We are human after all - Model systems to study human neuronal function

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URI: http://hdl.handle.net/10900/142181
Dokumentart: PhDThesis
Date: 2023-06-14
Source: -
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Lerche, Holger (Prof. Dr.)
Day of Oral Examination: 2023-04-04
DDC Classifikation: 500 - Natural sciences and mathematics
610 - Medicine and health
Keywords: Neurowissenschaften , Epilepsie , Induzierte pluripotente Stammzelle , Gehirn , Elektrophysiologie
License: http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=de http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=en
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In the work presented here, two model systems for neurosciences were established in order to better examine neuronal functions. In the first and second part of the project, skin fibroblasts from patients with variants in the KCNA2 gene (c.890G> A, p.R297Q and c.982T> G, p.L328V), which are the cause of the patients' epileptic encephalopathy (DEE), were reprogrammed into induced pluripotent stem (iPS) cells with the help of viral vectors. The characterization of the iPS cell lines was performed by karyotyping, by immunofluorescence staining against several pluripotency markers, a qRT-PCR to control the upregulation of the endogenous pluripotency markers and a differentiation of the iPS cells into cells from all three germ layers. In the third part of the project, these iPS cells were differentiated into human neurons through the virally mediated overexpression of the neuronal transcription factor NGN2. The subsequent electrophysiological characterization of the two cell lines and the comparison with healthy control lines showed no significant difference between the neurons of the KCNA2 p.R297Q variant carrier and the control cells. The differentiated neurons of the KCNA2 p.L328V variant carrier generated significantly smaller outward currents than the control neurons and responded to current injections with fewer action potentials than the control neurons. Because of the inadequate representation of this model system compared to the human brain, new alternatives were sought. In the fourth and fifth part of the project, it was possible for the first time to show that human cortical brain tissue outside the human body can survive for several weeks without the neurons or the entire neural network losing their basic morphological and electrophysiological properties. The decisive difference to previous approaches was the successful discovery that human brain tissue can be cultivated very well in human cerebrospinal fluid. The electrophysiological parameters compared were relatively stable between the acute, early (2-3 DIV) and a late-stage culture status (7-14 DIV). In addition, an efficient virus transduction is possible and allows fluorescence-mediated 3D reconstruction. A virally mediated GFP expression in neurons has no to date detectable influence on the morphology and the basic electrophysiological properties of the analyzed cells. Moreover, long-term live imaging of human spines could be performed, as well as the detection of an outgrowth of human axons. Both are methods that are difficult to perform in vivo. The integration of human organotypic brain slices presented here into existing model systems such as that of the iPS cell-derived neurons will be of great value for studies on living human neuronal networks, as well as for the screening of new drugs, but also for direct research into neurological diseases. This new model system gives us a great opportunity to learn more about the human brain and could help us to answer the question of what makes us human after all.

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