A cell therapy approach to prevent muscle atrophy and paralysis in an aggressive mouse model of ALS

doi:10.21203/rs.3.rs-1970365/v1

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A cell therapy approach to prevent muscle atrophy and paralysis in an aggressive mouse model of ALS

https://doi.org/10.21203/rs.3.rs-1970365/v1

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Breakdown of neuromuscular junctions (NMJs) is an early pathological hallmark of amyotrophic lateral sclerosis (ALS) that blocks neuromuscular transmission, leading to muscle weakness, paralysis and, ultimately, premature death. Currently, there are no available therapies to prevent progressive motor neuron degeneration, muscle denervation or paralysis in ALS. Here, we report important advances in the development of an optogenetic cell therapy for ALS that can restore innervation of targeted skeletal muscles and provide an interface to selectively control their function. Complete survival of intraneural grafts of channelrhodopsin-2 (ChR2) expressing motor neurons, derived from allogeneic embryonic stem cells (ESCs), is enabled by transient immunosuppression using a T-cell-β receptor targeting antibody. Importantly, the engrafted light-sensitive motor neurons robustly reinnervate target muscles in the highly-aggressive SOD1^G93A mouse model of ALS, even following engraftment after overt symptom-onset. Moreover, daily optical stimulation training of the engrafted ChR2-motor neurons reinforces de novo NMJs, resulting in a significant enhancement of the force generating capacity of the targeted muscle in response to optical stimulation and prevention of muscle fibre atrophy. These beneficial effects are maintained long term, even until late-stage disease in SOD1^G93A mice. Together, these advances pave the way for the development of a combinatorial therapeutic approach to restore function of paralyzed muscles and prevent muscle atrophy that could benefit all ALS patients, irrespective of the pathogenic or genetic trigger.

The progressive degeneration of motor neurons that occurs in amyotrophic lateral sclerosis (ALS), the most common adult motor neuron disease (MND), affects almost all cellular components of the neuromuscular system, including cortical and spinal motor neurons, interneurons¹, glial cells^2,3, as well as muscle⁴. Motor axons, contained within peripheral nerves, serve as the final common relay for transmission of motor signals that control all voluntary muscle contraction and movement. However, one of the earliest characteristic pathological features of ALS involves “die-back” of motor axon terminals⁵ and breakdown of neuromuscular junctions (NMJs), the specialized synapses between motor axon terminals and muscle fibres. Effectively, this represents a single point of failure that permanently blocks motor signal transmission, irrespective of the condition of central motor circuits involved in coordination of movement signals. This results in an inexorable progression of muscle weakness, atrophy and, ultimately, complete paralysis, eventually leading to premature death. The median survival time in ALS, from initial onset of symptoms to death, typically as a result of respiratory complications, is only 20-48 months⁶.

To date, over 20 monogenic mutations that cause ALS have been identified, although these still only account for ~10% of total cases, with the remaining ~90% being classified as sporadic ALS⁷. The downstream cellular and molecular pathomechanisms underlying neurodegeneration in ALS are extremely complex and include dysregulation of proteostasis, autophagy, RNA metabolism and axon transport, as well as excitotoxicity, oxidative stress and neuroinflammation⁸. Given the disparate causes and complex disease mechanisms, development of an effective therapy has proven extremely challenging and there are currently no effective treatments capable of arresting the progressive paralysis that occurs in ALS⁹. Even exciting emergent gene therapy approaches, such as antisense oligonucleotides (ASOs) that have shown early promise in clinical trials¹⁰, are unlikely to benefit the majority of patients with sporadic ALS and will not replace degenerated motor neurons or restore motor function once it has been lost. As a strategy to address the loss of motor neurons, pioneering clinical trials of stem cell-based therapies have largely focused on targeting the spinal cord in ALS patients^11-13. However, the primary aim of these approaches has been to deliver trophic support to protect surviving motor neurons and thereby slow disease progression. Unfortunately, the outcome of these trials has been modest, at best. It is also unclear whether they will benefit patients with overt motor function deficits. Therefore, there remains an urgent unmet need to develop novel therapies to rescue muscle innervation and maintain muscle function in ALS patients.

