Molecular mechanisms involved in epithelial differentiation of human induced pluripotent stem cells

The Skin is the largest organ in the mammalian body and serves as a barrier to protect the body from dehydration, mechanical trauma, and microbial invasion. In a healthy organism, the upper skin level - the epidermis regenerates constantly through the duration of life via continual proliferation, migration and differentiation. To date, skin replacements for patients with large burn injuries are generated with patients own keratinocytes, if possible. But the isolation, expansion in culture, and generation of the skin equivalent are time consuming. Decellularized cadaver skin is used to cover the wounds in the meantime. However, cadaver skin is rare and can cause an immune response. The application of induced pluripotent stem cells (iPSCs) in regenerative medicine and tissue engineering offers great possibilities. IPSCs display a nearly inexhaustible source of pluripotent cells, which can be differentiated into nearly every cell type of the mammalian organism. This enables to study differentiation processes during embryogenesis, especially in humans, which cannot be addressed in vivo. Furthermore, patient-specific iPSCs can be generated and used to establish patients-specific skin cultures. The differentiation of iPSCs into keratinocytes is a critical process. In order to use these iPSC-derived keratinocytes for skin replacements a pure cell population is required. Existing protocols for the differentiation of pluripotent stem cells into keratinocytes have been improved during the last years. Nevertheless, these protocols do not lead to a pure population of iPSC-derived keratinocytes. The aim of this project was to find new regulators, which are involved in the differentiation of pluripotent cells into the epithelial lineage. Therefore I generated hiPSCs from fibroblasts of healthy donors and validated their pluripotent state by investigating the expression of pluripotency associated markers on RNA and protein level. Furthermore, I performed the termatoma assay, the most stringent test for pluripotency. The generated iPSCs were differentiated by using a published protocol for 30 day, by culturing them in a defined medium supplemented with BMP4 and retinoic acid. The generated keratinocytes were shown to express basal and suprabasal keratinocyte markers on RNA and protein level. In order to find new factors which are involved in the development of early epithelial cells, I adapted a high-throughput siRNA screen to the iPSC differentiation. This screen enabled the knock-down of nearly 800 kinases during the differentiation of iPSCs into an epithelial lineage. HIPK4 was one of the top fifty candidates, which resulted in enhanced expression of the embryonic epidermal marker keratin 18. In further investigations, I could show that HIPK4 resulted not only in enhanced expression levels of keratin 18, but also the expression of other epithelial markers was increased. I could demonstrate that the knock-down of HIPK4 especially in the first few days has an enhancing effect on the differentiation and that this effect is only present in the presence of retinoic acid in the differentiation medium