Induction of Ventral Spinal V0 Interneurons from Mouse Embryonic Stem Cells

Abstract

The ventral spinal population of V0 interneurons (INs) contribute to the coordinated movements directed by spinal central pattern generators (CPGs), including respiratory circuits and left-right alternation. One challenge in studying V0 INs has been the limited number of cells that can be isolated from primary sources for basic research or therapeutic use. However, derivation from a pluripotent source, such as has been done recently for other IN populations could reduce this issue. However, there is currently no protocol to specifically derive V0 interneurons from embryonic stem cells or induced pluripotent stem cells. To generate an induction protocol, mouse embryonic stem cells (mESCs) were grown in suspension culture and then exposed to retinoic acid (RA) and collected at different time points to measure mRNA expression of the V0 progenitor transcription factor marker, Dbx1, and post-mitotic transcription factor marker, Evx1. The cultures were also exposed to the sonic hedgehog signaling pathway agonist purmorphamine (purm) and the Notch signaling pathway inhibitor N-{N-(3,5-difluorophenacetyl-L-alanyl)}-(S)-phenylglycine-t-butyl-ester (DAPT) to determine if either of these pathways contribute to V0 IN induction, specifically the ventral (V0V) subpopulation. From the various parameters tested, the final protocol that generated the greatest percentage of cells expressing V0V IN markers was an 8 day protocol using 4 days of suspension culture to form embryoid bodies followed by addition of 1 M RA from days 4 to 8, 100 nM purm from days 4 to 6, and 5 M DAPT from days 6 to 8. This protocol will allow investigators to obtain V0 IN cultures for use in in vitro studies, such as those examining CPG microcircuits, electrophysiological characterization, or even for transplantation studies in injury or disease models.