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Tissues of post mortem donors represent valuable alternative sources for the isolation of primary cells with mesenchymal stem/stromal cell (MSC)-like properties. However, the properties of primary cells derived from different tissues and at different post mortem times are poorly recognized. Here, we aim to identify the optimal tissue source between three knee and peri-knee tissues for the isolation of primary cells with MSC-like properties, and to define the influence of the time post mortem on the properties of these cells. We harvested tissues from subchondral bone marrow, synovium and periosteum from 32 donors at various post mortem times. Primary cells were evaluated using detailed in vitro analyses, including colony formation, trilineage differentiation, immunophenotyping and skeletal stem cell marker-gene expression profiling. These data show that the primary cells with MSC-like properties isolated from these three tissues show no differences in their properties, except for higher expression of CD146 in bone-marrow cells. The success rate of the primary cell isolation is dependent on the post mortem time. However, synovium and periosteum cells isolated more than 48 h post mortem show improved osteogenic and chondrogenic potential. This study suggests that knee and peri-knee tissues from donors even 3 days post mortem are strategic sources of MSCs for regenerative procedures.

Staranje je zapleten, več faktorski proces, ki prizadane celice, tkiva in organe. S staranjem organizma se stara tudi imunski sistem, ki je sicer ključen za obrambo organizma pred škodljivim okoljem. S starostjo povezane okvare imunskega sistema vodijo v nastanek večine s starostjo povezanih bolezni kot so rak, avtoimunske bolezni in srčno-žilne bolezni ter povročijo povečano dovzetnost posameznika za okužbe. Ena izmed možnih terapij, ki bi lahko izboljšala delovanje imunskega sistema v starosti, je presaditev mladih krvotvornih matičnih in progenitorskih celic (KMPC), ki bi jih posamezniku odvzeli in shranili v njegovi mladosti ter ponovno presadili v njegovi starosti. Sklepamo, da bi na ta način mlade in zdrave KMPC po presaditvi »pomladile« stari imunski sistem ter izboljšale njegovo delovanje, ob enem pa bi posamezniku podaljšali njegovo življensko dobo. Omenjeni princip se imunuje heterohrona avtologna presaditev mladih krvotvornih matičnih celic. V raziskavi smo v mišjem modelu uporabili nov način presaditve KMPC s predhodno mobilizacijo endogenih KMC v periferno kri. V ta namen smo uporabili molekulo CASIN, ki začasno izprazni nišo kostnega mozga prejemnika, na ta način pa omogoči večjemu številu presajenih celic pritrjanje vanjo. Z uporabo molekule CASIN smo po presaditvi mladih KMPC starim prejemnicam dosegli visok odstotek donorskega himerizma, dokazali nekatere izboljšave v imunskem sistemu in rahlo povišali mediano preživetja. Kljub temu pa bi bilo za prenos v humano medicino potrebno izvesti dodatne raziskave, ki bi potrdili ugodne učinke tovrstne terapije. Aging is a complex multi-factorial process that affects cells, tissues and organs. It also affects the immune system, which is crucial for the body's defense against the harmful environment. Age-related defects of the immune system lead to the development of most age-related diseases, such as cancer, autoimmune and cardiovascular diseases and cause an increased susceptibility to infections. One of the strategies that could improve the function of the immune system of an old individual is the transplantation of young hematopoietic stem and progenitor cells (HSCs) which would be collected from an individual in his/her youth and cryopreserved for a long time. After re-infusion later in life of the same individual, young and healthy HSPCs could »rejuvenate« the old immune system and improve its functioning. Furthermore, individuals lifespan could be prolonged. The process is called heterochronous autologous hematopoietic stem cell transplantation. In this study we used a new method of HSC transplantation with prior mobilization of endogenous HSCs into peripheral blood. For this purpose, we used the molecule CASIN, which temporarily empties the recipient's niche, thus allowing engraftment of a larger number of transplanted cells. With the use of the CASIN molecule, we achieved a high percentage of donor chimerism after transplantation of young HSPCs into old recipients. In the recipients of young HSPCs, we detected some improvements in the function of the immune system and a slighty higher median lifespan. Nevertheless, for translation into human medicine, additional research would be needed to confirm the beneficial effects of this type of therapy.

