The Pharmacology of Nerve and Muscle in Tissue Culture

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A remaining challenge, however, is to establish methods to evaluate the function of normal and diseased MNs obtained from these sources in a physiologically relevant setting. An important step towards this goal is the development of in vitro assays to measure the synaptic activity of MNs at neuromuscular junctions, as many studies have pointed to synaptic dysfunction as an early readout and possibly an initiating event in MN disease progression [14] , [15].

Moreover, when transplanted into the embryonic chick spinal cord [9] , [18] , [19] or peripheral nerve of mice [20] , these neurons appear to be capable of extending axons towards peripheral muscle targets.

Despite these successes, relatively little attention has been placed on direct measurements of the communication between stem cell-derived MNs and muscle cells. In part, this reflects the inherent difficulties in isolating connected pairs of cells in mass culture or transplantation settings.

In this study, we report the development of low-density culture conditions that encourage the formation of neuromuscular junctions between isolated ESC-derived MNs and muscle cells. This system enables the direct measurement of synaptic communication through dual patch clamp recordings. In this setting, MNs form neuromuscular junctions containing functionally importan synaptic proteins, and these synapses exhibit both spontaneous and stimulus-evoked transmitter release. Together, these findings constitute an important advance in validating the functional identity of stem cell-derived MNs and providing a platform for defining their synaptic properties under normal and diseased conditions.

To evaluate the synaptic activity of ESC-derived MNs, we first developed culture conditions that were amenable to patch clamp analysis of MN-muscle pairs. The initial step was to test whether cells could form synaptic contacts when plated at low density 1. We reasoned that such conditions might encourage the preferential growth of motor axons to nearby partners and minimize non-synaptic contacts made when cells are plated at high densities. At the point of contact between the axons and muscle cells there was a varicose enlargement of the terminal bouton Figs. This geometry of neuron-muscle pairing was sufficiently common that it enabled the reliable identification of nerve and muscle cells that were likely to have made functional synaptic contacts.

We next used immunofluorescence microscopy to investigate whether other macromolecules characteristic of cholinergic synapses were present at the nerve-muscle contacts. Concomitantly, synaptic vesicle proteins such as synaptophysin and SV2 were also present Fig. These results were representative of data obtained from at least two separate culture dishes for each antibody. Collectively, these data indicate that sites of contact between ESC-derived MNs and muscle cells contain components of the molecular machinery associated with cholinergic synapses.

To determine whether the nerve-muscle contacts formed in culture exhibit the functional properties of neuromuscular junctions, we sealed patch clamp pipettes onto MN-muscle pairs. Current injection into the ESC-derived MNs initially elicited passive membrane responses in the MNs, but no electrical response in muscle cells beyond the stimulus artifact Fig. However, once threshold was exceeded, the MNs fired an action potential. With a brief delay 1—3 msec , MN action potentials were followed by an excitatory post-synaptic current EPC in the muscle cells Fig. These EPCs are reminiscent of the classical electrophysiological signature of synaptic communication between MNs and muscle cells observed in en-bloc preparations [21].

A In each pair of recordings, the lower trace shows the MN response to depolarizing currents of increasing amplitude. For both MN1 and MN2, current was increased in 0.

Muscle Contraction Process [HD Animation]

Stimuli were delivered at 30 s intervals. MN responses were initially passive, but upon reaching threshold, MNs typically fired a single action potential with this stimulus duration. From 60 dishes examined, neuron-muscle pairs with geometries similar to that shown in Fig. The records in Fig. A—D Scatter plots show the variability of the following parameters as a function of days in co-culture: A EPC amplitudes, where each point is the largest EPC recorded at each synapse.

An important advantage of the dual patch configuration is that it enables one to conduct additional quantitative analyses of both spontaneous and evoked synaptic events at these neuromuscular junctions. The mEPC amplitude distribution Fig. Cumulatively, mEPC frequencies ranged from 0. Although we have not yet undertaken a systematic evaluation of the quantal content of the EPCs in this system, it is important to note that EPCs such as that shown in Fig. Taken together, these data indicate that mESC-derived MNs are capable of coupling action potentials to the synchronous release of multiple quanta at these nerve-muscle contacts to elicit muscle contractions.

