno zdravljenje so odgovorne glioblastomske matične celice (GMC) in njihova interakcija s tumorskim mikrookoljem, katerega del so tudi mezenhimske matične celice (MMC). Raziskave imajo nasprotujoče rezultate glede vpliva MMC na rast in invazijo tumorja, vendar imajo velik potencial kot vektorji pri dostavljanju terapevtikov v tumor. Vpliv ionizirajočega sevanja, ki se rutinsko uporablja za zdravljenje glioblastoma, na GMC in MMC smo določali v tridimenzionalnem (3D) modelu sferoidov monokultur in kokultur obeh vrst celic. Izvedli smo test klonogenosti, invazije in proliferacije z imunobarvanjem s protitelesi proti Ki-67 in spremljali celični cikel s Hoechst barvilom. Rezultati testa klonogenosti so pokazali, da so GMC odporne na sevanje za razliko od MMC. Rast sferoidov v obsevani kokulturi GMC z MMC se je zmanjšala v primerjavi z obsevano monokulturo GMC. Invazija MMC v monokulturi se je povišala po obsevanju, v kokulturi z GMC pa sevanje ni imelo vpliva na invazijo MMC. Ionizirajoče sevanje in prisotnost MMC v kulturi nista imela vpliva na invazijo GMC. V monokulturi so se GMC hitreje delile kot MMC, v kokulturi pa so MMC svojo proliferacijo pospešile. Obsevanje je znižalo proliferacijo MMC in GMC v kokulturi. Prisotnost GMC in ionizirajoče sevanje ni imelo vpliva na celični cikel MMC. Celični cikel obsevanih GMC se je v monokulturi ustavil v G2 fazi, v kokulturi z MMC pa v G1 fazi. Naši rezultati so pripomogli k boljšem razumevanju odziva GMC na ionizirajoče sevanje in vpliv MMC na odpornost GMC na ionizirajoče sevanje. Rezultati so dobra iztočnica za nadaljne raziskave mehanizmov odpornosti glioblastoma na standardno zdravljenje. Glioblastoma is the most aggressive brain tumour. Its resistance to standard therapy is due to glioblastoma stem cells (GSC) and their interaction with tumour microenvironment, part of which are also mesenchymal stem cells (MSC). Research on MSC effects on tumour growth and invasion shows seemingly contradictory results. Still, MSC have great potential as vectors for drug delivery to the tumour. Here, we studied how ionizing radiation affects GSC and MSC in monocultures and cocultures, both in tridimensional (3D) spheroid model. We used clonogenic assay, invasion assay, proliferation assay with antibodies for Ki-67 protein and we also analysed cell cycle with Hoechst dye. GSC were more clonogenic then MSC, when irradiated. When in coculture with MSC, GSC spheroid growth slowed down compared to GSC monocultures. When irradiated, MSC were more invasive in monoculture than in coculture. Irradiation and MSC didn't influence invasion of GSC. GSC were more proliferative then MSC in monoculture. Proliferation of MSC increased in coculture but decreased when they were exposed to radiation. Proliferation of GSC decreased in irradiated coculture with MSC compared to radiated monoculture of GSC. GSC and radiation didn't affect cell cycle of MSC. When we radiated GSC in monoculture cell cycle stopped in G2 phase, but not when they were irradiated in the presence of MSC. Our results contribute to understanding of effect of irradiation on GSC and effect of MMC on GSC radioresistance. Results of this study are useful for future research of mechanisms that are responsible for glioblastoma resistance to standard therapy.