Izhodišča: Akutna mieloična levkemija je maligna bolezen belih krvnih celic, usmerjenih v mieloično vrsto. Potek bolezni je hiter in zahteva takojšnje zdravljenje. Za zdravljenje se vedno bolj uporablja presaditev krvotvornih matičnih celic. Namen zaključnega dela je predstaviti presaditev krvotvornih matičnih celic kot obliko zdravljenja akutne mieloične levkemije in prikazati zdravstveno nego pacienta z akutno mieloično levkemijo, ki se zdravi s presaditvijo krvotvornih matičnih celic. Raziskovalne metode: Zaključno delo je nastalo na osnovi študija literature. Uporabljena je bila deskriptivna metoda dela, metoda analize in kompilacije ter metoda sinteze. Rezultati: Ugotovili smo, da najboljše uspehe zdravljenja akutne mieloične levkemije s presaditvijo krvotvornih matičnih celic prinaša alogenična presaditev krvotvornih matičnih celic. Avtologna presaditev krvotvornih matičnih celic se uporablja redko. S presaditvijo krvotvornih matičnih celic se lahko pacienta z akutno mieloično levkemijo popolnoma ozdravi. Vloga medicinske sestre in svojcev je ključna ter nepogrešljiva skozi celoten proces zdravljenja, saj je pacient podvržen raznim psihičnim in fizičnim problemom presaditve krvotvornih matičnih celic. Diskusija in zaključek: Pomembno je, da se ljudje odločajo za vpis v register potencialnih darovalcev krvotvornih matičnih celic, saj le tako obstaja možnost, da pri pacientu opravijo alogenično presaditev krvotvornih matičnih celic, ki je zelo učinkovit način zdravljenja akutne mieloične levkemije. Theoretical starting points: Acute Myeloid Leukemia is a malignant disease of white blood cells directed to the myeloid species. The course of disease is quick and requires immediate treatment. Haematopoietic Stem Cell Transplantation is increasingly used for treatment. The purpose of the diploma thesis is to present the Haematopoietic Stem Cell Transplantation as a form of Acute Myeloid Leukemia treatment and to demonstrate the patient health care with an Acute Myeloid Leukemia treated with Haematopoietic Stem Cell Transplantation. Research methods: Diploma thesis is based on literature studies. The descriptive method of work, the method of analysis, compilation method and synthesis method were used. Results: We found out that the best success of treating Acute Myeloid Leukemia with the transplantation of the Haematopoietic Stem Cells is the allogeneic transplantation of the Haematopoietic Stem Cells. Autologous transplantation of the Haematopoietic Stem Cells is rarely used. With the transplantation of the Haematopoietic Stem Cells, the patient with Acute Myeloid Leukemia can be completely cured. The role of a nurse and relatives is crucial and indispensable throughout the entire treatment process, as the patient is subjected to various psychological and physical problems during the Haematopoietic Stem Cells Transplantation. Discussion and Conclusion: It is important that people decide to enroll in the register of potential donors of Haematopoietic Stem Cells, as only in this way there is a possibility for the patient to undergo allogeneic transplantation of the Hematopoietic Stem Cells, which is a very effective way of treating Acute Myeloid Leukemia.

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.

Mezenhimske stromalne celice (MSC) so eden glavnih virov za celične terapije in tkivno inženirstvo. Sposobne so samoobnavljanja in diferenciacije, lahko uravnavajo imunski odziv in sodelujejo pri celjenju poškodb. S staranjem se njihove regenerativne sposobnosti zmanjšajo. Pri terapijah z MSC se zato poraja vprašanje, kako starost darovalca in celic vpliva na izid zdravljenja. V magistrskem delu smo skušali ugotoviti, kako starost miši BALB/c vpliva na lastnosti njihovih MSC. S pretočno citometrijo smo spremljali izražanje celičnih označevalcev med gojenjem mladih in starih celic, spremljali smo proliferacijo celic s testom MTT, določali smo sposobnost migracije celic s testom celjenja ran in prisotnost senescentnih celic v kulturi. Ugotovili smo, da se mlade celice hitreje delijo, imajo nižji delež senescentnih celic v kulturi in slabše migracijske sposobnosti v primerjavi s starimi celicami. Med izražanjem celičnih označevalcev med skupinama nismo opazili razlik. Mesenchymal stromal cells are one of the main sources for cell therapy and tissue engineering. They are capable of self-renewal and differentiation. They can regulate a body's immune response and participate in its wound-healing process. Their regenerative abilities reduce as they age. MSC therapies therefore raise a question of how the age of the donor affects the outcome of the treatment. In this master's thesis, we tried to determine how the age of BALB/c mice affects the properties of their MSC. We monitored the expression of cell markers during cell culture with flow cytometry. We monitored cell proliferation rate with the MTT assay and analyzed mouse MSC's migration capacities with wound-healing assay. By measuring the β-galactosidase activity we determined the presence of senescent cells in culture. We discovered that young cells divide more rapidly, have a lower proportion of senescent cells in culture and have lower migration capacity when compared to older cells. The expression of cell markers is the same for both cell groups.