Abstract:
The human retina is a complex tissue within the human eye and essential for the sense of sight. Retinal diseases, such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP) affect or destroy the sense of vision and impair the quality of life of patients drastically. As currently for many retinal diseases there are only few, or no treatment options available and animal models recapitulate the pathology of those diseases poorly. Therefore, adequate in vitro models of the human retina and the supporting retinal pigment epithelium (RPE) are urgently required.
In AMD, pathological changes do not exclusively involve cells of the neural retina, but initial disease manifestation takes place within the retinal pigment epithelium (RPE) and its near surroundings. Therefore, various protocols for the differentiation of RPE cells from human pluripotent stem cells (hPSCs) have been developed in recent years, showing the generation of mature and functional RPE cells.
In this thesis, two distinct approaches for the differentiation of hiPSC-RPE have been applied and were subsequently investigated for several essential characteristics of RPE cells. These essential RPE hallmarks included molecular characteristics, pigmentation, and morphology. Overall analysis demonstrated that both approaches equivalently generated RPE cells of good quality.
Nevertheless, RPE cells which are cultured on plastic surfaces in 2D pose several disadvantages, as those conditions promote e.g. transdifferentiation or detachment from the culture plate. Consequently, long-term cultivation over several month to years remains difficult. In this thesis, those drawbacks were aimed to overcome by implementing a novel three-dimensional RPE organoid (RPEorg) approach. RPE organoids were differentiated simultaneously with retinal organoids and cultured for more than 300 days.
A basic characterization of early (day 80-100), intermediate (day 191) and aged (day 280-360) RPEorg was performed by immunostainings and qRT-PCR analysis. RPEorg expressed several common RPE markers, some of them strongly increasing with age. Deeper analyses of RPEorg showed ultrastructural signs of mature RPE cells, such as apical microvilli and tight junctions, as well as melanosomes. Functionality of RPE cells comprised in RPEorg was demonstrated by phagocytosis of bovine photoreceptor outer segments (POS). Overall, maturation of RPE cells with age could be observed.
As a next step, RPE organoids were investigated for age-related changes in the context for AMD. Therefore, RPEorg were stained for drusen-associated proteins, such as APOE and TIMP3, lipids and hydroxyapatite. Those data were supported by qRT-PCR analysis and showed presence and partly upregulation of several disease relevant markers with aging. Additionally, transmission electron microscopy revealed ultrastructural signs of drusen formation in aged RPEorg.
To summarize, RPEorg are a suitable model for mature RPE and allows for investigations of age-associated changes related to AMD. Long-term cultivation of RPEorg enables studies of early stages of disease manifestation and drusen formation, which renders it suitable for pathomechanistic as well as drug developmental studies.
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