Investigation of intracellular Calcium-levels in iPSC derived neurons

Kurzfassung

Spaceflight conditions induce various physiological-adverse effects in space travelers, including significant alterations in the human brain, where microgravity can affect calcium homeostasis, a critical factor in neuronal function. Understanding these calcium dynamics is essential, as this process plays a fundamental role in cellular signaling, neurotransmission, and synaptic plasticity. Within this thesis the intracellular calcium levels in human induced pluripotent stem cell (hiPSC)-derived neurons, specifically focusing on induced Neurogenin (iNGNs) neurons, as a model to study these phenomena is investigated. CaMPARI2, a genetically encoded calcium indicator, was employed to measure calcium fluctuations in iNGNs. The CaMPARI2-constructs allowed for real-time monitoring of calcium concentrations within the cells, providing insights into the fluctuations that occur during cellular activities. The CaMPARI2 construct was transfected into iNGNs by using an optimized electroporation protocol. The primary goal of the study of this bachelor thesis was to establish a reproducible and efficient transfection protocol that ensures the permanent integration of CaMPARI2 in iNGNs, creating a stable cell line for future research purposes. Additionally, the study aimed to establish a foundational understanding of baseline calcium levels in human derived neurons, an area that has not been thoroughly explored. The findings of this study present significant advancements in both the methodology and application of iNGNs for neurobiological research. By successfully differentiating iPSCs into iNGNs and optimizing the transfection process, this thesis offers a novel approach to studying neuronal calcium dynamics, particularly in altered gravity conditions like those experienced during spaceflight. The use of iPSC-derived neurons also provides ethical advantages, eliminating the need for animal models and human stem cell harvesting. The results not only have implications for understanding the effects of spaceflight on the nervous system but also provide a platform for investigating calcium dysregulation in neurodegenerative diseases. By establishing a robust model for studying calcium dynamics, this research contributes to the growing field of space biology and neurodegenerative disease research, presenting a new method for analyzing the impact of environmental changes on human neuronal function.