We have previously demonstrated a novel proof-of-concept strategy to overcome muscle denervation and restore control of muscle contraction in a nerve injury model of muscle paralysis that could have major therapeutic value for restoring function of any targeted muscle or group of muscles in ALS patients¹⁴. Briefly, optogenetically-modified replacement motor neurons, derived from murine embryonic stem cells (mESCs), were engrafted into distal branches of peripheral nerves supplying specific lower hindlimb flexor and extensor target muscles in wildtype mice that had undergone a nerve ligation injury. Our results showed that the engrafted motor neurons were able to project axons from the graft site to the target muscles where they formed de novo NMJs. Due to the ectopic location of the engrafted motor neurons, outside the central nervous system (CNS), they do not receive endogenous motor signals, and must therefore be exogenously activated. Expression of the blue-light sensitive channelrhodopsin-2 (ChR2) protein¹⁵ in the engrafted motor neurons conferred the ability to selectively activate these engrafted neurons and thereby control the contractile function of the target muscle using acute optical stimulation¹⁴. The aim of this neural replacement strategy is therefore to provide a biological interface capable of rendering any target muscle receptive to control signals transmitted by optical stimulation to engrafted motor neurons¹⁶.

This novel approach to restore control of paralyzed muscles in ALS patients, using optical stimulation and cell replacement, has several key advantages over existing cell replacement and electrical stimulation strategies, including: i) the ability to engraft motor neurons peripherally, in close proximity to targeted muscles, which greatly accelerates the rate of reinnervation and delays the period of denervation, consequently reducing denervation-induced muscle atrophy; ii) avoidance of engrafting replacement cells into the neurotoxic environment that exists within the CNS of ALS patients and the necessity for reinnervating axons of CNS-engrafted motor neurons to overcome the inhibitory CNS:PNS barrier in order to exit the CNS and grow the often long distances to target muscles; iii) specificity of optical stimulation to the engrafted ChR2+ motor neurons avoids painful off-target activation of sensory afferents or aberrant activation of endogenous motor axons associated with electrical nerve stimulation (ENS); and critically, iv) the ability to recruit motor units in correct physiological order using optical nerve stimulation (ONS)¹⁷, avoids the problem of rapid muscle fatigue associated with ENS and incorrect, non-physiological motor unit recruitment. Furthermore, ENS-mediated control of muscle function depends on the presence of surviving motor axons and, since these are progressively lost during the course of disease progression in ALS, the ability of ENS to induce muscle contraction is steadily eroded. More importantly, it has recently been shown that ENS, applied to the phrenic nerve to assist respiratory function in two separate clinical trials in ALS patients^18,19, accelerated diaphragm muscle denervation, which likely contributed to a significant reduction in patient life-span²⁰. Therefore, it is unlikely that ENS will ever be suitable for artificial control of critical muscle function in ALS patients. In contrast, optogenetic stimulation, in combination with neural replacement, represents a safe alternative approach to artificially restore innervation and function of paralyzed muscles in ALS.

In the current study, we sought to optimize critical elements of this novel therapeutic strategy and to determine whether ChR2⁺ motor neurons can be used to successfully restore innervation, induce muscle contractions and prevent atrophy of targeted muscles in a model of ALS using the highly-aggressive SOD1^G93A mouse model of ALS.

Host-vs-graft response causes rapid rejection of most intraneural ESC-MNs allografts

ChR2⁺ motor neurons were derived from our previously characterized mouse embryonic stem cell (mESC) line¹⁴ and differentiated using a well-established protocol²¹. Since these donor ChR2⁺ motor neurons were generated from an mESC line originating from the 129S1SvImJ mouse strain (Table S1) and recipient mice SOD1^G93A mice were on a congenic C57BL6/J genetic background, they constitute an allogeneic source of donor cells. Importantly, allogeneic cells are likely to provide a more cost-effective, off-the-shelf cell therapy platform, compared to generating individual, patient-specific batches of cells suitable for autologous engraftment. Thus, an important initial objective of this study was to identify an immunosuppression regimen capable of preventing host-vs-graft rejection of allogeneic ChR2⁺motor neurons to enable their innervation of target muscles in recipient SOD1^G93A mice.

