Ph.D Thesis

Ph.D StudentBen-Yosef Oren
SubjectGeneration of Retinal Pigment Epithelial Cells from Human
induced pluripotent stem cells for the study of
Inherited Macular Degeneration
(Best Disease)
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Ido Perlman
Full Thesis textFull thesis text - English Version


Best Vitelliform Macular Degeneration (BVMD) is a rare autosomal dominant macular dystrophy, typically characterized by the presence of a vitelliform (yolk-like) lesion in the macula. The material is gradually absorbed, leaving an area of retinal pigment epithelium (RPE) atrophy and subretinal fibrosis, leading to loss of visual acuity and central scotoma, while peripheral visual fields remain normal. The flash electroretinogram is normal, but the electrooculogram shows a reduced or absent light rise, indicating a widespread dysfunction of the RPE. The mutated protein responsible for Best disease is Bestrophin-1 (hBest1), a protein that was shown to play several biological functions, including calcium dependent chloride channel, intracellular calcium homeostasis, and cell volume regulator. It is unclear how does mutated Bestrophin lead to photoreceptor degeneration and why the lesion is confined to the macula, as all RPE cells in the retina express the mutated Bestrophin protein.  The importance of this research is also as a potential model to Age related Macular Degeneration (AMD), an acquired disorder, leading to loss of visual acuity in people over 65 in the industrialized countries. Both diseases are macular diseases, despite having all the RPE layer affected, that are characterized by lipofuscin accumulation.

            We employed known technologies to turn fibroblasts from skin biopsies of healthy volunteers and Best disease patients into viable induced Pluripotent Stem Cells (iPSCs). The iPSCs were validated by various means including immunofluorscent staining and teratoma assays. They were then differentiated into RPE-like cells by medium manipulation. The RPE-like cells were allowed to mature and were examined genetically and functionally to ascertain their properties.

            Our findings showed that the Bestrophin mutation of the patient was consistent throughout the complex process leading from skin fibroblasts to RPE-like cells. The differentiated induced-RPE (iRPE) cells showed similar pigmentation, morphology and RPE markers as native RPE cells. The iRPE cells were grown in a special chamber to allow them to form a polarized monolayer. The layer clearly showed Bestrophin expression in the choroidal side and MerTK in the retinal side. We measured transepithelial potential (TEP) and transepithelial resistance (TER) across the RPE monolayer, and found lower TER and TEP in the layer of Best-iRPE compared to healthy-iRPE. We also tested the rate of phagocytosis of photoreceptors outer segments (POS), an important role of RPE cells, and found that Best-iRPE exhibited a higher rate of phagocytosis of POS then the healthy-iRPE.

            Our findings support our working hypotheses that it was possible to derive RPE-like cells from patients suffering from Best macular dystrophy, and that these RPE cells were very similar in their protein repertoire to the native RPE cells. We suggest that the increased phagocytosis of POS by the Best-iRPE cells may be the contributing factors to the lipofuscin accumulation in the RPE of patients with Best Disease, due to the incapability of the RPE to handle the rapid phagocytosis of POS. The decreased TEP and TER of the Best-iRPE indicate altered physiological role of Best-RPE layer.