Stretch Injury Alters Calcium Dynamics and Actin Organization in Human Astrocytes

Traumatic brain injury (TBI) is a major public health concern, linked to long-term neurodegenerative conditions such as Alzheimer’s disease. Although neurons are often the primary focus of TBI research, growing evidence highlights the critical role of astrocytes—especially their calcium signaling dynamics—in driving post-injury pathology. This thesis investigates how human induced pluripotent stem cell (hiPSC)- derived astrocytes respond to mechanical strain, aiming to reproduce changes in astrocyte behavior that can initiate neurodegeneration. In this research, a two- dimensional in vitro stretch model was used to apply controlled mechanical strain to monolayers of hiPSC-derived astrocytes cultured on flexible PDMS substrates. Cellular responses were assessed using a combination of live calcium imaging and immunofluorescence. Results showed a strain-dependent suppression of spontaneous calcium activity post-injury, mitochondrial dysfunction, and decline in cell viability. Additionally, injury induced changes in cytoskeleton structure and overexpression of Piezo1, a mechanosensitive ion channel. These findings support the hypothesis that astrocytic calcium dysfunction links immediate trauma pathology to chronic neurodegenerative pathology. Overall, this work captures consequential changes in astrocytes after trauma in an in vitro system that enables detailed investigation of them.