Magistrsko delo obsega pregled literature na temo interakcije mezenhimskih matičnih celic (MSC) z imunskim sistemom, stimulacije MSC za namen indukcije imunosupresivnega fenotipa in uporabe MSC v klinični aplikaciji. Eksperimentalni del obsega rezultate in vitro stimulacije MSC z izbranimi aktivacijskimi dejavniki (AD) in s supernatanti mešane limfocitne reakcije (MLR) za namen ugotavljanja, kateri AD in supernatanti MLR izzovejo imunosupresivni fenotip MSC, ter primerjave obeh načinov stimulacije. Uspešnost indukcije imunosupresivnega fenotipa MSC smo določali s povečanjem izražanja PD-L1 in IDO. Določali smo tudi HLA-DR kot pokazatelja večje imunogenosti stimuliranih MSC. Kot najboljše kombinacije AD pri indukciji imunosupresivnega fenotipa MSC so se izkazale rapamicin/MPLA/TNFα/IFNγ za indukcijo izražanja PD-L1 in kombinaciji rapamicin/TNFα/IFNγ ter MPLA/TNFα/IFNγ pri indukciji izražanja IDO. Kombinacije AD niso inducirale bistveno večjega izražanja HLA-DR. Pri teh kombinacijah AD sklepamo na njihovo sinergistično ali aditivno delovanje pri indukciji imunosupresivnega fenotipa in ob pregledu literature trdimo, da je to prvi objavljen poskus stimulacije MSC s takšnimi kombinacijami AD. Kot najboljši supernatanti pri indukciji imunosupresivnega fenotipa MSC so se izkazali supernatanti MLR, ki so potekali v gojišču z dodatkom MPLA, ki ojača imunski odziv. Supernatanti MLR so dosegli primerljive rezultate kot AD in so se izkazali kot smiselna alternativa stimulaciji MSC z AD. This thesis encompasses an overview of publications examining interactions of mesenchymal stem cells (MSC) with the immune system, the induction of an immunosuppressive phenotype by MSC stimulation and clinical applications of MSC. We present the results of in vitro MSC stimulation with different activating factors (AD) and supernatants from mixed lymphocyte reactions (MLR). The aim was to evaluate which AD and MLR supernatants induce an immunosuppressive phenotype in MSC, and to compare them. The induction of an immunosuppressive phenotype was determined by enhanced expression of PD-L1 and IDO, and the increased immunogenicity was determined by expression of HLA-DR. The best combinations of AD were rapamycin/MPLA/TNFα/IFNγ for inducing expression of PD-L1, and the combinations rapamycin/TNFα/IFNγ and MPLA/TNFα/IFNγ for inducing expression of IDO. These combinations of AD also did not induce higher expression of HLA-DR. The immunosuppressive phenotype induction by these combinations of AD might be caused by their synergistic or additive behaviour. To our knowledge this is the first publication of MSC stimulation with these combinations of AD. The MLR supernatants best at inducing an immunosuppressive phenotype were those that took place in media with added MPLA, which strengthens the immune response. MLR supernatants achieved similar results as AD and could be a potential alternative to MSC stimulation with AD.

Mezenhimske matične celice predstavljajo heterogeno skupino celic, ki jih lahko izoliramo iz številnih tkiv odraslega človeka. Zanimive so predvsem zaradi njihove sposobnosti migracije na mesto vnetja v telesu, kjer izločajo različne topne dejavnike kot so citokini, kemokini in rastni dejavniki, ki izboljšujejo angiogenezo, zavirajo vnetje in inhibirajo apoptozo. Zaradi številnih imunomodulatornih in regenerativnih lastnosti je vedno več zanimanja za uporabo mezenhimskih matičnih celic v kliniki pri številnih patoloških stanjih, za katere sta značilna vnetje in degeneracija tkiva. Vse večji obseg uporabe zahteva tudi vse boljše tehnike ločevanja celic, ki morajo biti hitrejše in bolj učinkovite. Namen naloge je bil raziskati potencialne materiale, ki bi bili ustrezni za ločevanje mezenhimskih matičnih celic od ostalih celic. V raziskovalni nalogi smo uporabili humane mezenhimske matične celice, izolirane iz kostnega mozga. Najprej smo okarakterizirali vzorce materialov z izbranimi tehnikami, in sicer smo merili zeta potencial, kontaktni kot ter hrapavost. V nadaljevanju smo ugotavljali vpliv lastnosti materialov na obnašanje celic – na njihovo adhezijo. Za najbolj učinkovit material se je izkazala mikrotitrska plošča s fibronektinom, na katero se je vezalo največ mezenhimskih matičnih celic. Na ostalih materialih nismo dobili ponovljivih rezultatov, zato nismo prišli do jasnih zaključkov. Ugotovili smo, da so interakcije med celicami in materiali zelo kompleksne in da je za njihovo razumevanje potrebno še precej raziskav. Iskanje novih metod ločevanja celic je težavno, saj je nemogoče določiti enostaven kriterij za uspešno adhezijo in nato ločevanje celic. Mesenchymal stem cells represent a heterogeneous group of cells that can be isolated from several adult tissues. In recent years the main interest is in their ability to migrate to the site of inflammation in the body, where they secrete various soluble factors such as cytokines, chemokines, and growth factors that improve angiogenesis, inhibit inflammation, and inhibit apoptosis. Their immunomodulatory and regenerative properties are the reason for increasing interest for clinical applications, where they could be used for treatment of many pathological conditions related to inflammation and tissue degeneration. Increasing use requires better cell separation techniques, which need to be faster and more efficient. The purpose of our research was to investigate potential materials which would be suitable for mesenchymal stem cell separation from other cells. We used bone marrow derived human mesenchymal stem. In the first part of the research, we characterized sample materials via measuring the zeta potential, contact angle and roughness. In the second part, we determined the influence of the material properties on the behavior of cells - on their adhesion. The best results were shown on the fibronectin coated microtiter plate, as the largest proportion of mesenchymal stem cells adhered to this material. We did not get consistent results on the other materials, therefore we did not come to clear conclusions. We have confirmed that cell-material interactions are very complex and still need to be researched in detail. Finding new methods for cell separation is difficult, as it is impossible to determine simple criteria for successful adhesion and following cell separation.

