Calcium imaging for stem cell grafts in mouse neocortex: continuous tracking and assessment of functional integration
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Neural stem cells have the capacity to self-renew and differentiate into multiple specialized neural phenotypes, providing a promising therapeutic strategy to replace damaged or lost neurons in neurological diseases. Preliminary clinical trials have demonstrated that grafting neural stem cells in brain tissue could achieve some therapeutic effects. However, the outcomes are highly variable with various adverse effects. Unfortunately, an understanding of which factors underlie success or failure remains elusive. To facilitate the development of safe and effective clinical therapies using grafted neural stem cells, it will be informative to improve our understanding of the environmental factors and means of controlling the process of integration. It is currently difficult to observe the characteristics of neural stem cell integration into host tissue continuously; the integration process is thus often deduced from snapshots of post-mortem tissue requiring sacrifice of transplanted animals at distinct time points, which is often inefficient and impractical to carry out. This dissertation addresses the problem by describing the development and use of a reliable experimental platform that enables continuous observation of stem cells grafted in mouse cortex throughout the course of integration. Current attempts to image neocortical regions on the surface of mouse brain typically use a small glass disc attached to the cranial surface. This approach, however, is often challenged by progressive deterioration in optical quality and permits limited tissue access after its initial implantation. I describe a design and demonstrate a two-stage cranial implant device developed with a remarkably versatile material, polydimethylsiloxane, which facilitates longitudinal imaging experiments in mouse cortex. The system was designed considering biocompatibility and optical performance. This enabled us to achieve sustained periods of optical quality, extending beyond a year in some mice, and allows imaging at high spatiotemporal resolution using wide-field microscopy. Additionally, the two-part system, consisting of a fixed headplate with integrated neural access chamber and optical insert, allowed flexible access to the underlying tissue. Finally, I demonstrate the technical feasibility of rapid adaptation of the system to accommodate varying applications requiring long-term ability to visualize and access neural tissue. Utilizing the two-part cranial window system, two distinct sources of neural stem cells dissected from distinct anatomical regions within mouse embryo and labeled with genetically encoded calcium indicators were transplanted into an adult mouse cortex. The cellular dynamics across hundreds of transplants were acquired periodically across several months using wide-field epifluorescence microscopy. This allowed longitudinal comparisons of cell and network activity from each animal. Immediately after transplantation, in both cell populations, the spontaneous network activity was dominated by a highly recurrent pattern of synchronous bursts, similar to the characteristic activity observed during early development of endogenous cells. Gradually, the network activity diversified and matured into complex activation patterns — network states with better information processing capacities. In an attempt to quantify functional integration of grafted cell-derived neurons with host neural network, several strategies were employed to capture the evolution in dynamic patterns of network activation, including cross-correlation, entropy, and information carrying capacity. Future work using such approach to analyze environmental factors on impacting neural stem cell integration in the native context will contribute to advanced stem cell therapy for neurological disorders.