Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications

Clements, Isaac P.; Millard, Daniel C.; Nicolini, Anthony M.; Preyer, A.J.; Grier, Robert E.; Heckerling, Andrew; Blum, R.A.; Tyler, Phillip; McSweeney, K. Melodi; Lu, Yifan; Hall, Diana; Ross, James D.

doi:10.1117/12.2213708

Cited by 10 publications

(7 citation statements)

References 34 publications

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“…We aimed to apply methods amenable to medium-throughput, longitudinal or drug screening studies using MEAs paired with the Lumos (Axion Biosystems) device, enabling simultaneous optogenetic and electrophysiological measurements (Clements et al, 2016). Previous studies have shown that primary rodent MN and sKM display similar extracellular action potential (EAP) characteristics (Langhammer et al, 2012;.…”

Section: Simultaneous Optogenetic and Electrophysiological Recordingsmentioning

confidence: 99%

“…Optogenetic control of the NMJ has been achieved via transgenic rodent or hiPSC-MNs paired with rodent, human primary sKM, and hiPSC-sKM in vitro (Steinbeck et al, 2015;Uzel et al, 2016, Osaki et al, 2018, (Afshar Bakooshli et al, 2019 and in vivo (Bryson et al, 2014). Optogenetics may also be paired with MEAs, enabling a system that can monitor electrophysiology of living cells in tandem with light stimulation, providing innovative applications for disease modeling or drug screening (Clements et al, 2016). Here, we leverage advances in hiPSC-differentiation, optogenetics, and MEAs to create a scalable, tunable, human patient-specific neuromuscular co-culture model system.…”

Section: Introductionmentioning

confidence: 99%

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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 Recordingsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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“…Optogenetic tools are being creatively incorporated into automated high-throughput drug-screening platforms (12,21,24,66). Using optogenetic tools dynamically could also potentially expand on the use of automated multichannel patch-clamp systems to multicellular preparations.…”

Section: Versatility and Flexibility Of Odcmentioning

confidence: 99%

Light-Activated Dynamic Clamp Using iPSC-Derived Cardiomyocytes

Quach

Krogh‐Madsen

Entcheva

et al. 2018

Biophysical Journal

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iPSC-derived cardiomyocytes (iPSC-CMs) are a potentially advantageous platform for drug screening because they provide a renewable source of human cardiomyocytes. One obstacle to their implementation is their immature electrophysiology, which reduces relevance to adult arrhythmogenesis. To address this, dynamic clamp is used to inject current representing the insufficient potassium current, I K1 , thereby producing more adult-like electrophysiology. However, dynamic clamp requires patch clamp and is therefore low throughput and ill-suited for large-scale drug screening. Here, we use optogenetics to generate such a dynamic-clamp current. The optical dynamic clamp (ODC) uses outward-current-generating opsin, ArchT, to mimic I K1 , resulting in more adult-like action potential morphology, similar to I K1 injection via classic dynamic clamp. Furthermore, in the presence of an I Kr blocker, ODC revealed expected action potential prolongation and reduced spontaneous excitation. The ODC presented here still requires an electrode to measure V m but provides a first step toward contactless dynamic clamp, which will not only enable high-throughput screening but may also allow control within multicellular iPSC-CM formats to better recapitulate adult in vivo physiology.

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“…Existing screening tools lack the capabilities for spatially and spectrally patterned illumination, and lack the temporal resolution, spatial resolution and sensitivity needed for high-speed measurements of neuronal firing. While optical stimulation capabilities are starting to be added to existing screening platforms [113], these capabilities remain rudimentary. This challenge will likely be addressed through a series of instruments with differing tradeoffs in throughput and information content.…”

Section: Challenges For Optogenetics-enabled Drug Discoverymentioning

confidence: 99%

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

Zhang

Cohen

2017

Trends in Biotechnology

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Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, and in particular tools for all-optical electrophysiology, now provide means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline, from target identification and validation, to target-based and phenotypic screens, to clinical trials.

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Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications

Cited by 10 publications

References 34 publications

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

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

Light-Activated Dynamic Clamp Using iPSC-Derived Cardiomyocytes

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

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