TEM-tabanlı çalışma Astrositlerin Fenotipi Induced Pluripotent Kök Hücrelerden Sağlıklı Bir Donörden ve Parkinson Hastalığı Olan Bir Hastadan Farklılaştırıldı.

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The aim of this study was to study the role of transmission electron microscopy (TEM) in assessment of the phenotype of astrocytes obtained with the directed differentiation technique from induced pluripotent stem cells (iPSCs) from a healthy donor and from a patient with a hereditary form of Parkinson's disease (PD).Materials and methodsMonolayer astrocyte cultures differentiated from iPSCs from a healthy donor and a PD patient having the G2019S mutation in the LRRK2 gene were used in the study. The obtained glial cultures were characterized using real-time PCR and immunocytochemical staining for glia-specific genes and proteins. TEM was used to examine astrocyte ultrastructure.ResultsPCR analysis and immunocytochemical staining demonstrated that cell lines received from a healthy donor and a PD patient expressed the required pattern of glia-specific genes and synthesized astrocyte-specific proteins. However, some glia-specific genes were expressed at reduced levels by mutant cells. One of the most typical ultrastructural features of astrocytes received from iPSCs from a PD patient was destructive changes in mitochondria, including mitochondrial clearing, swelling, and cristae destruction. In many cells, mitochondria were completely absent after a long culturing. Another characteristic feature of cells with a mutation in the LRRK2 gene was the accumulation of vacuoles with contents of varied electron density. Distinct changes in the ultrastructure of nuclei, protein-synthesizing organelles, and cytoskeletal elements were also seen in cultured astrocytes with a PD-associated LRRK2 mutation. Here, the morphometric study did not reveal any differences in the average cell area, nuclear area, cytoplasm area, or nuclear-cytoplasmic ratio between astrocytes of the control line and the PD mutation line.ConclusionReprogramming and obtaining of astrocytes from iPSCs received from a donor with a PD-associated mutation in the LRRK2 gene allow to assess the nature and dynamics of pathological morphochemical and ultrastructural changes caused by the mutation during gliogenesis. The use of combined techniques (PCR, immunocytochemistry, TEM) to compare cell cultures differentiated from iPSCs allow to assess, on the one hand, general culture parameters, such as the dynamics of culture differentiation based on changes in the expression level of specific genes and immunocytochemical markers, and on the other hand, morphofunctional changes at the level of individual cells. TEM demonstrates significant potential for studying cell cultures differentiated from iPSCs. This technique is instrumental for phenotyping the resulting cells based on their ultrastructure, assessing the degree of their morphological maturity, and identifying minor ultrastructural changes in cells, both pathological and differentiation-associated. The results of this TEM-based study indicate a pronounced decrease in mitochondrial viability and other ultrastructural abnormalities, thus confirming the idea of a significant role of astroglia in the development of the neurodegenerative process in the LRRK2-associated PD; hence, astroglia can be a basis for development of new approaches as well as for searching pharmacological targets in the pathogenetic therapy of the disease.

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