Možganski tumor glioblastom (GB) je najpogostejši in najagresivnejši primarni tumor osrednjega živčnega sistema pri odraslih. Zdravljenje je zaradi odpornosti tumorja na kemo- in radioterapijo ter nepopolne kirurške odstranitve neuspešno. K temu prispeva heterogenost GB. Rakave celice so v tumorju obdane s kompleksnim tumorskim mikrookoljem. Medcelična komunikacija v mikrookolju prispeva k razvoju tumorja. Glioblastomske matične celice (GMC) so odporne na terapijo in zaščitene v posebnem mikrookolju – niši. V niši sobivajo z nerakavimi stromalnimi celicami, kot so mezenhimske matične celice (MMC). Preučevali smo vpliv MMC na rast in invazijo GMC v možganih zarodkov cebric (Danio rerio). V možgane zarodkov smo vnesli GMC ter mešanice GMC in MMC, kar nam je omogočilo primerjavo tumorjev. S programom ImageJ smo kvantificirali relativno spremembo površine in dolžine tumorjev monokultur in sokultur celic v možganih in vivo. Razvili in optimizirali smo metodo priprave tkivih rezin možganov z vnesenimi označenimi humanimi celicami za imunohistokemijsko barvanje. Uporabili smo označevalce matičnosti in invazije GMC, s katerimi smo analizirali medsebojni vpliv celic. Ugotovili smo, da GMC in MMC v možganih cebric tvorijo mešane tumorje. Pokazali smo, da prisotnost MMC ne spremeni proliferacije, ne sproži diferenciacije in ne spremeni invazije GMC v možganih cebric. Prisotnost GMC je povečala rast in invazijo MMC. Glioblastoma (GB) is the most common and most aggressive brain tumor of primary central nervous system in humans. Treatment is unsuccessful due to the tumor's resistance to chemotherapy, radiotherapy and incomplete surgical removal. GB displays extensive cellular heterogeneity which represents a major obstacle for effective treatment. Cancer cells in a tumor are surrounded by a complex tumor microenvironment (TMO). Intercellular interactions within the TMO drive tumor progression. Glioblastoma stem cells (GMC) are resistant to therapy and protected in a special microenvironment - a niche. They coexist in the niche with non-cancerous stromal cells such as mesenchymal stem cells (MMC). We studied the impact of MMC on the proliferation and invasion of GMC in the zebrafish embryonic brain. We injected GMC and mixtures of GMC and MMC into the embryonic brain, which allowed us to compare tumors. The program ImageJ was used to quantify the relative difference of area and length of monoculture and coculture tumors in the brain in vivo. We developed and optimized a method of preparing zebrafish brain tissue sections, with injected labeled human cells, for immunohistochemistry. We used stem cell markers and markers of cancer cell invasion for interaction analysis. We found that GMC and MMC form mixed tumors in the zebrafish brain. We have shown that the presence of MMC does not alter proliferation, does not trigger differentiation, and does not alter invasion of GMC in the zebrafish brain. The presence of GMC increased the growth and invasion of MMC.

Matične celice so v zadnjih letih zaradi svojih edinstvenih lastnosti postale vir razvoja regenerativne medicine in tkivnega inženirstva. Ker različne lastnosti mezenhimskih matičnih celic (MSC) v pogojih in vitro lahko vodijo v potencialno različen terapevtski učinek, je temeljnega pomena odkrivanje vplivov različnih dejavnikov na značilnosti MSC. Uveljavljena podlaga za gojenje MSC je plastična osnova, vendar se zaradi boljšega posnemanja naravnega celičnega okolja za gojenje celic vedno bolj poudarja pomen uporabe celičnih nosilcev. V doktorski disertaciji smo raziskavo sistematično razdelili na dva dela. V prvem delu smo celice gojili na običajni plastični podlagi in proučevali vpliv vrste, spola in poznih pasaž na proliferacijski in diferenciacijski potencial MSC iz maščobnega tkiva (AMSC) psov in mačk. V drugem delu smo celice gojili na celičnih nosilcih iz svilnega fibroina (SF) in proučevali, kako SF vpliva na proliferacijski in diferenciacijski potencial AMSC. Rezultati prvega dela naše raziskave so pokazali, da AMSC mačk izkazujejo slabši proliferacijski in diferenciacijski potencial kot AMSC psov in da tudi pozne pasaže bolj negativno vplivajo na AMSC mačk kot na AMSC psov. Dodatno smo pokazali, da AMSC mačk v manjši meri izražajo preiskovane celične označevalce in v manj optimalnih pogojih izkazujejo slabšo živost. Z rezultati prvega dela raziskave smo pokazali, da vrsta živali lahko bistveno vpliva na značilnosti AMSC v pogojih in vitro in da bi živalsko vrsto morali upoštevati kot spremenljivko pri pripravi celic za regenerativno zdravljenje. Rezultati drugega dela naše raziskave so pokazali, da SF omogoča adhezijo