Patch pipettes were sealed onto MNs and muscle cells making contact as shown in Fig. C Scatter plot of mEPC frequency over different times in culture. The definitive feature of MNs is their ability to form functional neuromuscular junctions and thereby drive the contraction of skeletal muscle cells. Our study provides critical evidence that ESC-derived MNs can exhibit robust synaptic communication with muscle cells under simplified in vitro culture conditions.

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Empirically, this is a significant observation, as the use of stem cell-derived MNs for regenerative purposes or disease modeling requires that the cells faithfully mimic their natural counterparts in both molecular and functional properties. Our data show that ESC-derived MNs express several proteins, including nicotinic ACh receptors, Slc18a3 VAChT , the high affinity choline transporter Slc5a7, and SNARE proteins found at native neuromuscular junctions, and exhibit both spontaneous and action potential-dependent, multi-quantal secretion of ACh to trigger post-synaptic potentials and muscle contraction.

These results further provide an important extension to previous studies that have used bath application of glutamate to evoke post-synaptic potentials and muscle contraction in high-density MN-muscle cell cultures [13] , [16] , [22]. Moreover, the ability to quantify the functional properties of individual nerve-muscle contacts offers the opportunity to rigorously assess the impact of a variety of experimental manipulations on these synaptic events.

To define the synaptic activity of MN-muscle pairs, our investigation was intentionally restricted to the differentiated progeny of mESCs and C2C12 muscle cells. Our data indicate that MN-muscle synapses formed under these simplified conditions recapitulate many features of neuromuscular communication seen in vivo. For example, the frequency of spontaneous mEPCs 0. Similarly, the rise and decay times of EPCs are within the range observed for developing rat neuromuscular junctions [23]. It is important to note that functional MN-muscle synapses formed under these conditions typically survived for a maximum of 8—9 days in culture.

This limitation might reflect a lack of support provided in vivo by other cells including astrocytes and Schwann cells or presynaptic inputs to the MNs from spinal interneurons. Read the Safety Measures on Page 5 of the lab manual 1. If a department prepared the manual, use it for the name in the citation. It includ es essential measurement practices, troubleshooting hints for hardware, software and applications, maintenance procedures and a list of spare parts and accessories. Biology Textbook answers Questions. Exercise Structure of the Heart 1. Startup companies founded from research in the Mascal Lab and Olson Lab have been recently featured in the local news.

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Ads keep Slader free. However, without added myotube maturation through contraction regimes or otherwise, it should be noted that myotubes in culture are somewhat smaller than those in adult animals, which can introduce some difficulty in recording. Perhaps most strikingly, 3D neuromuscular cultures possess AChRs containing functional adult AChR epsilon subunit, which is, to our knowledge, the first report of a system that supports the de novo gamma to epsilon AChR subunit switch in culture.

Given challenges associated with maturing hPSC-derived skeletal muscle fibers beyond embryonic-like states, we hypothesize that our success may be due in part to the use of primary adult human myoblasts. In a proof-of-concept study, we demonstrate the application of our NMJ model to study adult NMJ activity by using a peptide that specifically blocks the epsilon subunit.

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Treatment with the peptide dampened glutamate-induced GCaMP6 calcium reporter activity in neuromuscular co-cultures demonstrating the utility of the system for adult NMJ studies. Tissue culture affords the opportunity to deconstruct the complexity of a tissue system and to systematically rebuild complexity as a means to identify physical and chemical factors that influence biological processes.

