Generation of V2a Interneurons from Mouse Embryonic Stem Cells

Brown, Carolyn S.; Butts, Jessica C.; McCreedy, Dylan A.; Sakiyama‐Elbert, Shelly E.

doi:10.1089/scd.2013.0628

Cited by 40 publications

(44 citation statements)

References 48 publications

(78 reference statements)

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“…Self-renewing pluripotent cells, such as ESCs, are an attractive alternative to sorting primary tissue because they can be differentiated into a variety of cell types in large quantities for in vitro study or transplantation. By adapting established motor neuron (MN) differentiation protocols (Wichterle et al, 2002), we have previously shown that directed differentiation of ESCs into V2a INs is possible by exposing embryoid bodies (EBs) to retinoic acid (RA); a weak sonic hedgehog (Shh) agonist, purmorphamine; and a Notch-inhibitor, DAPT (Brown et al, 2014). However, despite our ability to derive V2a INs from ESCs, post-mitotic Chx10+ cells constitute only ~15% of the total cell population post-induction, which is further diluted as glial cells proliferate with time (Brown et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…By adapting established motor neuron (MN) differentiation protocols (Wichterle et al, 2002), we have previously shown that directed differentiation of ESCs into V2a INs is possible by exposing embryoid bodies (EBs) to retinoic acid (RA); a weak sonic hedgehog (Shh) agonist, purmorphamine; and a Notch-inhibitor, DAPT (Brown et al, 2014). However, despite our ability to derive V2a INs from ESCs, post-mitotic Chx10+ cells constitute only ~15% of the total cell population post-induction, which is further diluted as glial cells proliferate with time (Brown et al, 2014). Methods including fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting have been used to isolate single cell populations, but they are limited by the availability of antibodies to lineage-specific surface antigens, which have not been identified for many ventral IN populations, and require dissociation processes that can be harmful to mature neurons.…”

Section: Introductionmentioning

confidence: 99%

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Generation of highly enriched V2a interneurons from mouse embryonic stem cells

Iyer

Huettner

Butts

et al. 2016

Experimental Neurology

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation of highly enriched V2a interneurons from mouse embryonic stem cells

Iyer

Huettner

Butts

et al. 2016

Experimental Neurology

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“…The application of both RA and Shh during embryoid body formation in murine ESCs was investigated by Wichterle et al [22], Miles et al [23], Kothapalli and Kamm [24], Brown et al [25], and in human ESCs by Li et al [26], Stacpoole et al [27], and Hu and Zhang [28], among others. Such studies have revealed the synergistic effects of these two factors.…”

Section: Introductionmentioning

confidence: 99%

“…For example, Kothapalli and Kamm found that the addition of RA alone to mESCs in 3D collagen matrices is optimal for dopaminergic neuron formation while the addition of RA and Shh to mESCs in 3 matrigel matrices is optimal for dopaminergic neuron formation [24]. Brown et al reported that the specific neuronal subtype V2a interneurons can be generated from mESCs through exposure to low concentration of RA (10 nM) and mild Shh agonist whereas high RA concentration (up to 10 μM) and a high potency Shh agonist limit these results [25]. In applications presented by Stacpoole et al as well as Hu and Zhang, hESCs are differentiated into spinal motor neurons by first exposing NPCs to RA to caudalize the cells, followed by the combination of RA and Shh (or Shh agonist) in the subsequent days to ventralize them [27,28].…”

Section: Introductionmentioning

confidence: 99%

Engineering personalized neural tissue by combining induced pluripotent stem cells with fibrin scaffolds

et al. 2015

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Induced pluripotent stem cells (iPSCs) are generated from adult somatic cells through the induction of key transcription factors that restore the ability to become any cell type found in the body. These cells are of interest for tissue engineering due to their potential for developing patient-specific therapies. As the technology for generating iPSCs advances, it is important to concurrently investigate protocols for the efficient differentiation of these cells to desired downstream phenotypes in combination with biomaterial scaffolds as a way of engineering neural tissue. For such applications, the generation of neurons within three dimensional fibrin scaffolds has been well characterized as a cell-delivery platform for murine embryonic stem cells (ESCs) but has not yet been applied to murine iPSCs. Given that iPSCs have been reported to differentiate less effectively than ESCs, a key objective of this investigation is to maximize the proportion of iPSC-derived neurons in fibrin through the choice of differentiation protocol. To this end, this study compares two EB-mediated protocols for generating neurons from murine iPSCs and ESCs: an 8-day 4-/4+ protocol using soluble retinoic acid in the last 4 days and a 6-day 2-/4+ protocol using soluble retinoic acid and the small molecule sonic hedgehog agonist purmorphamine in the last 4 days. EBs were then seeded in fibrin scaffolds for 14 days to allow further differentiation into neurons. EBs generated by the 2-/4+ protocol yielded a higher percentage of neurons compared to those from the 4-/4+ protocol for both iPSCs and ESCs. The results demonstrate the successful translation of the fibrin-based cell-delivery platform for use with murine iPSCs and furthermore that the proportion of neurons generated from murine iPSC-derived EBs seeded in fibrin can be maximized using the 2-/4+ differentiation protocol. Together, these findings validate the further exploration of 3D fibrin-based scaffolds as a method of delivering neuronal cells derived from iPSCs -an important step toward the development of iPSC-based tissue engineering strategies for spinal cord injury repair.

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“…Protocols using retinoic acid (RA) and sonic hedgehog (Shh) signaling to direct differentiation of ESCs into various ventral spinal cell identities including MNs [8] and V2a INs [9] by mimicking conditions found in the developing ventral neural tube have been reported.…”

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confidence: 99%

Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells

Sakiyama‐Elbert

2015

Stem Cells and Development

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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.

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Generation of V2a Interneurons from Mouse Embryonic Stem Cells

Cited by 40 publications

References 48 publications

Generation of highly enriched V2a interneurons from mouse embryonic stem cells

Generation of highly enriched V2a interneurons from mouse embryonic stem cells

Engineering personalized neural tissue by combining induced pluripotent stem cells with fibrin scaffolds

Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells

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