celic in vodi usodo AMSC v smeri hondrogene diferenciacije, ki smo jo potrdili z analizo celične morfologije, barvanjem zunajceličnega matriksa diferenciranih celic ter kvantifikacijo izražanja genov, značilnih za hrustančne celice. Z rezultati drugega dela raziskave smo pokazali, da je hondrogeno diferenciacijo celic moč doseči brez posebnih pogojev gojenja celic, ki so sicer potrebni za hondrogenezo MSC na običajni plastični podlagi. Rezultati doktorske disertacije omogočajo poglobitev pomena poznavanja razlik matičnih celic med živalskimi vrstami in hkrati predstavljajo pomembno osnovo za prihodnje študije in potencial za uvajanje novih zdravljenj z matičnimi celicami v veterinarski klinični medicini. Due to their unique properties, stem cells have become an important source for the development of regenerative medicine and tissue engineering in recent years. Since different properties of mesenchymal stem cells (MSCs) in vitro can lead to potentially different therapeutic effects, investigating the influence of various factors on the properties of MSCs is essential. The established basis for the cultivation of MSCs is a plastic surface, but due to the better mimicking of the natural cellular environment, the use of cell carriers for cell cultivation is being increasingly developed. In this dissertation, our studies are systematically divided into two parts. In the first part, cells were grown on a conventional plastic surface and the influence of species, sex and late passages on the proliferation and differentiation potential of canine and feline adipose derived MSCs (AMSCs) were investigated. In the second part, cells were grown on silk fibroin (SF) cell carriers and influence of SF on the AMSCs was investigated. The results of the first part of our study showed that feline AMSCs had poorer proliferation and differentiation potential than canine AMSCs and that late passages had a more negative effect on feline AMSCs than on canine AMSCs. In addition, feline AMSCs expressed fewer examined cell surface markers and exhibited lower viability under less optimal conditions. The results of the first part of the study showed that animal species can significantly affect the properties of AMSCs in vitro and that animal species should be considered when preparing cells for regenerative treatment. The results of the second part of our study showed that SF enables cell adhesion and directs AMSC fate towards chondrogenic differentiation, which was confirmed by the analysis of cell morphology, staining of extracellular matrix of differentiated cells and quantification of genes, characteristic of chondrocytes. With the results of the second part of the study, we have shown that chondrogenic AMSC differentiation can be achieved without the special cell culture conditions that are otherwise required for chondrogenesis of AMSCs on a conventional plastic surface. The results of the dissertation allow us to deepen the knowledge about the importance of the stem cell differences between species and at the same time represent basis for further studies and the potential for the introduction of new stem cell therapies in clinical veterinary medicine.

Several plant polyphenols have been shown to reduce osteoarthritis symptoms due to their antioxidant, anti-inflammatory and immunomodulatory properties. We investigated the effects of two different polyphenolic extracts (Belinal, Pycnogenol) and two different polyphenols (resveratrol, quercetin) on the chondrogenic potential of bone-derived mesenchymal stem/stromal cells (MSCs) from healthy donors and patients with osteoarthritis. Our main aim was to determine whether Belinal, a commercially available polyphenolic extract from silver fir (Abies alba L.) branches, has comparable chondrogenic potential with the other tested extract and the polyphenols under inflammatory and non-inflammatory conditions. In our study, Belinal promoted significantly greater chondrogenesis compared to the untreated (p = 0.0289) and resveratrol-treated (p = 0.0468) MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Under inflammatory conditions, chondrogenesis was significantly enhanced for MSCs treated with Belinal compared to the control (p = 0.0483). The other extract and the polyphenols did not show any significant effects on chondrogenesis under non-inflammatory or inflammatory conditions. None of the tested extracts and polyphenols showed significant effects on chondrogenesis in healthy donors, under either non-inflammatory or inflammatory conditions. Our data show that Belinal can boost the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions.

