Directed Differentiation and Characterization of Spinal V3 Interneurons from Mouse Embryonic Stem Cells
Date of Award
Doctor of Philosophy (PhD)
Neuronal populations involved in locomotion controlling central pattern generators within the spinal cord hold great potential for spinal cord injury therapy. Spontaneous recovery in rodent models suggests that a population that can reorganize around an injury site could be useful for functional recovery therapeutics after spinal cord injury. The glutamatergic, commissural, long-extending V3 interneurons shown to balance locomotor rhythm regularity and robustness within central pattern generators in vivo are both an ideal population for spinal cord injury therapeutics and a vital population to study as a part of locomotor circuitry. Unfortunately, due to the scarcity of these cells in the spinal cord, in vitro studies of dissociated V3 interneurons are technically challenging. Embryonic stem cells provide a bountiful cell source for the study of different cell types and regenerative medicine. While there are extensive reports on mouse embryonic stem cell derived spinal motoneurons, many other spinal neuronal populations have not been derived. This dissertation focuses on the induction and characterization of V3 INs from embryonic stem cells. In the first study, an induction protocol for V3 interneurons from mouse embryonic stem cells was established. A motoneuron protocol was driven towards a more ventral fate by lowering retinoic acid concentration during induction and increasing the induction duration of morphogen sonic hedgehog signaling. In the second study, a selectable V3 interneuron cell line was generated by knocking the puromycin resistance enzyme, puromycin N-acetyltransferase, into the Sim1 locus on one allele within the mouse genome, allowing native Sim1 gene regulatory elements to drive puromycn N-acetyltransferase expression. Puromycin selection highly enriched for the V3 interneuron population, allowing the cultures to be characterized by electrophysiology and immunocytochemistry. Selected cells survived for four weeks and exhibited synaptic function as well as glutamatergic marker expression. This work establishes a method and a tool for high throughput, low labor acquisition of V3 interneurons for future studies.
Dennis Barbour, Jianmin Cui, James Huettner, Vitaly Klyachoko