This method is particularly powerful in studies of the NMJ where decoupling nerve and muscle influences during development and in the adult, within the context of an animal model, is confounded by tissue death. Through an iterative comparison of 2D and 3D muscle alone and neuromuscular co-cultures, we found that CHRNE transcript expression is upregulated in both 2D and 3D neuromuscular co-cultures. Since our PSC-derived motor neurons express neuregulin-1 protein, but we do not observe appreciable NMJ activity in our 2D neuromuscular co-cultures, we speculate that if CHRNE transcript induction is reliant on NRG-1, then localized MN-mediated delivery of the protein may be necessary to achieve physiologically relevant concentrations of the protein, and that transmission via the NMJ is not required.

Importantly, epsilon protein levels further increased and its function was detected WTX-responsivity only in the context of 3D neuromuscular co-cultures. Indeed, our observations that muscle fibers established in 3D culture are more mature Figure 1 and Figure 1—figure supplement 1 and 2 and that 3D neuromuscular co-cultures exhibit spontaneous endogenous endplate potentials Figure 3 and Video 7 fit well with studies linking muscle fiber maturation state and activity to AChR subunit conversion and stability Caroni et al. Through the availability of a methodology supporting de novo adult NMJ development, it is now possible to delve deeper into the mechanisms regulating metabolic stability of the epsilon subunit in normal development and in disease states.

Studies aimed at understanding the intricacies of subunit integration, recycling, and stability are poised for exploration upon the availability of antibodies that allow for immunostaining studies of the human epsilon protein, or the generation of genetically modified lines in which subunits are fluorescently tagged. In summary, this approach to model the adult human NMJ in a dish provides a versatile and simple way to study skeletal muscle and NMJ development, but more importantly, constitutes the first report of a method to study adult, rather than embryonic, human NMJ activity in as early as two weeks of co-culture time.

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  6. Our calcium reporter neuromuscular tissues can easily be integrated with other optogenetic methods Steinbeck et al. Finally, our method is amenable to modeling diseases that target the adult NMJ e. Primary myoblast and fibroblast-like cell lines were established and maintained as previously described Blau and Webster, The cell suspension was passed multiple times through a 20 G needle to facilitate the release of the mononucleated cell population and subsequently depleted of red blood cells with a brief incubation in red blood cell lysis buffer Table 2.

    Subsequent experiments utilized low passage cultures P4—P9. DMEM was aspirated the next day just prior to cell culture. The growth media was exchanged 2 days later with myoblast differentiation medium Table 2. Half of the culture media was exchanged every other day thereafter. Plates were then placed within a vacuum chamber for a minimum of 10 min to remove bubbles from the liquid PDMS.

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    At this point, plates were parafilm sealed, and stored at room temperature. Pluronic acid solution was aspirated and molds were rinsed with a PBS solution before seeding human muscle tissues. Remark: The integrity of nylon hooks, as anchor points, on the Velcro pieces is critical for the successful culture of 3D tissues and preventing their immature rupture. As such, we recommend careful inspection of the Velcro pieces before to ensure latch and hooks are without defects, and after to ensure pieces are well adhered in the dish.

    These are critical steps pre-tissue seeding to ensure successful tissue remodeling and culture. Three-dimensional 3D human skeletal muscle tissues were generated in culture as previously described Madden et al. Briefly, cells at these defined ratios were resuspended in the hydrogel mixture Table 2 in the absence of thrombin.

    Thrombin Sigma was added at 0. Myoblast growth media Table 2 lacking bFGF, but containing 1. In both cases agrin, neuregulin , treatment began when tissues were switched to differentiation medium and recombinant proteins were added to exchange media at 2-fold concentration. James Thomson. Remark: Batch-to-batch variability of small molecules and growth factors used in the motor neuron differentiation process can affect the success of the differentiation. As such, we recommend testing each new batch. As such, the cell pellet post thaw should be transferred from the conical tube to the culture plate with passage through a pipette no more than three times.