In the absence of any immunosuppression, the survival rate of ChR2⁺ motor neuron grafts at 35 d post engraftment, was only 2.3% (3/129) in SOD1^G93A mice. However, in the rare cases where the engrafted ChR2⁺ motor neurons did survive, we observed robust intramuscular axon growth and partial reinnervation of NMJs, even at very late-stage disease (Fig. 1a-c and Supplementary Video 1). Furthermore, acute optical stimulation of the engrafted ChR2⁺motor neurons was able to induce tetanic contraction of the target muscle in these SOD1^G93A mice (Fig. 1d), although these were weak in magnitude. Interestingly, assessment of shorter timepoints revealed that engrafted ChR2⁺ motor neurons were capable of surviving for up to 14 days (data not shown). It is therefore likely that the poor long-term survival was due to host-vs-graft rejection of the engrafted cells, rather than disease-related toxicity. These findings suggest that if the engrafted motor neurons can evade the host immune response, they can functionally reinnervate target muscles for a therapeutically relevant timescale in this highly aggressive model of ALS.

Tacrolimus (FK506) overcomes ESC-MN allograft rejection but inhibits muscle innervation

In an effort to enhance graft survival, we first tested the calcineurin inhibitor, tacrolimus (FK506), which is not only routinely used to facilitate solid organ allograft survival in humans but has also been reported to promote axon regeneration after nerve injury²². Cohorts of wild-type and SOD1^G93A mice (table S2) were treated with FK506 (5mg, kg^-1, d^-1) from the time of intraneural ChR2⁺ motor neuron engraftment; graft survival and muscle reinnervation were then assessed 30-35d post-engraftment. Although FK506 did enable robust ChR2⁺ motor neuron allograft survival, at the graft site, in all animals examined (Table S2), we identified three major problems with this immunosuppressant agent. Firstly, although the engrafted motor neurons were to survive and project axons along peripheral nerve branches and within the targeted lower hindlimb muscles (Fig. 2a,b), FK506 completely prevented muscle fibre reinnervation, evidenced by lack of response to acute ONS (data not shown) and complete absence of ChR2⁺/YFP⁺ NMJs (Fig. 2c) in both SOD1^G93A (n=8) and nerve-ligated wild-type mice (n=4). Secondly, exuberant growth of carry-over pluripotent stem cells led to intraneural tumour formation in most mice (Fig. 2d and Table 2), which resulted in overt hindlimb motor deficits (Fig. 2e and Supplementary Video 2). Thirdly, in partial agreement with previous reports in rats²³, FK506 prevented body mass increase and/or induced body mass decline in a subset (44.4%; 8/18) of SOD1^G93A mice (Fig. 2f); this effect was less evident in wild-type mice (Supplementary Fig. 1). Onset of body mass decline in SOD1^G93A mice typically occurs at ~115 days in²⁴, indicating that FK506 may exacerbate the disease phenotype.

Since calcineurin inhibitors are known to be associated with peripheral neuropathy²⁵, it is possible that FK506 treatment alone may adversely affect endogenous or engrafted motor axons. Indeed, examination of the cross-sectional area distribution of total (ie. sensory + motor) and motor neuron axons in branches of the sciatic nerve in FK506-treated SOD1^G93A and WT mice (Fig. 3a) revealed a significant loss of axons that affected most axonal calibres in the tibial nerve in wild-type and SOD1^G93A mice (Fig. 3b,c). A more pronounced loss of total and motor axons, spanning medium to large sized axonal calibres, was observed in the common peroneal nerve branch (Fig. 3d,e), which clearly shows that FK506 can not only exacerbate ongoing motor axon loss in SOD1^G93A mice but can also induce motor axon loss even in wild-type mice. Importantly, these neuropathy-like effects appear to be specific to FK-506, since an alternative immunosuppressant, H57-597 mAb (discussed in detail below), did not significantly alter total or motor axon size distribution or total axon counts (Supplementary Fig. 2), compared to untreated SOD1^G93A mice.

