On-chip 3D neuromuscular model for drug screening and precision medicine in neuromuscular disease

Osaki, Tatsuya; Uzel, Sebastien G. M.; Kamm, Roger D.

doi:10.1038/s41596-019-0248-1

Cited by 98 publications

(87 citation statements)

References 56 publications

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“…The devices produced with SLA 3D printing include the simultaneous presence of millimetric and micrometric features, a combination hardly possible with techniques such as photolithography, which is confined to the realization of structures within hundreds of micrometers. [ 5–10 ] To replicate such structures in PDMS, here for the first time the highly elastic polymer Ecoflex [ 30,31 ] is applied as a reusable replica molding substrate for tissue engineering applications. To avoid accidental damage during demolding, conventional procedures typically require supplementary steps solely to fix caps on each pillar [ 9,10,33 ] or serial replica molding stages with high risk of failure.…”

Section: Discussionmentioning

confidence: 99%

“…[ 1 ] Unsurprisingly, more and more research groups started investing resources and expertise in the generation of 3D in vitro models for various tissues. [ 2–10 ] Adopting this paradigm is particularly significant for contractile and load‐bearing tissues, such as tendons, cardiac, and skeletal muscle. [ 11–14 ] However, since the criteria for a transition from monolayer cultures to 3D systems were first outlined more than ten years ago, [ 15,16 ] cost‐effective ways for easy production of 3D cell culture systems are still lacking.…”

Section: Introductionmentioning

confidence: 99%

“…[ 5–7 ] However, this approach carries a limitation in the production of features larger than few hundreds of micrometers and lacks versatility: producing a single master mold is time‐consuming and expensive, requiring most of the time dedicated facilities such as clean rooms. To avoid multistep photolithography, molds have been generated using single‐step lithography [ 8–10 ] or milled Teflon. [ 19 ] This was followed by manually gluing a thin square of PDMS on top of each pillar for obtaining the T‐shape, making this method time‐consuming and not prone to high throughput.…”

Section: Introductionmentioning

confidence: 99%

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Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

Iuliano

Wal

Ruijmbeek

et al. 2020

Adv Materials Technologies

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The transition from 2D to 3D engineered tissue cultures is changing the way biologists can perform in vitro functional studies. However, there has been a paucity in the establishment of methods required for the generation of microdevices and cost‐effective scaling up. To date, approaches including multistep photolithography, milling and 3D printing have been used that involve specialized and expensive equipment or time‐consuming steps with variable success. Here, a fabrication pipeline is presented based on affordable off‐the‐shelf 3D printers and novel replica molding strategies for rapid and easy in‐house production of hundreds of 3D culture devices per day, with customizable size and geometry. This pipeline is applied to generate tissue engineered skeletal muscles in vitro using human induced pluripotent stem cell‐derived myogenic progenitors. These production methods can be employed in any standard biomedical laboratory.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

Iuliano

Wal

Ruijmbeek

et al. 2020

Adv Materials Technologies

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“…As HOX play a 281 central role in instructing cell diversification in the three lineages, controlled manipulation of the 282 HOX clock has a great potential for cell engineering besides MN subtypes. Our strategy is likely 283 expandable to other axial stem cell derivatives such as paraxial mesoderm that generate the 284 different muscles of the body with clear applications for the modeling of neuromuscular diseases 285 (Bakooshli et al, 2019;Diaz-Cuadros et al, 2020;Faustino Martins et al, 2020;Frith et al, 286 2018;Machado et al, 2019;Matsuda et al, 2020;Osaki et al, 2020;Pourquié et al, 2018;287 Steinbeck et al, 2015;Verrier et al, 2018). More broadly, the temporal generation of distinct 288 types of neurons or glia from the same progenitor domain is a widely used strategy to increase 289 cell diversity in the nervous system (Dias et al, 2014;Kohwi and Doe, 2013;Oberst et al, 2019a;290 Rossi et al, 2017).…”

Section: Discussion 248mentioning

confidence: 99%

Dynamic extrinsic pacing of theHOXclock in human axial progenitors controls motor neuron subtype specification

Mouilleau

Vaslin

Gribaudo

et al. 2020

Preprint

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Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX 1 genes within axial stem cells that fuel axial embryo elongation. Whether HOX genes sequential 2 activation, the "HOX clock", is paced by intrinsic chromatin-based timing mechanisms or by 3 temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in 4 human pluripotent stem cells differentiating into spinal cord motor neuron subtypes which are 5 progenies of axial progenitors. We show that the progressive activation of caudal HOX genes in 6 axial progenitors is controlled by a dynamic increase in FGF signaling. Blocking FGF pathway 7 stalled induction of HOX genes, while precocious increase in FGF alone, or with GDF11 ligand, 8 accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated 9 appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The 10 HOX clock is thus dynamically paced by exposure parameters to secreted cues. Its manipulation 11 by extrinsic factors alleviates temporal requirements to provide unprecedented synchronized 12 access to human cells of multiple, defined, rostro-caudal identities for basic and translational 13 applications. , et al. (2013). Accelerated highyield generation of limb-innervating motor neurons from human stem cells. J. Neurosci. 33, 574-586. ., et al. (2019). Stem cell-derived cranial and spinal motor neurons reveal proteostatic differences between ALS resistant and sensitive motor neurons. Elife 8,.

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“…Advances in hiPSC differentiation have shown formation of functional NMJs in vitro between hiPSC-MN and rodent sKM (Umbach et al, 2012;Yoshida et al, 2015), and between hiPSC-MNs and primary human sKM (Santhanam et al, 2018;Steinbeck et al, 2015;Afshar Bakooshli et al, 2019) or hiPSC-sKM (Puttonen et al, 2015, Osaki et al, 2018, Osaki et al, 2020 . Recent demonstrations have shown that 3D neuromesodermal organoids can generate functional NMJs that consist of sKM, MNs, and Schwann cells (Faustino Martins et al, 2020) .…”

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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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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On-chip 3D neuromuscular model for drug screening and precision medicine in neuromuscular disease

Cited by 98 publications

References 56 publications

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

Dynamic extrinsic pacing of theHOXclock in human axial progenitors controls motor neuron subtype specification

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

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