Glioblastom (GB) je najpogostejši in najagresivnejši možganski tumor pri človeku. Glioblastomske matične celice (GMC) so odgovorne za rezistenco na radio- in kemoterapijo, ohranjanje in širjenje GB ter njegovo ponovitev. V tumorskem mikrookolju so prisotne tudi mezenhimske matične celice (MMC), ki z interakcijami s celicami GB pospešijo razvoj GB. Zaenkrat še ni znano, kako MMC vplivajo na rast ter invazijo GMC. Vpliv celičnih interakcij med GMC in MMC na rast in invazijo celic GB smo določali v 3D modelu sferoidov in vitro ter v možganih zarodkov rib cebric (Danio rerio). Pokazali smo, da MMC vplivajo na morfologijo GMC v sferoidih sokultur. Z uporabo konfokalne mikroskopije smo razlike v morfologiji med GMC v monokulturi in sokulturi z MMC potrdili tudi v modelu in vivo. Naši rezultati kažejo, da prisotnost MMC poveča rast GMC v modelih tumorja in vitro ter in vivo. MMC so bile v primerjavi z GMC bolj invazivne, v sokulturi pa so GMC zmanjšale invazijo MMC in vitro. MMC na invazijo GMC in vitro ter in vivo niso imeli učinka. Nasprotno smo v modelu in vivo pokazali večjo invazijo GMC v primerjavi z injiciranimi MMC. Razlog za razlike v invaziji pri dveh različnih uporabljenih modelih je v mikrookolju, ki je v možganih cebric kompleksnejše. Pokazali smo, da so MMC v tumorskem mikrookolju pomembne, saj pospešijo rast GMC. Glioblastoma (GB) is the most common and most aggressive brain tumour in humans. Glioblastoma stem cells (GMC) are responsible for resistance to radiotherapy and chemotherapy, the maintenance and spread of the tumour, and its recurrence. Mesenchymal stem cells (MMC) are also present in the tumour microenvironment, which, through interactions with glioblastoma cells, promote GB development and increase the self-renewal capacity of GMC and their invasion. It has not been studied so far how MMC affect GMC growth and invasion. We determined the influence of cellular interactions between GMC and MMC on the growth and invasion of glioblastoma cells in a 3D model of spheroids in vitro and in the brains of zebrafish embryos (Danio rerio). We showed that MMC affected the morphology of GMC in the spheroids of co-culture. Using confocal microscopy, we also showed differences in morphology between GMC in monoculture and co-culture with MMC in vivo. We demonstrated that the presence of MMC increased GMC growth in in vitro and in vivo tumour models. MMCs were more invasive than GMCs, but in co-culture GMCs reduced MMC invasion in vitro. In contrast, GMC invasion was significantly higher in the in vivo model compared to injected MMCs. The reason for the differences in invasion in different models used is in the microenvironment, which is more complex in the brains of zebrafish. We showed that MMCs are important in the tumour microenvironment as they promote GMC growth.

Glioblastom (GBM) je rak, ki nastane predvidoma iz astrocitov, z najslabšo prognozo med vsemi, tudi možganskimi raki – v povprečju med 14 do 16 mesecev – in je med temi tudi najbolj pogost. Veliko raziskav se osredotoča na iskanje učinkovitejših možnosti zdravljenja, zato smo v tem delu raziskali vlogo in mehanizme delovanja kemokina CCL5 in njegovega receptorja CCR5 pri GBM. Kemokini so funkcionalna družina citokinov, ki sprožijo kemotakso in so odgovorni za migracijo, tj. usmerjeno gibanje, in infiltracijo imunskih celic, v kontekstu raka pa tudi tumorskih celic. CCL5 in CCR5 imata dokazano pro-tumorsko vlogo pri mnogih vrstah raka, tudi pri GBM, a tu njuna vloga in način medcelične signalizacije še nista bila razjasnjena. Z imunofluorescenco smo na tkivnih rezinah GBM proučili kolokalizacijo med proteini, ki so označevalci določenih zvrsti celic v tumorju in kemokina CCL5 in/ali receptorja CCR5. Ugotovili smo, da CCR5 izražajo tudi GBM matične celice (GSC), kar nakazuje specifične parakrine in avtokrine mehanizme signalizacije. Osredotočili smo se tudi na proučevanje invazivnosti celic GBM v sokulturi z mezenhimskimi matičnimi celicami (MSC) in dokazali signifikantno povečanje invazivnosti tumorskih celic. Ta invazivnost je bila tudi posledica signalizacije CCL5/CCR5 med GBM in MSC celicami, saj je bila po dodatku antagonista receptorja CCR5 znatno inhibirana. Kot antagonist smo uporabili spojino maraviroc (MRV), ki je sicer že odobrena učinkovina za zdravljenje pri okužbi z virusom HIV. Poleg izločane oblike citokina CCL5 smo z mikroskopsko analizo GBM tkivih rezin CCL5 zaznali tudi v jedru. Z bioinformatsko analizo potencialne vloge CCL5 v jedru smo ugotovili, da obstaja podlaga za morebitno vezavo proteina CCL5 na molekulo DNA. Pomen naše raziskave je morebitno izboljšanje oz. kombinirano zdravljenje GBM učinkovitost sedanjih pristopov (operacija, obsevanje, kemoterapija, imunoterapija) bi namreč lahko učinkovito izboljšali z uporabo oz. repozicioniranjem MRV za dodatno zdravljenje GBM, ki inhibira os CCL5/CCR5 in posledično inhibira od strome odvisno invazijo celic GBM. Glioblastoma (GBM) is a cancer that presumably originates from astrocytes, and is the most common brain cancer and a cancer with the worst prognosis of all – on average 14-16 months. Many studies seek to find ways to improve GBM therapy, which was also the