Direct Conversion of Human Fibroblasts to Induced Neurons
Sammanfattning: During direct cellular reprogramming, forced expression of key transcription factors (TFs) directly converts one terminally differentiated cell type into that of another fate, exemplified in this theses by the conversion of fibroblasts into functional induced neurons (iNs). Direct conversion of mouse fibroblasts to functional neurons was established in 2010, and the starting point of my doctoral thesis was the aim to transfer iN-technology to human cells and to explore the potential of this technique in regards to generate subtype-specific neurons as well as to use these cells for transplantation studies. In Paper I, we described the possibility to convert human fetal and postnatal fibroblasts into functional human induced neurons (hiNs). Generating a mixed population of glutamatergic and GABAergic hiNs, we were exploring the ability to direct hiNs towards a distinct neuronal subtype and therefore selected TFs, which are expressed in midbrain dopaminergic (mDA) neurons and their progenitors. When over-expressing these fate-specifying TFs during direct neuronal conversion, we could show for the first time, that hiNs acquire a dopaminergic (DA) subtype. This led us to explore, whether functional hiNs could be generated from adult sources. In Paper II, we succeeded in generating functional hiNs from fibroblasts of different adult donors; in addition we found that the donor age does not affect the conversion potential of the fibroblasts. The successful generation of functional hiNs in vitro caught our interest to whether new neurons could be obtained also via direct conversion in vivo. In Paper III, we demonstrated that transplanted fibroblasts convert to hiNs in the adult rat brain and that residing astrocytes in the mouse brain can be converted into iNs. For hiNs to become a clinically relevant cell source for cell therapy, additional analysis regarding their survival and maturation after transplantation but also the improvement of conversion efficiencies is important. In Paper IV, we addressed this in part by devising a more efficient conversion protocol as well as by examining the impact of different maturation times of hiNs in culture prior to grafting. Besides using hiNs for transplantation studies, in Paper V, we demonstrated that hiNs could be used to develop a phenotypic screening assay for the identification of small molecules and associated signaling pathways, important for direct neuronal conversion. Overall, this thesis work has opened up possibilities to generate hiNs for brain repair, to obtain patient-specific hiNs for disease-modeling in vitro and to utilize hiNs in high content screening assays.
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