    Half of the culture media was exchanged every other day thereafter and included both neurotrophic factors at 2-fold concentration. Thrombin was added and tissues were seeded into dumbbell-shaped molds as described above. Co-cultures were analyzed at time points indicated in the figures and legends. For a subset of experiments , Figure 2—figure supplement 2F , Figure 3—figure supplement 2A , Figure 4—figure supplement 1C—D , and Videos 11 — 12 , immortalized myogenic progenitor cells AB, from fascia lata muscle of a healthy 20 year old male , were employed.

    Briefly, human-derived skeletal muscle cell hSMC lines used in this work were derived from healthy subjects and were then immortalized by transduction with human telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, as previously described Mamchaoui et al. The cell population was then sorted for GFP expression to enrich transduced cells and were then further expanded in myoblast growth media Table 2.

    Methods to produce 3D neuromuscular tissues using immortalized myogenic progenitor cells were exactly as those described above for primary human muscle progenitors with the exception that fibroblast-like cells were excluded. Neurites are dissected using the forceps and the released motor neuron cluster is carefully transferred to a 1.

    Attention should be given to visually inspect and avoid collection of undifferentiated cells in this step. Qualitatively, motor neuron clusters possessing a densely packed cluster of nuclei yielded reproducible results. Remark: High levels of spontaneous contractile activity of myofibers is observed in 3D muscle-motor neuron co-cultures post day 10 differentiation which might lead to their premature rupture at an earlier time before day As such, 3D co-culture tissues should be inspected carefully post-day 10 differentiation. Following fixation, samples were incubated in blocking solution Table 2 for at least 1 hr.

    After several washes in blocking solution, samples were incubated with appropriate secondary antibodies diluted in the blocking solution for 30 min at room temperature. Confocal images were acquired with Fluoview software using an Olympus IX83 inverted microscope. Western blot was performed using a Bio-Rad Power Pac and Trans-Blot Turbo Transfer System to transfer the proteins from the polyacrylamide gel to a nitrocellulose membrane. Samples were then washed with PBS and 40X images, all 0.

    AChR cluster outline drawings were binarized to facilitate downstream analysis. The Sub Sample and Particle Analyzer method was used with FracLac along with default settings and four grid locations. Lacunarity was measured for each AChR cluster within an experiment and then averaged. To ensure accurate and reproducible conditions for electrical stimulation, a custom-made stimulation chamber was produced using a 35 mm petri-dish, two carbon rods, and platinum wires.

    At day 14 of differentiation, an individual tissue was transferred to the chamber and covered in differentiation medium. Platinum wires were hooked up to a commercial function generator Rigol DGU. A Rigol DSE digital oscilloscope was used to confirm the frequency and amplitude of signals before connecting the pulse generator to the platinum wires. Cells were then sorted to purify the infected cells based on GFP expression. Human skeletal muscle progenitor cultures expressing GCaMP6 were imaged using an Olympus IX83 microscope equipped with modules to control the temperature and CO 2 concentration.

    To assess the effect of Waglerin-1 on glutamate-stimulated calcium transients, a video was recorded for each tissue before and after glutamate stimulation. Video segments, equal in length, representing pre- and post-glutamate GcAMP6 signals were each projected into a 2D image. The 2D projected images were then subtracted to eliminate spontaneously active fibers from our analysis.

    Background from different imaging sessions were normalized. To quantify neuromuscular tissue contraction, videos were assembled into stacks using ImageJ software and 3 regions of interest were traced within each stack. Maximum movement distance for each trace was determined and averaged for each sample. Data are presented as movement distance in pixels. In this studies, neuromuscular co-cultures were generated using GCaMP6 transduced human myogenic progenitor cells and a single motor neuron cluster.

    Videos were captured at a frequency of 12 frames per second for at least 15 s before and after stimulation. Videos were processed exactly as described above calcium transient analysis to eliminate the spontaneously active fibers from the analysis. The location of the motor neuron cluster was identified from a bright field image, which was used to outline the structure with a circle in the epifluorescent images.