Since FK506 is known to suppress myoblast proliferation and differentiation²⁶ and can cause rare cases of myopathy in humans²⁷, it is also possible that the FK506-dependent prevention of muscle reinnervation by engrafted ChR2⁺ motor neurons is due to a muscle specific effect. In any case, these findings clearly show that FK506 is unsuitable as an immunosuppressant to support ChR2⁺ motor neuron allograft survival and, indeed, suggest it should be contra-indicated for use in ALS patients. Importantly, however, the complete protection of engrafted ChR2⁺ motor neurons by FK506 confirms that host-vs-graft rejection was responsible for the poor long-term graft survival observed in the absence of immunosuppression, rather than disease related neurotoxicity.

T-cell modulatory immunosuppression confers graft survival and target muscle innervation

In light of the deleterious effects of FK506, and given our aim of conferring compatibility of allogeneic ChR2⁺ motor neurons as a universal cell therapy for ALS, we sought to identify a more specific form of immunosuppression that avoids the negative effects of FK506 yet supports long term graft survival. Therefore, we investigated the T-cell receptor-β (TCR-β) targeting monoclonal antibody, mAb H57-597, which has previously been shown to effectively promote long-term heart allograft survival in mice²⁸. In addition, since our findings indicated that immunosuppression results in a greater risk of tumour formation from carry-over pluripotent stem cells, differentiated ChR2⁺ motor neurons were also pre-treated with mitomycin-C (MMC; 2 μg/ml for 2 hrs) prior to engraftment, to eliminate tumorigenic cells ²⁹, to further enhance the translational potential.

MMC-treated ChR2⁺ motor neurons were unilaterally engrafted into the tibial nerve of symptomatic SOD1^G93A mice (aged 95.7 ±4.6 days) in conjunction with transient H57-597 mAb delivery (1μg, kg^-1; i.p. injection at 0, 1-, 3-, 7- and 14-days post-engraftment). The extent of reinnervation and the ability to optically control the function of the triceps surae (TS) muscle group in the lower hindlimb of SOD1^G93A mice was then assessed at late-stage disease (133 ±6.9 d; n = 12) by determining the physiological response of the reinnervated muscles to acute ONS of the engrafted motor neurons followed by histological analysis of muscle and nerve.

Histological analysis confirmed that in mAb H57-597 treated animals, engrafted ChR2⁺ motor neurons were present in all recipient SOD1^G93A mice (n > 78 to date). Importantly, there was significant axonal projection within intramuscular nerve branches and robust reinnervation of muscle fibres in the targeted TS muscle (Fig. 4a,b; Supplementary Video 3 and 4). As we previously reported in a wild-type mouse nerve ligation model¹⁴, some de novo NMJs in SOD1^G93A mice exhibited signs of immaturity, including poly-innervation (Fig. 4c), as well as collateral and terminal sprouting of motor axons (Fig. 4d). Since the peripherally-engrafted reinnervating motor neurons are inactive during the post-engraftment period and progressive muscle atrophy is ongoing, only 10.5% of endplates are innervated by engrafted ChR2⁺ motor neurons (Fig. 4e). Importantly, these reinnervated endplates are functional, since acute in vivo ONS of the engrafted ChR2⁺ motor neurons in the exposed sciatic nerve of late-stage disease SOD1^G93A mice at 133 ±7.2 days of age (37.7 ±5.1 days post-engraftment; n = 12), induced positive contractile responses in all animals, although the amplitude of the maximal contractile force was still weak (0.8 ± 0.2 g; n = 11).

