title: Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells.
abstract: Excitatory commissural V3 interneurons (INs) of the ventral spinal cord have been shown to balance locomotor rhythm regularity and robustness in vivo. Unfortunately, due to the scarcity of these cells in the spinal cord, in vitro studies of dissociated V3 INs have yet to be reported. In this study, we developed an induction protocol for V3 INs from mouse embryonic stem cells. The effect of the concentration of a strong sonic hedgehog (Shh) agonist (smoothened agonist [SAG]) and retinoic acid (RA) on expression of progenitor p3 and postmitotic V3 IN transcription factor markers (ie, Nkx2.2 and Sim1) was examined. Cells were differentiated toward a more ventral fate by increasing the duration of SAG exposure from 4 days in a previously established motoneuron induction protocol to 6 days. At the end of the induction period, transcription factor expression was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, in situ hybridization, and flow cytometry. Lower concentrations of RA and a longer duration of SAG exposure led to increased levels of p3 and V3 marker expression. This novel induction protocol reveals the importance of Shh signaling duration in the dorsal-ventral patterning of the neural tube, and it provides a method to obtain V3 INs for future studies to allow better understanding their role in rewiring and regeneration after spinal cord injury.
authors.affiliation: Department of Biomedical Engineering, Center of Regenerative Medicine, Washington University in St. Louis. , St. Louis, Missouri.
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Biomedical Engineering, Center of Regenerative Medicine, Washington University in St. Louis. , St. Louis, Missouri.
authors.forename: Shelly E
authors.initials: SE
authors.lastname: Sakiyama-Elbert
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007395:Interneurons / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D055495:Neurogenesis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin; D029961:Zebrafish Proteins
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014212:Tretinoin; D029961:Zebrafish Proteins; C000715627:Nkx2-2 protein, mouse; C573100:SAG protein, mouse; C101750:Sim1 protein, mouse; C000715630:nkx2.2b protein, zebrafish