motivation for our research on the role and mechanism of action of chemokine CCL5 and its cognate receptor CCR5 in GBM. Chemokines are a functionally distinct family of cytokines that induce chemotaxis and are responsible for physiological cell migration and immune cell trafficking, as well as tumour cell migration in cancer. CCL5 and CCR5 are proven to have pro-tumour roles in many cancers, including GBM, but the exact role and cell cross-talk mechanism have not yet been determined. Using immunofluorescence on GBM tissue slides, we studied the colocalization of proteins that are markers of various cel types within the tumour and CCL5 and CCR5 we observed that also GBM stem cells (GSC) express CCR5, which indicates specific paracrine and autocrine signalisation mechanisms. Our work was also focused on the studying of the invasivness of GBM cell in coculture with MSC cells and we have proven a significant increase in tumour cell invasiveness. The invasiveness was a result of the CCL5/CCR5 signalisation between GBM cells and mesenchymal stem cells (MSC), as the adding of the CCR5 receptor antagonist Maraviroc caused it to be notably inhibited. Maraviroc is already an approved drug used in the therapy of HIV patients. Along the secreted form of CCL5, using microscopic analysis we observed CCL5 also in the nucleus. Our bioinformatic analysis of the potential role of CCL5 in the nucleus has shown a basis for the possible DNA-binding ability of CCL5. The role of our findings is possibly a better GBM therapy the effectiveness of current methods (surgery, radiation, chemo- and immunotherapy) could be effectively improved with repositioning MRV for an additional GBM therapy, which inhibits the CCL5/CCR5 axis and consequently inhibits the stroma-induced GBM cell invasion.

Mezenhimske matične celice zaradi svojega širokega potenciala diferenciacije v različne vrste celic in produkcije rastnih faktorjev in različnih komponent zunajceličnega matriksa, ponujajo številne možnosti uporabe v terapevtske namene. Poleg njihove uporabe pri regeneraciji tkiv in modulaciji imunskega sistema, lahko služijo tudi kot dostavni sistemi pri zdravljenju rakavih obolenj, saj raziskave kažejo, da mezenhimske matične celice pogosto migrirajo k tumorskim tkivom. Citokin interlevkin 12 (IL-12) zaradi svoje sposobnost aktivacije naravne in pridobljene imunost ter stimulacije produkcije citokina interferona ?, ki koordinira protitumorsko obrambo, predstavlja idealnega kandidata za tumorsko imunoterapijo. Cilj naloge je bil z ne-virusnimi metodami pridobiti linijo stabilno transfeciranih pasjih matičnih celic, ki bi v zadostni meri proizvajale IL-12, da bi lahko potencialno služile v namen protitumorske terapije pri psih. V nalogi smo uporabili metodo elektroporacije in lipofekcije. Z uporabo reporterskega plazmida pEGFP N1 smo optimizirali pogoje elektroporacije, ki smo jih kasneje uporabili pri elektroporacijah s terapevtskim plazmidom pcDNA3.1zeo+sccaIL12. V optimizacijo smo vključili elektroporacijske parametre, število celic/elektroporacijo, količino plazmidne DNA/elektroporacijo in vrsto elektroporacijskega pufra. Stabilne linije pasjih matičnih celic, ki bi izražale caIL-12, nam po večkratnih poskusih elektroporacije in selekcije ni uspelo pridobiti, vendar smo s qPCR metodo uspeli dokazati prehodno izražanje caIL-12 na mRNA nivoju. Rezultati magistrskega dela so postavili dobre temelje za transfekcijo pasjih matičnih celic pridobljenih iz maščobnega tkiva. Mesenchymal stem cells, due to their wide potential for differentiation into different cell types and the production of growth factors and various components of the extracellular matrix, offer many possibilities for therapeutic use. In addition to their use in tissue regeneration and in the modulation of the immune system, they can also serve as delivery systems for the treatment of cancer, as studies have shown that mesenchymal stem cells often migrate towards tumor tissue. Cytokine interleukin 12 (IL-12), due to its ability of activation natural and adaptive immunity and stimulation of the production of cytokine interferon ?, which coordinates anti-tumor defense, is an ideal candidate for tumor immunotherapy. The goal of our work was to obtain a stable transfected line of canine stem cells with non-viral methods, that adequately express IL-12 and could potentially be a part of anti-tumor therapy in dogs. We used the method of electroporation and lipofection. Using the reporter plasmid pEGFP N1, we optimized the conditions of electroporation, which were later used in electroporations with the therapeutic plasmid pcDNA3.1zeo + sccaIL12. The optimization included the electroporation parameters, the number of cells/electroporation, the amount of plasmid DNA/electroporation, and the type of electroporation buffer. We were not able to obtain stable transfected canine stem cells that express caIL-12 despite repeated attempts of electroporation and selection. However, with the qPCR method we successfully proved transient expression of caIL-12 at the mRNA level. The results of the master thesis have laid out good foundation for the transfection of canine stem cells derived from fat tissue.