Motor neuron subtype identity does not affect response to optical stimulation

In an effort to increase the amplitude of the contractile response of the target muscle to optical stimulation we next tested whether engraftment of motor neurons with a fast-firing subtype identity may be more suitable than engraftment of predominantly slow-firing motor neurons by using alternative differentiation protocols³⁰. The stronger, predominantly fast-twitch, gastrocnemius component of the TS muscle group is usually innervated by fast-firing/fast-fatigable (FF) motor neurons, which are known to have the capacity to innervate many more individual muscle fibres per motor unit than slow-firing motor neurons, which normally innervate a much smaller number of weaker, slow-twitch muscle fibres, predominantly within the soleus and plantaris regions of the triceps surae. MMC-treated ChR2⁺ motor neurons, differentiated to yield FF subtype identity motor neurons, were engrafted into the tibial nerve of (106 ±7.2 days) SOD1^G93A mice, in combination with transient H57-597 mAb treatment. Maximum isometric muscle contraction of the TS muscle in response to acute optical stimuli was then determined at the same age (133.9 ±7.2 days, n = 13) as previous grafts of predominantly slow-firing motor neurons (133 ±6.9 days, n = 11). This physiological analysis revealed that the motor neuron subtype identity did not significantly affect amplitude of the muscle response to acute optical stimulation (Supplementary Fig. 3a) and that the maximum contractile force elicited by ONS remained extremely weak compared to supra-maximal ENS (Supplementary Fig. 3b). This result implies that, unlike during normal neuromuscular development, motor neuron subtype identity is not an important determinant of muscle fibre innervation in the mixed fibre type triceps surae muscle. This finding has significance for future clinical translation, since only a single subtype of motor neuron may be required to innervate a variety of different muscles. Only MMC-treated motor neurons with a slow-firing medial motor column identity were used in the subsequent experiments reported here.

Since modification of motor neuron subtype identity did not enhance contractile response, our next challenge was to identify an effective method to enhance reinnervation and force generating capacity of the targeted muscle in response to optical stimulation.

Optical stimulation training significantly enhances target muscle force generation

NMJ formation is known to be an activity-dependent process, thus, without regular stimulation, although peripherally engrafted ChR2⁺ motor neurons may survive, they are unlikely to form mature NMJs and will therefore have little effect on declining muscle function and atrophy in SOD1^G93A mice. Moreover, paralysis and atrophy of affected muscles proceed unchecked in the SOD1^G93A mouse model of ALS. We therefore investigated whether regular activation of the engrafted ChR2⁺ motor neurons, in conjunction with H57-597 mAb treatment, could enhance NMJ maturation and maximize the ability of target muscles to generate contractile force in response to acute optical stimulation in late-stage SOD1^G93A mice. To do this, we adapted a wireless, fully implantable optical stimulation system³¹, in order to implement a daily optical stimulation training regimen for engrafted mice.

First, we modified the PCB design to simplify assembly (Fig. 5a), implemented a new encapsulation method to ensure long term survival of the devices after implantation (Fig. 5b) and incorporated an RF signal switch and pulse controller to deliver precisely timed RF pulses to power a 470nm light emitting diode (LED) connected to the implantable device (Fig. 5c).

The modified optical stimulation devices were then surgically implanted in SOD1^G93A mice concomitantly with intraneural engraftment of ChR2⁺ motor neurons, with the trailing LED positioned in close apposition to the graft site (Fig. 6a). Commencing at 14 d post-engraftment, to allow growing ChR2⁺ motor axons sufficient time to reach the target muscle, the mice were transferred to a custom built chamber located above a resonance-frequency induction cavity for 1 hr per day, in order to undertake optical stimulation training (OST; Fig. 6b and Supplementary Video 5), using a bespoke pulse pattern that was empirically determined to elicit maximum contraction (Supplementary Figs. 4 – 6), followed by a 2 s rest interval. Following daily OST in engrafted SOD1^G93A mice for 21 days, isometric muscle tension physiology was performed at late-stage disease to determine the maximal contractile force of the TS muscle elicited by acute ONS of the exposed sciatic nerve graft site.

