On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

Rabieh, Noha; Ojovan, Silviya M.; Shmoel, Nava; Erez, Hadas; Maydan, Eilon; Spira, Micha Ε.

doi:10.1038/srep36498

Cited by 37 publications

(39 citation statements)

References 69 publications

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“…We quantified evoked spike counts, or those falling within a 500ms window post-stimulation, over a 5 minute recording session. Treatment with 1-heptanol led to abolishment of evoked spike counts that could be partially rescued at lower concentrations, suggesting that recorded EAPs originated from electrotonically-coupled sKM that are recorded as bursting activity, as reported by others (Rabieh et al, 2016) ( Figure 4C-E ). Support for this model was shown by heptanol treatment in C2C12 myotube cultures that yielded similar results ( Figure S4A ).…”

Section: Simultaneous Optogenetic and Electrophysiological Recordingssupporting

confidence: 79%

Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

Shintani

Wan

et al. 2020

Preprint

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The failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72 -associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease. Results Creation of stable optogenetic motor neuronshiPSC lines were reprogrammed from fibroblasts collected from a healthy control (Line C), a C9orf72 G 4 C 2 expansion carrier diagnosed with behavioral variant FTD (bvFTD, Line A), and genetically corrected isogenic clones (isoA80, isoA51). All hiPSC lines were previously characterized Pribadi et al., 2016). To generate MNs, we followed a previously published protocol (Du et al., 2015) to produce populations of Hb9 + MNs via an adherent-or suspension-based culture protocol within 25 days, and abundant ChAT expression by day 30. ( Figure 1A-E ). We additionally generated isogenic, optogenetic reporter lines via co-transduction with two lentiviruses to enable optogenetic control of Hb9 + MNs ( Figure S1A ). iPSC clones were screened (see Methods, Figure S1B-E ), resulting in selection of a single pair of stable isogenic clones (Opto-A1, Opto-isoA80-8). Suspension-based methodology yielded highly pure, Hb9 + MN spheres ( Figure 1F-G ), and was adopted in subsequent experiments. Spontaneous myotube contractions in 2D culture are mediated via gap junctionsWe generated hiPSC-sKM following our previously published protocol . Differentiated sKM showed mature sarcomeric organization by Titin staining and electron microscopy ( Figure 1H-I ), and spontaneous contractions in dense cultures. Prior to establishing co-cultures, we posited that sKM would need to be dissociated in order to distinguish NMJ-mediated contractions from spontaneous contractions. Upon dissociation and re-plating of contractile sKM, we observed a cessation in contractile activity of re-plated myotubes, although some sKM still displayed spontaneous calcium oscillations ( Figure S2A-B ). We reasoned this cessation may be due to the loss of gap junction connections, whose proteins are expressed in sKM during development (Merrifield and Laird, 2016), and were putatively observed via electron microscopy ( Figure S2C-D ).Evidence for gap junctions in sKM wa...

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Section: Simultaneous Optogenetic and Electrophysiological Recordingssupporting

confidence: 79%

Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

Shintani

Wan

et al. 2020

Preprint

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“…High‐aspect‐ratio nanostructured electrodes reduce the contact resistance and improve the signal‐to‐noise ratio of planar electrodes . Motivations include studying the fundamental cell electrophysiology of cardiac, neural, and skeletal‐myotube cells, as well as developing platforms for high‐throughput drug screening . The spatially resolved, parallel, intimate interface of high‐aspect‐ratio nanostructures with cells and tissue is well suited for sensing complex neural network behaviors .…”

Section: Bioelectronic Stimulation and Sensingmentioning

confidence: 99%

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

et al. 2020

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Materials patterned with high‐aspect‐ratio nanostructures have features on similar length scales to cellular components. These surfaces are an extreme topography on the cellular level and have become useful tools for perturbing and sensing the cellular environment. Motivation comes from the ability of high‐aspect‐ratio nanostructures to deliver cargoes into cells and tissues, access the intracellular environment, and control cell behavior. These structures directly perturb cells' ability to sense and respond to external forces, influencing cell fate, and enabling new mechanistic studies. Through careful design of their nanoscale structure, these systems act as biological metamaterials, eliciting unusual biological responses. While predominantly used to interface eukaryotic cells, there is growing interest in nonanimal and prokaryotic cell interfacing. Both experimental and theoretical studies have attempted to develop a mechanistic understanding for the observed behaviors, predominantly focusing on the cell–nanostructure interface. This review considers how high‐aspect‐ratio nanostructured surfaces are used to both stimulate and sense biological systems.

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“…Besides the morphologically tight adhesion, the membrane curvature caused by vertical nano-protrusions may also induce the aggregation of ion channels, increasing the local ionic current. 31,32 Furthermore, treated using hydrophobic bands, nanopillars can even enhance their seal resistance to the GU range, which is comparable to that of patch-clamp seals (Fig. 4b).…”

Section: Enhancement Of Seal Resistancementioning

confidence: 65%