Photoreceptors are sensory neurons in our eyes’ retinas that convert light into electrochemical signals thus allowing us to see the world around us. Human retinas have two types of photoreceptors: rods important for night vision and cones important for high-acuity daylight vision. In some retinal diseases, photoreceptors degenerate leaving patients visually impaired or even blind. One of the promising therapeutic approaches is cell therapy, which acts by supporting surviving or by substituting lost photoreceptors by transplanting donor photoreceptors produced in vitro. Human induced pluripotent stem cells (hiPSCs) represent a favorable donor cell source for transplantation, as they are patient-specific and have the ability to self-renew. While photoreceptors isolated from human retinal organoids represent bona fide cells for cell therapy, long time needed for their production, which coincides with developmental time, hampers their clinical application. Another approach for hiPSC differentiation is by overexpressing different transcription factors (TFs), the so-called forward programming. Although proved fast and efficient in producing multiple neuronal cell types, efficient forward programming protocols for engineering photoreceptors were so far not established. Therefore, aim of my thesis was to find TFs that drive in vitro photoreceptor differentiation from hiPSCs and to establish a fast forward programming protocol for producing photoreceptors in high yields. To find TFs that drive photoreceptor differentiation, I performed a TF-library-on-library screening in a reporter hiPSC line. The reporter hiPSCs expressed fluorescent markers only if synthetic photoreceptor-specific promoters were activated, i.e. in case of photoreceptor differentiation. The specificity of the reporter was confirmed in human retinal organoids. For the screening, I transduced the reporter cell line with two lentiviral libraries: a biased one consisting of 16 TFs known from in vivo photoreceptor development and an unbiased one consisting of 1756 TFs. After overexpressing TFs, cells that activated photoreceptor promoters were fluorescently sorted and analyzed. As 80 % of the sorted cells were positive for photoreceptor-specific genes, the screening was highly specific. Furthermore, the screening identified TFs that I validated in the reporter cell line as single, double and triple combinations to find the most efficient one in driving photoreceptor differentiation. The double combination of OTX2 and NEUROD1, known players in photoreceptor development, activated the cone reporter in 10 % of the cells, while additional overexpression of GON4L increased the activation to 25 %. GON4L was never before associated with photoreceptor development showing that in vitro differentiation might be uncoupled from its in vivo counterpart. The cone differentiation efficiency was increased to 50% by treating the cells with AraC, a cell cycle inhibitor that removes all proliferating cells from the cultures. Whether the cell will activate the cone reporter depended on expression levels of individual TFs. Higher and unequal levels, with NEUROD1 having the highest expression, were favorable for obtaining cells with activated cone reporter. Thus, by producing monoclonal cell lines, I identified competent clones with differentiation efficiencies higher than that of a polyclonal cell line and going up to 58%. Except activating the cone reporter, the cone precursor-like cells differentiated from hiPSCs by overexpressing the three TFs OTX2, NEUROD1 and GON4L acquired neuronal morphology and expressed photoreceptor precursor markers. As precursors are the optimal developmental time point for transplantation studies, the engineered cells were transplanted into mouse model of cone degeneration to assess their possible therapeutic potential. Some of the transplanted cells survived in vivo in the subretinal space but did not show any maturation or integration into the remaining retinal circuitry. Thus, further maturation of the cells in vitro is needed before the transplantation. So far, cone precursor-like cells showed ability to mature in vitro when co-cultured with retinal pigment epithelial cells derived from hPSCs and when cultured in presence of additional previously published growth factors. Therefore, changing culturing conditions from stem cell to photoreceptor-specific showed beneficial for further in vitro maturation and paved the way for further research. In conclusion, this study advanced TF-mediated cone photoreceptor engineering. It showed that overexpressing the three TFs OTX2, NEUROD1 and GON4L is enough to push differentiation of more than 50% of hiPSCs into cone precursor-like cells in only 10 days. Additional research to improve maturity and homogeneity of engineered cells – overexpressing additional TFs and changing culturing conditions is ongoing. Fast and efficient protocol established in this study is beneficial for bringing in vitro differentiated cone photoreceptors closer to their commercial application. Such engineered cones could be used as biomedical testbeds for drug discovery and research and represent a promising donor material for cell transplantation to treat blindness.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:89196 |
| Date | 23 January 2024 |
| Creators | Zuzic, Marta |
| Contributors | Ader, Marius, Busskamp, Volker, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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