Estudio de los cambios funcionales, anatómicos y moleculares en el sistema nervioso central en respuesta a distintos tipos de trasplante de células madre

Abstract

INTRODUCTION The retina, part of the CNS, offers an easily accessible window into the study of neuronal degeneration. Optic nerve crush (ONC) is a well-studied rodent model of RGC degeneration. Neuroprotective therapies attempt to prevent or delay neurodegenerative processes. However, cell therapies are not having the high success rates as expected. OBJECTIVES The general objectives of this thesis are: 1. To evaluate the use of perinatal tissues in ophthalmology by means of a bibliographic search of preclinical models. 2. To study the effect of BM-MSC cells in four types of transplants on the functionality and architecture of the healthy retina. 3. To evaluate the importance of the type of transplantation on the neuroprotective and neuroregenerative effect of BM-MSC. 4. To study the neuroprotective capacity of BM-MSC-derived vesicles after ONC in syngeneic transplantation. 5. To analyse the neurotoxic and neuroprotective effect of the TEG3 clone in healthy retina and after optic nerve crushing. 6. To evaluate differences in gene expression in microglia cells. MATERIAL AND METHODS In the experiments of this thesis, C57/BL6 pigmented, BALB/c albino, and C57BL/6-Tg(CAG-EGFP) transgenic mice were used. Sprague Dawley (SD) rats were also used. The cells chosen for the experiments were murine and human bone marrow-derived mesenchymal stromal cells (BM-MSCs) and the immortalised olfactory ensheathing glia line TEG3. RGC degeneration was induced by ONC at 0.5 mm from the eyeball. Injection of cells and vehicle was performed by intravitreal injection. Minocycline was delivered by intraperitoneal injection as was dexamethasone which was combined with oral cyclosporine A in water. In vivo experiments were performed for SD-OCT and function by ERG. Molecular studies of gene expression by qPCR and quantification of cytokines by immunosorbent assays (ELISA) and scRNA sequencing were performed. Immunofluorescence experiments were also performed on both slice and flat retinas. RGC quantification was performed automatically and their distribution was studied using isodensity maps and/or neighbour maps. A systematic literature search on the use of perinatal tissues (PnD) in preclinical models in ophthalmology was performed. Statistical analyses were performed in GraphPad® Prism v6. Differences were considered significant when p-value < 0.05. RESULTS In the literature review on the use of PnD we observed that most studies used hAM as whole tissue to treat corneal defects. In the study of syngeneic, allogeneic and xenotransplantation of hAM-MSC in the uninjured retina, we found that xenotransplantation is the most aggressive, decreasing the functionality of the tissue and altering the structure. Regarding the neuroprotection of BM-MSCs, syngeneic transplantation was the only one able to significantly protect both RGC subpopulations from death, and the number of regenerated axons was higher. Intravitreal injection of murine BM-MSC-derived extracellular vesicles showed better neuroprotective effects than the cells themselves at 5 days after ONC. In the study of intravitreal injection of the TEG3 clone, we observed that it activates microglia cells and causes a loss of RGCs at 21 days, but neuroprotects against death after ONC at 7 days. In the study of gene expression in xenotransplanted microglia cells, we found that 12 active cell groups (microglia/macrophages) are involved in this immune response. CONCLUSIONS There is a lack of homogeneity in preclinical studies of PnD in ophthalmology. The type of BM-MSC transplantation affects the structure and functionality of the healthy retina. Extracellular vesicles derived from BM-MSCs enhance the neuroprotective properties of BM-MSCs. Transplantation of TEG3 cells results in a reduction of the RGC population and neuroprotection after ONC in allogeneic. BM-MSC xenotransplantation into intact retinas activates the adaptive immune response pathway.