Sağlıklı İnsan Uyarılmış Pluripotent Kök Hücrelerinden (uPKH’lerden) NGN2, LHX3 ve ISL1 Üçlü Kasedi Kullanarak Motor Nöron Eldesi

Abstract

Neurodegenerative diseases are progressive and fatal diseases that reduce the standard of living of individuals and cause significant functional losses. Amyotropic lateral sclerosis (ALS) is a disease caused by motor neurone degeneration and affects upper and lower motor neurons. In addition to the Superoxide dismutase (SOD1) gene, approximately 40 gene mutations have been found to be effective in ALS. Spinal muscular atrophy (SMA) is an inherited disease caused by mutations in the SMN1 and SMN2 genes. SMA affects motor neurons and causes muscular dystrophy. It affects the controlled muscle movements of the individual's central, peripheral and skeletal musculature and is characterised by symptoms of progressive weakness and loss of muscle mass. Various methods have been used to study neurodegenerative diseases, but human induced pluripotent stem cells (iPSCs) represent a powerful alternative to understand the course of human diseases and to develop personalised therapies. In this thesis, motor neurons were obtained from healthy human iPSCs using Neurogenin 2 (NGN2), Islet 1 (ISL1), LIM homeobox 3 (LHX3) triple piggyBac plasmid cassette. iPSCs were transfected with the piggyBac triple cassette by lipid-mediated transfection method and stably transfected cells were selected by puromycin selection. In doxycycline-stimulated cells, transcription factors were overexpressed and iPSCs were transferred to neural lineage. The obtained motor neurons were characterized by analyzing neuronal gene expression and immunofluorescent staining with neuronal markers. With this method, preliminary data was provided and the method was optimized to be used to model motor neuron diseases for future drug trials.