TY - JOUR
T1 - Generation of highly enriched V2a interneurons from mouse embryonic stem cells
AU - Iyer, Nisha R.
AU - Huettner, James E.
AU - Butts, Jessica C.
AU - Brown, Chelsea R.
AU - Sakiyama-Elbert, Shelly E.
N1 - Funding Information:
Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under grants R01 NS090617 (S.E.S), R01NS051454 (S.E.S) and NS30888 (J.E.H.). N.I. was supported by an Individual Predoctoral National Research Service Award ( F31NS090760 ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under grants R01 NS090617 (S.E.S), R01NS051454 (S.E.S) and NS30888 (J.E.H.). N.I. was supported by an Individual Predoctoral National Research Service Award (F31NS090760). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/3/1
Y1 - 2016/3/1
N2 - 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.
AB - 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.
KW - Electrophysiology
KW - Neuronal differentiation
KW - Puromycin selection
KW - Spinal cord injury
KW - Transcription factor
UR - http://www.scopus.com/inward/record.url?scp=84956676330&partnerID=8YFLogxK
U2 - 10.1016/j.expneurol.2016.01.011
DO - 10.1016/j.expneurol.2016.01.011
M3 - Article
C2 - 26784005
AN - SCOPUS:84956676330
VL - 277
SP - 305
EP - 316
JO - Experimental Neurology
JF - Experimental Neurology
SN - 0014-4886
ER -