Ailesel Akdeniz ateşi hastalığına spesifik uyarılmış pluripotent kök hücrelerin üretimi ve karakterizasyonu

Familial Mediterranean Fever (FMF) is a recessively inherited auto inflammatory disease that is characterized by periodic fevers and local abdominal and joint pain. Mutations in the MEFV gene are responsible for the disease phenotype and encodes the Pyrin protein which has a role in regulating the inflammasome and the pro-inflammatory cytokine IL-1β . IL-1β is secreted by various immune cells to recruit other immune cells upon pathogen recognition. While secretion of IL-1β is strictly controlled in healthy people, immune cells of FMF patients secrete high levels of IL-1β. Even though it is demonstrated that various mutations in MEFV gene cause abnormal IL-1β secretion, the molecular basis of FMF is still unclear. To elucidate the mechanism of FMF, reliable in vitro models of the disease is needed. Induced pluripotent stem cells (iPSCs) are stem cells that can self-renew while retaining the ability to differentiate. The potential of differentiation, limitless proliferation and the ability to generate them in a patient-specific manner make iPSCs an important source for disease modeling. Disease modeling is the method to generate large numbers of disease-related cell types in vitro to analyze the underlying molecular mechanisms. Somatic cells of a patient can be reprogrammed into iPSCs which can then be differentiated into disease-relevant cell types. IPSC-based disease models allow for the investigation of disease-associated phenotypes by comparing healthy and patient-derived cells. Such models can also constitute in vitro platforms for the discovery of new drugs to treat the diseases in question. To develop a model of FMF, in this thesis, I generated iPSCs from an FMF patient bearing the homozygous M694V mutation and from the patient's healthy carrier parent as a control. Patient-derived fibroblasts were reprogrammed using episomal reprogramming vectors to generate integration-free iPSCs. FMF-iPSCs were characterized by gene expression analyses, immunofluorescence staining and teratoma formation assay all of which indicated that reprogrammed cells are bona fide iPSCs. To generate macrophage which is one of the FMF-relevant cell type from these iPSCs, three different methods including embryoid body formation, co-culture with OP9 cells and viral transfection have been tested but were unsuccessful. Further experiments are needed to generate in vitro derived functional macrophage from iPSCs. FMF-specific iPSCs can be used in the future studies to model this disease. Both healthy and patient iPSCs can be differentiated into FMF-related cell types including macrophages and neutrophils to discover the underlying mechanism of disease. In addition, these cells lines can be utilized in combination with drug screening and genome targeting strategies to open up new approaches to treat FMF disease.