Dataset Information

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.

SUBMITTER: Iyer NR

PROVIDER: S-EPMC4761286 | biostudies-other | 2016 Mar

REPOSITORIES: biostudies-other

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Generation of highly enriched V2a interneurons from mouse embryonic stem cells.

Iyer Nisha R NR Huettner James E JE Butts Jessica C JC Brown Chelsea R CR Sakiyama-Elbert Shelly E SE

Experimental neurology 20160116

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 enri ...[more]

PMID: 26784005

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Project description:The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGF?) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

Dataset Information

Generation of highly enriched V2a interneurons from mouse embryonic stem cells.

Publications

Generation of highly enriched V2a interneurons from mouse embryonic stem cells.

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