Date of Award
Doctor of Philosophy (PhD)
Functional recovery following spinal cord injury has been attributed to plasticity in local interneuron populations, which are able to create novel circuits that bypass the inhibitory lesion to activate pre-existing motor pathways. Cell replacement strategies and growth factor delivery platforms often attempt to enhance these natural mechanisms of regeneration. The work of this thesis sought to overcome the major obstacle to manipulating specific interneurons for therapy: an inability to obtain large, enriched interneuron subpopulations for drug screening and transplantation. We focused on the generation of V2a interneurons, which normally coordinate a wide range of locomotor tasks in both the spinal cord and respiratory centers of the hindbrain. They are an ipsilaterally projecting excitatory glutamatergic inteneuron population that is defined by expression of the homeodomain protein Chx10 during development. Using CRISPR/Cas9-mediated homologous recombineering, we generated a transgenic mouse embryonic stem cell (ESC) line that enabled us to positively select for Chx10+ ESC-derived V2a interneurons (ESC-V2as) by treating differentiated cells with puromycin. ESC-V2a cultures remained free of proliferative cells, matured into normal glutamatergic neurons, and were capable of forming synapses onto motor neurons in adherent monolayer culture. For further in vitro investigation and transplantation studies, we cultured ESC-V2as and progenitor populations as spherical aggregates to generate a cellular microenvironment more closely related to that in vivo. We used this platform to test whether V2a IN axon growth could be stimulated in a dose-dependent manner by glial-derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and platelet-derived neurotrophic factor (PDGF) and/or by co-culture with ESC-derived progenitor motor neurons (ESC-pMNs). Although ESC-V2as did not variably respond to the growth factors applied when cultured in isolation, co-culture with ESC-pMNs improved ESC-V2a neurite extension, survival, maturation, and electrophysiological activity. Application of optimized concentrations of GDNF and PDGF, which act specifically on ESC-pMNs but not ESC-V2a INs, enhanced these positive effects. Finally, we sought to determine the feasibility and efficacy of transplanting neural progenitor cells (NPCs) or ESC-pMNs enriched with ESC-V2as in a respiratory model of spinal cord injury. Despite the historic difficulty in transplanting post-mitotic neuronal populations, ESC-V2as were able to survive and migrate from injury site when co-transplanted with progenitors, and in the case of V2a/NPC groups, resulted in improved recovery of diaphragm activity compared to control groups. Taken together, this thesis establishes in vitro and in vivo methodologies to investigate possible mechanisms of V2a interneuron rewiring following spinal cord injury and a platform to optimize combinatorial treatment strategies moving forward.
Shelly Sakiyama-Elbert, James E. Huettner
Dennis Barbour, Rob Mitra, Paul S. Stein, Steven George