    Binned data from three independent experiments were then reported on a histogram to report the average number of glutamate responsive fibers as it relates to the distance i. Data were digitized at 10 kHz and filtered at 2. Data were quantified using MiniAnalysis Synaptosoft. Each fiber was allowed to recover for a few minutes, to allow its resting membrane potential to stabilize before recordings were performed.

    For electrophysiological recordings following optogenetic stimulation, 3D muscle tissues were generated using human skeletal muscle progenitors transduced with a lentiviral vector encoding humanized ChR2 with HR mutation fused to EYFP and driven by EF1a AddGene plasmid Cells were sorted to purify the infected cells based on the EYFP signal. In glutamate stimulation experiments, glutamate was pipetted by hand into the edge of the bath and allowed to diffuse to the tissue.

    Remark: To facilitate electrophysiological recordings, 3D neuromuscular tissues were removed from their culture well by popping the entire circular piece of PDMS containing the channel and co-culture tissue out of the well plate and transferring it into a 35 mm culture dish. Addition of the neurotransmitter glutamate generates ripples and disturbs the equilibrium in the culture bath that may lead to the loss of sharp microelectrode recording. Therefore, the bolus of the neurotransmitter should be added from the side of the culture bath gently to then allow for its diffusion the tissue.

    Remark: Spontaneous contractions of the neuromuscular tissues complicate the sharp microelectrode recordings. Neuromuscular tissues are most spontaneously active once removed from the incubator. As such, resting the tissue at room temperature for 5—10 min after transferring from the 37 C incubator reduces the spontaneous activity and facilitates the sharp microelectrode recordings. In these experiments, IgG was added once and the media was not exchanged thereafter. After 3 days of treatment, the Day 14 co-cultures were stimulated with glutamate followed by an acetylcholine stimulation and calcium transients were captured by imaging the GCaMP6 signals using an Olympus IX83 microscope.

    Video segments, equal in length, representing GCaMP6 signals from glutamate and ACh serially stimulated tissues were each projected into a 2D image. Motor neuron clusters were separated from undifferentiated single cells using Accutase ThermoFisher and transferred to a Geltrex coated 6-well plate on Day 14 of differentiation. Clusters were washed with HBSS for at least one hour at room temperature before imaging. Videos were analysed for fluorescence intensity before and after stimulation at each indicated time-point using NIH ImageJ software. GAPDH gene expression served as the loading control.

    All oligo sequences are summarized in Table 4. In , two muscle tissue cultures technical replicates were treated with Agrin for each biological replicate six samples in total. In and 7 technical replicates from three muscle patient donors was analyzed at the 1 week culture time point. For all other neuromuscular co-culture studies, each primary myoblast line was co-cultured with MNs established from separate human pluripotent stem cell derivations.

    Statistical analysis was performed on data obtained from technical replicates using GraphPad Prism 6. Statistical differences between experimental groups were determined in most studies by unpaired t-test. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction" for consideration by eLife.

    Your article has been reviewed by three peer reviewers, including Andrew Brack as the Reviewing Editor and Reviewer 1, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The manuscript from the Gilbert lab provides the first demonstration of a robust method to generate mature muscle fibers with functioning 'adult' NMJ in vitro. This has been a significant hurdle for many years.

    This new approach will have significant utility for basic scientists working in the muscle or NMJ field, and those that model disease in vitro. The experiments have been performed to the highest standard, the data are compelling, the methods thorough, and the statistics appropriate. This work is a significant technical advance. Reviewers felt that the authors provide compelling evidence for the use of 3D muscle cultures. While there is general excitement for the manuscript, there were two main issues raised.

    Is activity required for the maturation of the muscle fibers? Below are the main points to consider. These points are given more context within the reviewer's individual comments. Please provide information outlining the ease of adaptability and the technical hurdles that are critical for success.

    Do they get more fully formed NMJs? Do the authors see action potentials prior to glutamate application? If the authors have data that could be incorporated within the 2-month revision window, please do so. If not, the manuscript would benefit from a discussion on this issue. I have one request for the authors, which stems from the fact that the take away message is the ease of the method.

