title: Generation of highly enriched V2a interneurons from mouse embryonic stem cells.
abstract: Challenges in parsing specific contributions to spinal microcircuit architecture have limited our ability to model and manipulate those networks for improved functional regeneration after injury or disease. While spinal interneurons (INs) have been implicated in driving coordinated locomotor behaviors, they constitute only a small percentage of the spinal cord and are difficult to isolate from primary tissue. In this study, we employed a genetic strategy to obtain large quantities of highly enriched mouse embryonic stem cell (ESC)-derived V2a INs, an excitatory glutamatergic IN population that is defined by expression of the homeodomain protein Chx10 during development. Puromycin N-acetyltransferase expression was driven by the native gene regulatory elements of Chx10 in the transgenic ESC line, resulting in positive selection of V2a INs after induction and treatment with puromycin. Directly after selection, approximately 80% of cells are Chx10(+), with 94% Lhx3(+); after several weeks, cultures remain free of proliferative cell types and mature into normal glutamatergic neurons as assessed by molecular markers and electrophysiological methods. Functional synapses were observed between selected ESC-derived V2a INs and motor neurons when co-cultured, demonstrating the potential of these cells to form neural networks. While ESC-derived neurons obtained in vitro are not identical to those that develop in the spinal cord, the transgenic ESCs here provide a unique tool to begin studying V2a INs in isolation or for use in in vitro models of spinal microcircuits.
authors.affiliation: Department of Biomedical Engineering, Washington University, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130, USA.
authors.forename: Nisha R
authors.initials: NR
authors.lastname: Iyer
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, Campus Box 8228, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Huettner
authors.affiliation: Department of Biomedical Engineering, Washington University, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130, USA.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Butts
authors.affiliation: Department of Biomedical Engineering, Washington University, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130, USA.
authors.forename: Chelsea R
authors.initials: CR
authors.lastname: Brown
authors.affiliation: Department of Biomedical Engineering, Washington University, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130, USA. Electronic address: sakiyama@wustl.edu.
authors.forename: Shelly E
authors.initials: SE
authors.lastname: Sakiyama-Elbert
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism; D000200:Action Potentials / Q000187:drug effects / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D007395:Interneurons / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C489555:Evx1 protein, mouse; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C499523:Lhx1 protein, mouse; C094898:Lhx3 protein; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D011500:Protein Synthesis Inhibitors; D014157:Transcription Factors; C088975:Vsx2 protein, mouse; D011691:Puromycin; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase
keywords: Electrophysiology; Neuronal differentiation; Puromycin selection; Spinal cord injury; Transcription factor