Enrichment for V1 interneurons from mouse embryonic stem cells and evaluation of de novo network formation

Spinal Cord Injury (SCI) can be a life-altering injury that leads to functional impairments in both sensory integration and motor function. Upon insult to the spinal cord, numerous endogenous repair mechanisms are employed to mitigate the spread of damage, however, this in some cases leads to lifelong loss of functionality. Often pursued as a transplantation source, embryonic stem cells (ESCs) have proven themselves capable of being derived into many of the populations that are damaged following SCI. Numerous studies have explored this in pre-clinical applications, with relatively robust success, however, there remains a lack of determination on which population to use. This determination is further obfuscated by the lack of understanding of complex spinal circuits. To mitigate these issues, I proposed a bottom-up approach, wherein using mixed cultures of enriched ESC-derived spinal neurons. Using these mixed cultures, we could then produce models of spinal circuits on microelectrode arrays (MEAs) to understand how these networks form. As much of the literature has focused on excitatory phenotypes, here, I explored methodologies to direct the differentiation of ESCs into distinct subtypes of inhibitory V1 interneurons (INs). I found, contrary to the literature I was only able to produce FoxP2⁺ V1 INs using my protocol. As there were remnant proliferative cells in my derived V1 INs, which would lead to heterogeneity, I used genomic engineering to produce a transgenic mouse ESC line that can produce enriched cultures of V1 INs. The cell line generated reduces the percentage of proliferative cells produced, while increasing the number of En1⁺ V1 INs produced. To evaluate MEAs as a platform to study network formation, I then proceeded to evaluate enriched cultures of V2a and V3 INs using cross-correlation and conditional Granger causality analysis. The work in this dissertation provides tools and methods that should enable future research into individualizing potential cell transplantation options for SCI.