Developmental insights and biomedical potential of human embryonic stem cells : modelling trophoblast differentiation and establishing novel cell therapies for age-related macular degeneration

Sammanfattning: Understanding the molecular pathways responsible for lineage segregation in the preimplantation human embryo is critical in order to fully elucidate the mechanisms involved in pluripotency and differentiation of embryonic stem cells. A significant increase in our comprehension of such processes will lead to an improvement in the quality and efficiency of these cells for applications requiring stem cell maintenance and differentiation, such as regenerative medicine. Through responsible and ethical research, such new knowledge can then be translated effectively and efficiently into future advancements in health and medical practices. This thesis focuses on two different applications of human embryonic stem cells (hESC): first, as an in-vitro model to investigate the genetic requirements for human trophoblast formation and second, as a cell replacement therapy for age-related macular degeneration (AMD) through the establishment of efficient, scalable, and clinically compliant protocols for their differentiation into retinal pigment epithelium cells (RPE). In paper I, we used human embryonic stem cells to model trophoblast establishment and differentiation in order to better understand the mechanisms governing trophectoderm segregation in the embryo. Combining this in-vitro model with the use of pharmacological inhibitors and CRISPR/Cas9 genome editing, we demonstrated that blockade of the YAP1/WWTR1-TEAD complex impairs not only trophoblast differentiation, but also survival of undifferentiated stem cells. Furthermore, through systematic targeting of the different components of the complex, we described a dominant role for YAP1 in these processes and a striking genetic and functional redundancy of the function of TEAD proteins. Altogether, the findings indicate a role for the Hippo signaling pathway, both in human trophectoderm segregation and in maintaining human pluripotency. In papers II and III, we developed xeno-free and defined methodologies for the differentiation of human embryonic stem cells into RPE with the potential for use in replacement therapies for common retinal degenerative diseases, such as age-related macular degeneration. These invitro derived cells exhibited specific morphological and molecular features and functional properties that are typical of native RPE. In addition, upon subretinal transplantation into a large-eyed animal model, hESC-derived RPE cells were able to integrate and survive for extensive periods of time and rescued the neuroretina from the damage induced at the moment of injection. Finally, we identified a set of unique cell surface markers that were able to distinguish the RPE from other potential contaminating cell types or undifferentiated remnants and demonstrated their utility in monitoring differentiation efficiency and in increasing the purity and homogeneity of the final cell product. Through this work, we demonstrate that human embryonic stem cells hold enormous potential for modeling specific aspects of human development, which can help to elucidate the complex mechanisms governing lineage segregation and support the production of bona fide differentiated cell types to serve in future replacement therapies.