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Stem cells persist throughout life, replacing cells lost to homeostatic turnover, injury, and disease. However, their functions decline with age, which contributes to degeneration and dysfunction. The molecular mechanisms involved in the aging of stem cells are the same as the ones involved in the aging of somatic cells, including telomere shortening, oxidative stress, epigenetic dysregulation, miRNAs changes, alterations of DNA, RNA, proteome, and various cellular organelles. Aging impacts various pathways, such as insulin/insulin-like growth factor 1 (IGF-1), mTOR, FoxO, AMP-activated protein kinase (AMPK), sirtuin, and many others, resulting in senescent stem cells that exhibit functional and numerical impairment. Stem cells have developed special mechanisms to prevent age related damage accumulation and to sustain their stemness properties, however, these mechanisms lose their effectiveness over time. The most fatal consequence of this is found in the immune system, where both innate and adaptive immunity are affected, exhibiting a plethora of defects, including increased autoimmune disease occurrence, elevated tolerance to cancer and chronic inflammatory status. Stem cell therapies call for the best quality of stem cells grafts. Stem cell products should be devoid of cells containing a senescent phenotype, thus a comprehensive knowledge of the biology behind the senescence of stem cells should be taken into account in every cell based therapy.

As a precious biological substance, donated blood has to be used in a sustainable, safe and efficient manner. Therefore good transfusion practice in modern surgery, anaesthesiology and intensive care is of key importance. New therapeutic methods, operations, transplantation of organs and hematopoietic stem cells often require intensive and long term supportive therapy including transfusions of blood components. Nowadays, transfusion service is confronted with continuously increasing demand for sufficient quantity of safe high quality blood, and on the other hand with limited resources of blood. The article deals with the current modern approaches for optimization of transfusion practice and Patient Blood Management that have to be implemented in Slovenian hospitals.

Extracellular membrane vesicles are fragments shed from plasma membranes off all cell types that are undergoing apoptosis or are being subjected to various types of stimulation or stress. Even in the process of programmed cell death (apoptosis), cell fall apart of varying size vesicles. They expose phosphatidylserine (PS) on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cellular origin. Extracellular membrane vesicles have been isolated from many types of biological fluids, including serum, cerebrospinal fluid, urine, saliva, tears and conditioned culture medium. Flow cytometry is one of the many different methodological approaches that have been used to analyze EMVs. The method attempts to characterize the EMVs cellular origin, size, population, number, and structure. EMVs are present and accumulate in blood products (erythrocytes, platelets) as well as in fresh frozen plasma during storage. The aim of this review is to highlight the importance of extracellular vesicles as a cell-to-cell communication system and the role in the pathogenesis of different diseases. Special emphasis will be given to the implication of extracellular membrane vesicles in blood products and their clinical relevance. Although our understanding of the role of EMVs in disease is far from comprehensive, they display promise as biomarkers for different diseases in the future and also as a marker of quality and safety in the quality control of blood products.

Starostne spremembe imunskega sistema so vzrok za zmanjšano sposobnost obrambe proti okužbam, za pojav vnetnih bolezni in povečano incidenco raka. Obstaja več teorij, ki skušajo razložiti številne istočasne in prepletene procese staranja. Po teoriji staranja matičnih celic je za staranje tkiv in organov odgovoren upad sposobnosti matičnih celic, da bi uspešno obnavljale tkiva. Namen naše raziskave je bil na modelu laboratorijskih miši raziskati, ali lahko presaditev celic kostnega mozga (KM) mladih živali izboljša delovanje imunskega sistema starih živali ter s tem izboljša njihovo splošno zdravje in morda celo podaljša življenjsko dobo. Neobsevanim samicam BALB/c smo v 14., 16. in 18. (19.) mesecu starosti presadili celice KM mladih samcev. Vsem prejemnicam celic KM smo določili stopnjo himerizma v KM, v izoliranih populacijah limfocitov T, limfocitov B in nevtrofilcev ter v CFU kolonijah, pripravljenih iz KM prejemnic. Analizirali smo spremembe števila celic prirojenega in pridobljenega imunskega sistema ter funkcij imunskih celic po presaditvi (sposobnost endocitoze, citokinski odziv in test stimulacije proliferacije). Manjše število živali smo imunizirali s hemocianinom iz školjke (KLH) ter z analizo titra protiteles testirali njihov imunski odziv na antigen. Pri vseh miših smo redno ocenjevali 30 različnih parametrov ter določili indeks krhkosti. Spremljali smo njihovo življenjsko dobo ter ob smrti opravili raztelesbe ter beležili patoanatomske spremembe. Rezultati so pokazali, da so imele stare miši, ki so prejele celice mladega KM, izboljšane nekatere imunske parametre prirojenega in pridobljenega imunskega sistema v primerjavi s kontrolno skupino, vendar teh ni bilo dovolj, da bi lahko hipotezo raziskave potrdili. Prejemnice KM niso imele vidno boljšega splošnega zdravja, indeks krhkosti ni pokazal razlik med poskusno in kontrolno skupino in miši tudi niso živele dlje od kontrolne skupine miši. Age-related changes in immune functions are the cause of the increased vulnerability to infections, inflammatory diseases, and cancer incidence. There are many existing theories that try to explain the numerous, simultaneous, and interconnected aging processes. The stem cell theory of aging postulates that stem cells become inefficient at maintaining the original functions of the tissues. In our study, we used a mice model to observe whether non-conditioned bone marrow (BM) cell transplantation from young donors to old recipients would improve their immune system functions, improve their general health, and possibly even extend their lifespans. Female BALB/c mice received BM cells from young donors at 14, 16 and 18(19) months of age. We determined chimerism levels in the BM, isolated T cell, B cell and neutrophil populations, and in colony-forming units (CFU) in BM. We analysed the changes of relative cell number of the innate and adaptive immune systems, and conducted functional assays (endocytic capability, cytokine response, and cell proliferation). We immunized a small group of mice with keyhole limpet haemocyanin (KLH) antigen and tested their immune response to antigen. All mice were regularly assessed using 30 parameters to calculate a frailty index (FI). We noted their lifespans, conducted post-mortem examinations, and recorded any pathoanatomical changes. Our results showed that old mice, transplanted with the BM cells of young donors, had some improvement in the immune parameters of their innate and adaptive systems, however, the evidence was insufficient for us to confirm our hypothesis. Recipient mice did not experience a noticeable improvement in their general health, and the FI showed no significant differences between the transplanted and control mice. In addition, the transplanted group did not live longer than the control group.