In confirmation of our hypothesis, there was a highly significant, 9.4-fold, increase in the maximal tetanic force (7.5 ±0.94 g versus 0.8 ±0.2 g; p = <0.000001) elicited by ONS in the engrafted OST group of SOD1^G93A mice at late-stage disease, compared to age-matched untrained SOD1^G93A mice (132.4 ±6.8 days versus 133 ±6.9 days; n = 7 and 11, respectively; Fig. 6c-g), although OST did not alter contractile rate characteristics (Supplementary Fig. 7a-c). Moreover, quantitative comparison of the maximum force elicited by ONS compared to ENS of TS muscles in late-stage SOD1^G93A mice (Fig. 6h), showed that in mice that underwent OST, acute ONS elicits up to 22.7 % (± 4.5) of total residual muscle force produced by supra-maximal ENS, which activates both endogenous and engrafted motor neurons (Fig. 6i), in contrast to only 1.46 % (± 0.18) in untrained SOD1^G93A mice (Supplementary Fig. 8). This represents a remarkable >13-fold improvement in force generation. In engrafted SOD1^G93A mice that did not undergo OST, the extremely weak twitch contractions in response to ONS precluded interrogation of individual motor unit force values and determination of motor units number estimates (MUNE) in most mice. In contrast, in SOD1^G93A mice that underwent OST, the significantly increased contractile response to ONS enabled clear separation of individual motor unit values (Fig. 6j), enabling MUNE values to be determined (Supplementary Fig. 7d). Furthermore, as we previously reported in nerve-ligated WT mice¹⁴, delivery of repetitive ONS pulses (250 ms bursts of 20 Hz illumination, every 1 s, for 180 s duration) to induce rhythmic, submaximal contraction of the TS muscle did not induce rapid muscle fatigue, whereas equivalent pulses of ENS stimulation of the contralateral TS muscle did result in rapid muscle fatigue (Fig. 6k). This observation has significant implications for the ability to control repetitive motor functions in ALS patients.

Optical stimulation training prevents atrophy of reinnervated muscle fibres

Finally, having established that optical stimulation training significantly enhances the maximal force elicited by optical stimulation of engrafted ChR2⁺ motor neurons in late-stage SOD1^G93A mice, we examined whether long-term optical stimulation training could also prevent atrophy of reinnervated muscle fibres. Since NMJs comprise an extremely small volume of the entire muscle, high-resolution 3D imaging of the entire muscle to determine muscle fibre innervation status and fibre diameter information is not feasible. Therefore, we developed a novel technique, termed digital Cross-sectional area Analysis from Longitudinal Muscle Sections (dCALMS), in order to assess these properties. This involved 3D reconstruction and analysis of regions of interest, obtained from 30μm thick longitudinal TS muscle sections (Fig. 7a) from ChR2⁺ motor neuron engrafted, late-stage SOD1^G93A mice that had undergone OST. Each region contained at least one NMJ innervated by a ChR2⁺ motor neuron, along with randomly captured neighboring fibres (Fig. 7b, Supplementary Fig. 9 and Supplementary Video 6). The 3D reconstructions were then digitally re-sliced in the transverse orientation (Fig. 7c, top panel), in order to obtain data on muscle fibre cross-sectional area (CSA) using a semi-automated process (Fig. 7c, lower panel). The dCALMS analysis revealed that the average CSA of muscle fibres innervated by engrafted ChR2⁺ motor neurons were similar in size to fibres still innervated by residual endogenous motor neurons (922.3 versus 1018.3 μm²; p = ≤0.85; Fig. 7d). Importantly, the CSA of muscle fibres innervated by ChR2⁺ motor neurons was significantly greater than fibres with completely denervated endplates (average CSA = 525.4 μm²; p = ≤0.00001) or fibres whose innervation status could not be determined (average CSA = 668.8 μm²; p = ≤0.0003), since the endplate was outside the scanned region of interest.

This study shows for the first time that replacement stem cell-derived motor neurons can robustly and reliably reinnervate target muscles in the highly aggressive SOD1^G93A mouse model of ALS, even when engrafted after onset of overt symptoms; moreover, the restored innervation can be maintained even until extremely late-stage disease. Furthermore, our findings show that engrafted ChR2⁺ motor neurons can not only provide an interface to safely and selectively control the function of, potentially, any targeted muscle but, also, that regular optical stimulation training (OST) can be used to: i) reinforce connectivity between engrafted motor neurons and muscle fibres; ii) significantly enhance the maximal force elicited by optical stimulation of the targeted muscle; and iii) prevent atrophy of muscle fibres that are reinnervated by engrafted motor neurons. The highly significant improvements in muscle innervation, atrophy prevention and maximum contractile force, as a result of the daily OST regimen, confirms that stimulation-induced activity is necessary to maximize connectivity between engrafted motor neurons and their target muscles.

