Estudio de la viabilidad y diferenciación de las células madre mesenquimales de tejido adiposo en condiciones de estrés oxidativo y envejecimiento. Posibilidad de su uso terapéutico e influencia del factor de elongación 2

Abstract

The present thesis is a compilation of several experiments about the use of adiposederived mesenchymal stem cells (ADSCs) in cell therapy. The study focus on the behavior, molecular mechanisms and environment changes of these cells during aging and under oxidative stress, that could determine its efficacy in Regenerative Medicine. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. In the first part, we presented and described in detail the isolation methods, expansion, differentiation and cryopreservation of adipos-derived stem cells (ADSCs). The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define human mesenchymal stem cells (hMSCs) for laboratory investigations and preclinical studies. ISCT believes this minimal set of standard criteria will foster a more uniform characterization of MSCs and facilitate the exchange of data among investigators. These criteria are adherence to plastic in standard culture conditions; “in vitro” differentiation into osteoblasts, adipocytes, and chondroblasts; specific surface antigen expression in which 95% of the cells express the antigens CD105, CD73, and CD90, with the same cells lacking (2% positive) the antigens CD45, CD34, CD14 and CD11b, CD79a or CD19, and HLA-DR. In case of murine models these criteria have not been established yet, though many authors try mimic them. There is considerable evidence that cellular senescence and replicative exhaustion impair the regenerative potential of adult stem cells, a characteristic they share with normal somatic cells. The effects of oxidative stress on MSCs are still unknown. Reactive oxygen species (ROS) are oxygen-derived small molecules, which react readily with a variety of chemical structures such as proteins, lipids, sugars, and nucleic acids, that lead to cellular damage in aging. Nowadays, it is increasingly recognized that ROS are involved in the regulation of cell function despite the fact that for many years they were considered to be harmful elements in biological systems. Despite the enormous effort that has thus far been invested into clinical trials, the importance of ROS in the outcome has not sufficiently considered. It has been proposed that the high antioxidant capacity of MSCs makes them ideal for the treatment of pathologies in which tissue damage is linked to oxidative stress. Nonetheless, not all levels of ROS cause cell damage by oxidation and nitration of macromolecules. It is currently believed that only unregulated levels of ROS are harmful, while regulated ROS production promotes essential signaling pathways, which regulate cell functions such as cell proliferation, differentiation, survival, and apoptosis. Redox regulation or controlled ROS generation is the net effect of a subtle balance between ROS generation and neutralization/utilization by cellular antioxidant systems. Thus, oxidative stress represents an unbalanced situation in which ROS generation exceeds antioxidant systems leading to tissue damage. The responses of adult human stem cells to different stress stimuli such as oxidative stress, heat shock, and γ-radiation have been widely studied in the context of tissue repair, tissue engineering, and transplantation. As far as this thesis is concerned, we show how MSCs promote the increased adipogenic fate and suppresses the osteogenic lineage mediated by ROS, due to a deregulation of pluripotencial factors Nanog and Sox2. Recently, MSCs therapies have come under criticism as, despite decades of research, relevant translational questions of MSCs biology and function remain unanswered. Previous studies showed that MSCs suffer from several drawbacks hampering clinical applications including, decreased number and quality of cells with donor age, and loss of proliferation and differentiation potential upon expansion “in vitro”. These observations pose a significant challenge that must be overcome in order to enable cellular therapies for older patients, the population mostly in need for tissue replacement. Hereby, aged-related variations could be identified, with correlation analyses of functional properties like protein homeostasis, resistance to oxidative stress, differentiation capability and pluripotentiality. Besides the individual age of the cell, stem and progenitor cells functions are influenced by the cellular environment, i.e. the niche and the architecture of the tissue they reside in. An other issue, we have addressed how to track the processes that happen before, during and after the stem cell engraftment using gene reporters in investigation. Optical imaging technologies combined with the use of genetically encoded fluorescent and luminiscent proteins have enabled the visualization of stem cells over periods of time in vivo and ex vivo. Bioluminiscence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo. Our results show that multimodal tracking of adipose-derived stem cells labeled in central nervous system, allows localization as well as cell identification after engraftment. This system has the advantages of avoiding the dilution with each cellular division, accurate to assess cell viability to track engraftment, have a linear relationship between substrate and photon emission and mark cells to track them by immunofluorescence and immunohistochemistry cells. Some disadvantages are gene reporter is inserted in DNA in unspecific places, not good promoters not good expression and signal, and the substrate distribution depends on many factors like delivery route, type of anesthesia and the host environment can also influence. To end up, MSCs should have the potential to stimulate the endogenous neurogenesis. It is commonly assumed that MSCs exert their therapeutic effects through immunomodulatory and trophic factor release rather than through cell replacement. It has been shown that ADSCs can ameliorate Parkinson disease symptoms by autologous transplantation into the rat. Thus far, the mechanisms of potential neuroprotective and regenerative effects of ADSCs are not fully understood because they have only been investigated several weeks after transplantation. In our case, we have studied the effect in a later period of time (four months). Although the acute effects would be particularly interesting, given that MSCs are often not detectable anymore at later time points, raising the question of how these cells actually achieve their results.