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.

Presaditev alogenskih krvotvornih matičnih celic je način zdravljenja določenih malignih in nemalignih bolezni. Stanje po alogenski presaditvi, pri katerem so v prejemnikovem telesu sočasno prisotne celice prejemnika in darovalca krvotvornih matičnih celic, imenujemo himerizem. Z določitvijo himerizma lahko zgodaj zaznamo zaplete kot so ponovitev bolezni, bolezen presadka proti gostitelju in zavrnitev presadka. Sledenje himerizma je zato pomembno za oceno in napoved uspešnosti presaditve krvotvornih matičnih celic in omogoča hitrejše ukrepanje ter poveča možnost ozdravitve bolnika. V nalogi smo za sledenje himerizma po presaditvi krvotvornih matičnih celic uporabili metodo PCR v realnem času in ocenili primernost kompleta setov AlleleSEQR (Celera, ZDA) ter programske opreme AlleleSEQR Suite v1.1 Chimerism Analysis Software (Celera, ZDA) in KMRengine Chimerism Analysis Software (GenDx, Nizozemska). Za testiranje smo uporabili vzorce genomske DNA, izolirane iz periferne krvi 23 bolnikov in njihovih 23 darovalcev. Pri vseh parih smo med 34 bialelnimi polimorfizmi insercij/delecij določili informativne označevalce, ki omogočajo razlikovanje med prejemnikovo in darovalčevo genomsko DNA. Nato smo s testom za kvantitativno določanje himerizma v več zaporednih vzorcih prejemnika, odvzetih v različnih časovnih intervalih po presaditvi krvotvornih matičnih celic, določili delež prejemnikove genomske DNA. Rezultate določitve himerizma smo primerjali s klinično sliko bolnika. Informativnost nabora označevalcev je bila 100 % za vse pare prejemnikov in darovalcev ter je bila po pričakovanju za nesorodne pare večja kot za sorodne. Pri 19 od 23 bolnikov se je klinična slika popolnoma ujemala z rezultati določitve himerizma. Pri štirih bolnikih se klinična slika ni vedno ujemala z rezultatom določitve himerizma. Pri petih bolnikih bi bilo za popolno interpretacijo rezultatov določitve himerizma potrebnih več zaporednih vzorcev. Bolezen presadka proti gostitelju je bila prisotna pri večjem deležu bolnikov s popolnim himerizmom kot pri bolnikih z mešanim himerizmom. Občutljivost metode je bila visoka, saj smo zaznali že 0,02 % himerizem, kar daje zgodnjo informacijo o dinamiki deleža prejemnikove genomske DNA. S testnim sistemom smo uspešno opravili tudi zunanjo kontrolo kakovosti. Zaključimo lahko, da je testni sistem primeren za sledenje himerizma po presaditvi alogenskih krvotvornih matičnih celic pri slovenski populaciji prejemnikov krvotvornih matičnih celic. Allogeneic hematopoietic stem cell transplantation is used in treating certain malignant and non-malignant diseases. Chimerism after allogeneic haematopoietic stem cell transplantation is a condition, in which recipient and donor cells are present in recipient’s body. Chimerism analysis enables early detection of complications, such as disease relapse, graft-versus-host disease and graft rejection. Chimerism monitoring is therefore important for assessment and prediction of successfulness of hematopoietic stem cell transplantation and enables earlier intervention and thus the possibility of a patient being cured is higher. In our study we determined the usefulness of the AlleleSEQR (Celera, USA) kit and software AlleleSEQR Suite v1.1 Chimerism Analysis Software (Celera, USA) and KMRengine Chimerism Analysis Software (GenDx, Netherlands) for chimerism monitoring after hematopoietic stem cell transplantation with the use of quantitative PCR. Genomic DNA samples isolated from peripheral blood of 23 patients and their 23 donors were used for testing. Among 34 biallelic insertion/deletion polymorphisms informative markers were determined, which enables differentiation between donor and recipient genomic DNA. The proportion of recipient genomic DNA in several successive recipient samples, collected in different time intervals after transplantation, was then determined using the test for quantitative chimerism monitoring. The chimerism results were compared with patient’s clinical picture. The informativity of the assay was 100 % for all included donor/recipient pairs and was higher for unrelated pairs than for related. The results of the determination of proportion of genomic DNA matched with the patient’s clinical picture in 19 patients out of 23. The chimerism results did not always match with the clinical picture in four patients. In five patients we would need more successive samples to completely interpret the chimerism results. A smaller proportion of patients with mixed chimerism had graft-versus-host disease compared to the patients with complete chimerism. The sensitivity of the assay is high, as we detected 0,02 % chimerism, which enables earlier information about the change in proportion of recipient genomic DNA. The external quality control was successfully passed with the tested system. We can conclude that the test system is suitable for chimerism monitoring after allogeneic haematopoietic stem cell transplantation for Slovenian population of haematopoietic stem cell recipients.

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.