    And while the data support this notion, I would ask the reviewers to provide more information in commentary or data as to where the limitations or difficulties in the method lie. For example, what is the day-to-day variability between neuromuscular samples, or the variability between dishes? In essence, it would be helpful to any adopter of this method if they understood the limitations and potential roadblocks.

    The paper by Bakooshli et al. The paper is fairly comprehensive and mostly quite well-done. I think it is suitable for eLife and should be published after suitable revisions. I have three general comments and then several specific questions detailed below. First, the authors have compelling data illustrating differences in the speed of muscle development. Can any lessons be learned see also below from 3D that could be applied to 2D cultures? Second, while the authors do an admirable job of comparing their new 3D systems with a more conventional 2D systems always difficult to do fairly since there is always a clear preference for one vs.

    First of all, it's not clear to me how easy the system described in this manuscript would be to establish in a non-engineering based lab. A guide to its use or an outline of the type of future developments that would make this system more routine would be useful. Many of the authors' measurements are made on "bulk" cultures, with fewer being done on individual fibers or individual NMJ's. So, certainly one downside may be that 2D does make some of that more straightforward many of my questions listed below relate to what can be observed at a single fiber level in 3D and a few require additional straightforward experiments.

    My final general comment is that the myasthenia gravis work seems like an almost unnecessary add-on. Was this done to compare the current system to that used by Steinbeck et al. In vitro studies of skeletal muscle can be problematic because the in vivo function of these cells requires interaction with multiple other cell types including motor neurons, however these interactions are difficult to replicate ex vivo.

    This article describes a method for generating de novo differentiated muscle fibers 3D culture in vitro, coupled with generation of pluripotent stem cell-derived motor neurons, which will self-assemble into functional units in coculture. To validate the technique, the authors showed that these muscle fibers express myosin heavy chain and acetylcholine receptor isoforms specific to mature adult muscle which are not usually upregulated in vitro and demonstrated that the neuromuscular synapses NMJs respond in a physiologically appropriate fashion to electrical and chemical stimulation, as measured by muscle contractile activity, calcium flux, and electrophysiology.

    These muscle fibers and motor neurons were generated from human cells, so as a final proof-of-principle the authors treated the matured neuromuscular units with serum from patients with myasthenia gravis MG , an autoimmune disease which targets the mature isoform of NMJ receptors and diminishes their function. Within two weeks of treatment, they observed alterations in the in vitro generated NMJs mimicking clinical signs in patients, supporting the potential utility of these in vitro cultures for modeling human disease.

    This paper is a fairly straightforward paper; the experiments are well designed and documented and the paper itself is clearly written. The authors are correct in stating that there is an unmet need in the field for an in vitro model of the mature NMJ, and the culture system they describe is better than anything currently in use in the field. As this is primarily a technique paper, I don't think additional studies beyond what is already included are necessary, although there are several that could be done which come to mind- if this is published, I think it will be of significant use to the field and those experiments will be carried out by this group and others.

    We thank the reviewers for the opportunity to provide more detail related to our personal experiences with this co-culture method and platform. Indeed, this information is key to ensuring widespread adoption, together with our proactive efforts in hosting labs from across the world for hands-on training.

    The Pharmacology of Nerve and Muscle in Tissue Culture The Pharmacology of Nerve and Muscle in Tissue Culture
    The Pharmacology of Nerve and Muscle in Tissue Culture The Pharmacology of Nerve and Muscle in Tissue Culture
    The Pharmacology of Nerve and Muscle in Tissue Culture The Pharmacology of Nerve and Muscle in Tissue Culture
    The Pharmacology of Nerve and Muscle in Tissue Culture The Pharmacology of Nerve and Muscle in Tissue Culture
    The Pharmacology of Nerve and Muscle in Tissue Culture The Pharmacology of Nerve and Muscle in Tissue Culture

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