The prevailing view in the ALS field is that spinal motor neuron pathology first manifests at the nerve terminal and that muscle fibres are likely to actively contribute to degeneration of endogenous motor neurons in ALS³². Our findings clearly demonstrate that affected muscles, even in the highly aggressive SOD1^G93A ALS model, remain receptive to reinnervation by healthy engrafted motor neurons, even until late-stage disease. Moreover, once target muscles have been reinnervated, this approach enables implementation of a safe muscle training/exercise regimen that can be used to preserve muscle integrity and prevent irreversible muscle wasting that otherwise occurs as a result of progressive neurodegeneration in ALS³³. Since skeletal muscles are not simply biomechanical actuators, but have complex functions in overall metabolic homeostasis, thermoregulation, venous return and maintenance of blood volume, the ability to prevent muscle atrophy using OST, is likely, by itself, to have major health benefits for ALS patients. In contrast, the use of electrical nerve stimulation (ENS) to control muscle function or, indeed, exercise programs is likely to accelerate degeneration of remaining motor axon terminals²⁰, and is therefore unlikely to be safe. Unlike ENS, the highly selective nature of ONS does not activate or interfere with endogenous motor neuron function and due to its ability to recruit motor units in physiological order, ONS has the added significant benefit of avoiding rapid muscle fatigue¹⁷. The ability to combat muscle atrophy, using OST, could extend the ability of targeted to execute functionally useful movements, potentially indefinitely in ALS patients.

Since differentiation methods that yield either fast-firing or slow-firing motor neurons did not appear to affect the ability of engrafted motor neurons to innervate the mixed fibre type TS muscle group in SOD1^G93A mice, motor neuron subtype identity appears to be redundant in this case. Therefore, a single type of motor neuron, produced at scale, could potentially be used to target a large number of different muscles in each patient. This has advantages in terms of simplifying the regulatory approval process, since the donor motor neurons would be produced in exactly the same way, irrespective of the graft site or recipient.

Indeed, another key finding of this study that could streamline future translation of this therapeutic strategy, is the identification of a TCR-β targeting antibody, H57-597 mAb, as an effective mediator of allograft survival. Existing T-cell targeting monoclonal antibodies, such as OKT-3, have been clinically approved³⁴ and, importantly, this form of immunosuppression overcomes severe adverse effects associated with the commonly used CNI-based immunosuppressant, tacrolimus (FK506). Our data shows that H57-597 mAb treatment was well tolerated during transient administration from symptom-onset up until late-stage disease in SOD1^G93A mice, however, the aggressive disease progression in this model precludes investigation of longer-term effects and it remains to be seen how long-term TCR-β-based immunosuppression may be tolerated in ALS patients. A similar immunosuppression regimen involving a CD25 (IL2) targeting monoclonal antibody, Basiliximab, along with glucocorticoid and tacrolimus maintenance therapy, has been shown to be safe but poorly tolerated in ALS patients, as an independent investigative approach³⁵, as well as part of a separate clinical trial assessing intraspinal grafts of neural precursor cells in ALS patients¹¹. Therefore, immunosuppression per se, is not an impediment to this strategy.

The use of allogeneic donor cells, with a safe and effective immunosuppression regimen, means that a future cell therapy could potentially be universally compatible with all ALS patients, which would significantly reduce costs and simplify the regulatory approval process, compared to individually tailored autologous cell grafts. Of course, further studies will be required to ensure that human-compatible, induced pluripotent stem cell (iPSC)-derived donor motor neurons are able to function in the same manner as allogeneic mouse ESC-derived motor neurons. The generation of HLA-matched super donor hiPSC lines may further mitigate the need for immunosuppression³⁶, however, immunogenicity of the ChR2 protein could mean that some form of immunosuppression may be necessary for any optogenetic therapy in the peripheral nervous system, including viral delivery³⁷.