Mezenhimske matične celice MSC so celice, ki nam v kliniki omogočajo zdravljenje različnih poškodb in bolezni. Zaradi svoje imunosupresivnosti, so pomemben dejavnik pri zdravljenju bolezni presadka proti gostitelju GvHD, ki lahko nastane po alogenski transplantaciji tkiva. Iz buffy coata, suspenzije levkocitov, granulocitov in nekaj eritrocitov, smo izolirali imunske celice PBMC dveh različnih darovalcev. Ustvarili smo mešano limfocitno reakcijo, ki smo ji dodali po pet različnih koncentracij s 30 Gy obsevanih MSC. Reagent timidin se je vgradil v imunske celice in na podlagi količine, ki jo izmerimo, sklepamo na proliferacijo PBMC celic. Z njo smo ugotavljali inhibitorni učinek MSC na imunske celice. Optimalno število MSC je bilo 10⁵, če bi bila večja, bi prišlo do kontaktne inhibicije. Proliferacija z višanjem števila MSC premosorazmerno pada. Mesenchymal stem cells MSC are cells, which help us in treating various injuries and deseases in clinic. Because of their immunosupressiveness, they are important factor in GvHD treatment, which may occur after allogenic tissue transplantation. We isolated immune cells PBMC from two different donors. Isolation was made from the buffy coat, a suspension of leukocytes, granulocytes and still some erytrocytes. We created a mixed lymphocyte reaction, where we aded five different concentration of 30 Gy irradiated MSC. The thymidine reagent, which was used, was incorporated into the immune cells. From an amount which was mesured, we concluded proliferation of PBMC. With proliferation, we detected an inhibitory effect of MSC to immune cells. Optimal MSC number was 10⁵. In the case of higher quantity, there would be a contact inhibition, which would cause cells to die. Proliferation with increased MSC quantity, was dropping disproportinately.

V zadnjem desetletju postajajo mezenhimske matične celice (MMC) zaradi sposobnosti diferenciacije v različne celice in obsežnega parakrinega delovanja vse bolj uporabne v regenerativni medicini. Uspešno so bile izolirane iz skoraj vseh vrst tkiv, vendar problem predstavlja njihova maloštevilčnost, in pomankanje standardiziranih postopkov za določanje koncentracije MMC v sveže izoliranih vzorcih. Namen naše raziskave je bil vpeljati postopek za štetje MMC v vzorcih kostnega mozga, stimulirane periferne krvi in maščobnega tkiva s pretočnim citometrom, z uporabo protiteles proti celičnim označevalcem CD271, CD45 in fluorescentnih kroglic za natančno štetje celic. Po vpeljavi postopka smo naše rezultate uspešno primerjali z rezultati testa določanja števila fibroblastov, ki tvorijo kolonije (CFU-F). V vzorcih smo vzporedno določali tudi koncentracijo krvotvornih matičnih celic. Največjo koncentracijo MMC smo določili v vzorcih maščobnega tkiva, najmanjšo v vzorcih periferne krvi. Za razliko od koncentracije krvotvornih matičnih celic in števila CFU-F je bila korelacija med koncentracijo MMC in starostjo darovalcev vzorcev statistično neznačilna, vendar bi bilo potrebno v nadaljno raziskavo vključiti večje število in starostno bolj raznolike darovalce. In the last decade Mesenchymal Stem Cells (MSC) are being used more extensively in regenerative medicine. This is due to their ability to differentiate into various cells and due to their extensive paracrine function. They have been successfully isolated from almost every type of tissue, the problem, however, lies in their small numbers and the lack of standardised procedures to determine MSC concentration in freshly isolated samples. The aim of our research was to introduce a procedure for counting MSC in the samples of bone marrow, stimulated peripheral blood and fat tissue with a flow cytometer, using antibodies against cell markers CD271, CD45 and florescent beads for precise cell count. After introducing the procedure, we successfully compared our results with the test determining the count of colony-forming units - fibroblasts (CFU-F). At the same time, we used the samples to determine the concentration of haematopoietic progenitor cells. The highest concentration of MSC was found in the sample of fat tissue, the lowest in those of peripheral blood. Unlike the concentration of haematopoietic progenitor cells and the count of CFU-F, the corelation between the concentration of MSC and the age of the donor samples was statistically insignificant. To get more conclusive results, we should expand our research to a larger group of donors from many different age groups.

