Immunoelectron microscopy labeling for the identification of oligodendrocytes and their precursor cells

Laburpena

Oligodendrocytes are the myelinating cells of the central nervous system. Besides myelination, they provide metabolic and trophic support to neurons. These cells originate from oligodendrocyte progenitor cells (OPCs) that are widely distributed in the white and gray matter. Oligodendrocytes and OPCs are severely affected in different pathologies in the human brain. These impairments are caused by several different pathogenic mechanisms such as genetic mutations, infections, neoplastic mutations, autoimmunity and traumatic injuries. Most of these dysfunctions result demyelination. Among the multiple techniques to study demyelination/remyelination, transmission electron microscopy (TEM) has emerged as the gold standard. However, in human tissue and in some cases in rodent tissue, the lack of optimal fixation difficult TEM characterization of the cells and their analysis. Hence, in the present thesis presented as a compendium of publications, we have used TEM to study several aspects of oligodendrocytes and OPCs. In the first article presented we studied and characterized the fine structure of human oligodendrocytes and OPCs via immunogold labeling. Our results suggest that there are 3 different ultrastructural differentiation stages in oligodendrocytes. The second publication is a book chapter in which we developed a tyramide-based amplification method to enhance immunogold labeling to detect oligodendrocyte lineage markers such as PDGFRa, NG2 and BCAS1. This method displays a better specificity than standard pre-embedding immunogold and can be performed in less time and using lower concentrations of antibodies. The third publication describes the existence of a clustered population of BCAS1-positive cells in a case of human oligodendroglioma for the first time. This points out the importance of oligodendrocyte progenitor markers in glial neoplastic entities. The last article, shows how immunoelectron microscopy can be used for subcellular tracking of the novel CRISPR system in hard to manipulate cells such as OPCs. Our results show how nuclear transportation is hardly achieved in primary OPCs compared to control standardized cell lines. Interestingly, OPCs package and process the enzyme Cas9 into endosomal compartments rather than directing it for nuclear transportation.