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

Falta palabras clavesThe 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

Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs). Over the last four decades, an extensive body of literature regarding lipid peroxidation has shown its important role in cell biology and human health. Since the early 1970s, the total published research articles on the topic of lipid peroxidation was 98 (1970–1974) and has been increasing at almost 135-fold, by up to 13165 in last 4 years (2010–2013). New discoveries about the involvement in cellular physiology and pathology, as well as the control of lipid peroxidation, continue to emerge every day. Given the enormity of this field, this review focuses on biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE), summarizing not only its physiological and protective function as signaling molecule stimulating gene expression and cell survival, but also its cytotoxic role inhibiting gene expression and promoting cell death. Finally, overviews of in vivo mammalian model systems used to study the lipid peroxidation process, and common pathological processes linked to MDA and 4-HNE are show

Folic acid supplementation: some practical aspects

Since 1956, when Harman first postulated the free radical theory of aging, numerous studies have been carried out to test the protective action of antioxidants. One of these protective compounds used in antioxidant therapy is folic acid (FA). Folate deficiency can lead to several pathologies and its protective role is very well known. Because the negative effects of the synthetic form on the metabolism of folates and the controversy about the role of folic acid in cancer, the question is whether or not folic acid is good for everyone. In this paper we summarize some aspects of the biochemistry of folic acid and we show some precautions that should be taken into consideration when supplementing with this compound

The Neurokinin-1 Receptor Is Essential for the Viability of Human Glioma Cells: A Possible Target for Treating Glioblastoma

Background. Glioblastoma or glioma is the most common malignant brain tumor. Patients have a prognosis of approximately 15 months, despite the current aggressive treatment. Neurokinin-1 receptor (NK-1R) occurs naturally in human glioma, and it is necessary for the tumor development. Objective. The purpose of the study was to increase the knowledge about the involvement of the substance P (SP)/NK-1R system in human glioma. Methods. Cellular localization of NK-1R and SP was studied in GAMG and U-87 MG glioma cell lines by immunofluorescence. The contribution of both SP and NK-1R to the viability of these cells was also assessed after applying the tachykinin 1 receptor (TAC1R) or the tachykinin 1 (TAC1) small interfering RNA gene silencing method, respectively. Results. Both SP and the NK-1R (full-length and truncated isoforms) were localized in the nucleus and cytoplasm of GAMG and U-87 MG glioma cells. The presence of full-length NK-1R isoform was mainly observed in the nucleus, while the level of truncated isoform was higher in the cytoplasm. Cell proliferation was decreased when glioma cells were transfected with TAC1R siRNA, but not with TAC1. U-87 MG cells were more sensitive to the effect of the TAC1R inhibition than GAMG cells. The decrease in the number of glioma cells after silencing of the TAC1R siRNA gene was due to apoptotic and necrotic mechanisms. In human primary fibroblast cultured cells, TAC1R silencing by siRNA did not produce any change in cell viability. Conclusions. Our results show for the first time that the expression of the TAC1R gene (NK-1R) is essential for the viability of GAMG and U-87 MG glioma cells. On the contrary, the TAC1R gene is not essential for the viability of normal cells, confirming that NK-1R could be a promising and specific therapeutic target for the treatment of glioma.Junta de Andalucía BIO-15

Treatment with mesenchymal stem cells in an animal model of parkinson´s disease

Consejería de Economía, Innovación y Ciencia, Junta de Andalucía P10-CTS-649

Potential use of adipose tissue stem cells in the control of aging

Cell therapy with adult stem cells is a new battle front for the control of aging. Before being used for this purpose, we need to answer several basic questions about the biochemistry and physiology of these cells. This paper presents some aspects and preliminary results obtained in our laboratory using stem cells from adipose tissue.Ministerio de Ciencia e Innovación BFU 2010 2088

Effect of Age and Lipoperoxidation in Rat and Human Adipose Tissue-Derived Stem Cells

A wide range of clinical applications in regenerative medicine were opened decades ago with the discovery of adult stem cells. Highly promising adult stem cells are mesenchymal stem/stromal cells derived from adipose tissue (ADSCs), primarily because of their abundance and accessibility. These cells have multipotent properties and have been used extensively to carry out autologous transplants. However, the biology of these cells is not entirely understood. Among other factors, the regeneration capacity of these cells will depend on both their capacity of proliferation/differentiation and the robustness of the biochemical pathways that allow them to survive under adverse conditions like those found in damaged tissues. The transcription factors, such as Nanog and Sox2, have been described as playing an important role in stem cell proliferation and differentiation. Also, the so-called longevity pathways, in which AMPK and SIRT1 proteins play a crucial role, are essential for cell homeostasis under stressful situations. These pathways act by inhibiting the translation through downregulation of elongation factor-2 (eEF2). In order to deepen knowledge of mesenchymal stem cell biology and which factors are determinant in the final therapeutic output, we evaluate in the present study the levels of all of these proteins in the ADSCs from humans and rats and how these levels are affected by aging and the oxidative environment. Due to the effect of aging and oxidative stress, our results suggest that before performing a cell therapy with ADSCs, several aspects reported in this study such as oxidative stress status and proliferation and differentiation capacity should be assessed on these cells. This would allow us to know the robustness of the transplanted cells and to predict the therapeutic result, especially in elder patients, where probably ADSCs do not carry out their biological functions in an optimal way

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)