Prikazan je pomen uporabe mezenhimskih matičnih celic v terapiji različnih bolezni in poškodb kjer zaradi njihovega imunomodulatornega, antiapoptotičnega delovanja in diferenciacijskega potenciala pripomorejo k hitrejši regeneraciji. Za njihovo uspešno uporabo je potrebno znanje o postopkih izolacije, karakterizacije, diferenciacije in shranjevanja celic, ter o različnih dostavnih sistemih, ki se razlikujejo med različnimi terapijami. Diplomsko delo prav tako vsebuje prednosti in slabosti uporabe biomaterialov kot nosilcev pri različnih načinih aplikacije celic, ki lahko zaradi odprtega sistema pripomorejo k njihovemu boljšemu nadzoru in diferenciaciji. Na koncu je delovanje mezenhimskih matičnih celic pri psu prikazano na primeru osteoartritisa, periodontalnih bolezni, regeneracije kože, kroničnih poškodb, kroničnega vnetja črevesja in tumorja. The importance of therapeutic use of mesenchymal stem cells is outlined through their immunomodulatory and antiapoptotic effects as well as differentiation potential, which all contribute to faster regeneration. For successful therapeutic use the knowledge about isolation of mesenchymal stem cells, their characterization, differentiation and preservation is needed. Due to their specific effects different application systems which are adapted to different therapies play an important role. Strengths and weaknesses of biomaterials for different applications are discussed as they can be beneficial because of better control over the cells and their differentiation in an open system. At the end the therapeutic effect of mesenchymal stem cells are presented in the case of osteoarthritis, periodontal disease, skin regeneration, chronical wounds, chronic bowel disease and tumor.

Terapija prionskih bolezni še ni poznana, se pa v zadnjem času raziskuje veliko novih principov zdravljenja. Eden od teh je zdravljenje s protitelesi proti prionom, ki bi lahko preprečila širjenje PrPSc. Mezenhimske matične celice (MMC) so potencialni vektor za dostavo teh protiteles v možgane zaradi lastnosti samoobnavljanja, diferenciacije, imunomodulacije, sposobnosti prečkanja krvno-možganske pregrade ter pato-tropizma. V naši raziskavi smo skušali vstaviti zapis za fragment scFv protitelesa V5B2 v mišje MMC s pomočjo elektroporacije. V prvem delu smo optimizirali pogoje elektroporacije, in sicer smo uporabili 8 kvadratnih pulzov, dolgih 1 ms, z močjo električnega polja 1,0 kV/cm in frekvenco 1 Hz. Celice v koncentraciji 3 x 106 celic/ml smo porirali v visoko prevodnem pufru. Koncentracija plazmida z vstavljenim GFP je bila 30 µg/ml. Elektroporacijo smo ponovili s plazmidi z vključki za znotraj- in zunajcelično izražanje scFv V5B2. Celice smo okarakterizirali s pretočno citometrijo in ugotovili, da ima elektroporacija le majhen vpliv na fenotip MMC. Prisotnost scFv V5B2 v celicah in gojišču smo skušali dokazati z imunocitokemijsko analizo in testom ELISA, a nismo bili uspešni. Glede na naše rezultate se scFv V5B2 ne izraža v mišjih MMC. Za uspešno izražanje bi bila potrebna nadaljnja optimizacija metode transfekcije, morebitna zamenjava vektorja konstruktov ter optimizacija analitičnih metod za detekcijo izražanja. There is no known therapy for treatment of prion diseases, but recently a lot of new principles of treatment have been discovered. One of these are anti-prion antibodies which could prevent spreading of PrPSc. Mesenchymal stem cells (MSC) are potentially a delivery system for these antibodies to the brain, due to their self-renewal, differentiation, immunomodulation properties, pathotropism and ability to cross the blood-brain barrier. In this thesis, we attempted to insert a molecular construct for scFv fragment of anti-prion antibody V5B2 into mouse MSC by electroporation. In the first part, we optimized electroporation parameters, by using 8 square wave pulses, 1 ms long, with 1,0 kv/cm electric field strength and 1 Hz frequency. Cells were resuspended in high conductivity electroporation buffer to a final concentration of 3 x 106 cells/ml and 30 µg/ml of GFP plasmid was added. Electroporation was repeated with plasmids containing intracellular and secretory scFv V5B2. The cells were characterized by flow cytometry where, after electroporation, only a small difference in phenotype of mouse MSC was observed. We tried to determine the expression of scFv V5B2 in and outside of cells by immunocytochemistry and ELISA, but were not successful. According to our results, the scFv V5B2 is not expressed in mouse MSC. Further optimization of transfection methods, possible replacement of vectors and optimization of analytical methods for detection of expression is required.