The anatomical separation of specific nerve branches in humans means that this approach could be used to target and independently control large numbers of muscles in each individual ALS patient. However, it will be first necessary to demonstrate safety and efficacy in a relatively low risk muscle to restore a simple motor function. For example, the common peroneal nerve is highly accessible from a neurosurgical perspective and existing ENS devices, developed to correct foot drop for other neurological disorders³⁸, could be readily adapted to assist ambulatory function in early-stage ALS patients. In the longer term, existing multichannel ENS devices, which have been developed to control more complex ADLs in high-level spinal cord injury (SCI) patients³⁹, could also be adapted into a minimally invasive, transcutaneous optical stimulation device⁴⁰. This combinatorial therapeutic strategy, comprising allogeneic donor cells, an effective immunosuppression regimen and optical stimulation device, is highly compatible with the rapidly evolving field of brain computer interface (BCI) technology. BCI could be used to decode a paralyzed patient’s intention to perform a given movement in order to control the activity of the engrafted motor neurons, which provide the necessary interface to execute the intended movement via a wearable optical stimulation device. This novel approach would entirely bypass the severe damage that occurs throughout the entire CNS in ALS patients and enable autologous control of movement. Moreover, this strategy also has broad utility for a wide range of other neurogenic causes of paralysis, such as spinal cord injury and stroke.

Although the findings of this study clearly demonstrate that our combinatorial cell therapy approach is effective in a highly aggressive mouse model of ALS, further investigation is required in order to confirm that the strategy can be applied to alternate model of ALS, with a longer lifespan, in order to fully explore the long-term efficacy of the approach, particularly in terms of chronic allograft survival using a transient immunosuppression approach. It is possible that some form of maintenance therapy may also be required to confer long term graft survival. Of course, the biggest challenge will be to demonstrate that human optogenetically-modified motor neurons are capable of reinnervating target muscles in the same manner as we have demonstrated for mESC-derived motor neurons. It will also be necessary to scale this cell therapy strategy up, using larger animal models that more accurately recapitulate human-scale anatomy.

Although challenges remain, the findings of this study provide strong support for this novel cell therapy, which if successful could finally begin to deliver major health benefits for ALS patients.

Mouse embryonic stem cell (mESC) generation, culture and differentiation:

The mESCs used in this study were generated as previously described¹⁴. Briefly, mESC underwent successive rounds of gene-targeting to insert a Hb9::GFP-IRES-CD14 reporter construct to enable motor neuron purification, insertion of a CAG::Channelrhodopsin2-YFP (ChR2-YFP) construct using Tol2 transposition to enable optogenetic control of neural activity^15,41 and insertion of a CAG::Glial derived neurotrophic factor (Gdnf) construct using PiggyBac targeting to promote motor neuron survival; this resulted in generation of clone #C9G.

Undifferentiated mESCs, from clone #C9G, were expanded on irradiated C57BL/6 mouse embryonic fibroblasts (iMEFs) [Gibco] according to standard cell culture protocols. For maintenance, briefly, freshly thawed mESC aliquots were plated on iMEF feeder layers, grown on gelatinized 24-well or 6-well plastic tissue culture treated dishes (approx. 2.5 x 10⁵ cells/cm²), in mESC media, consisting of Knockout-DMEM [Gibco], supplemented with 15% ES-qualified FBS, 1x nucleosides, 1x non-essential amino acids, 1x L-glutamine + penicillin/streptomycin, 0.1mM 2-mercaptoethanol [all supplied by Gibco] and 100U/ml ESGRO® Recombinant Mouse LIF Protein [Merckmillipore]; cells were incubated at 37°C, 5% CO₂, cells were fed daily and passaged or differentiated prior to colonies reaching 70% confluence.

Motor neuron differentiation:

Motor neuron differentiation was carried out as previously described ^21,30. Briefly, mESC colonies were dissociated using TryplE solution [Gibco], 7 mins at 37°C, and transferred to non-tissue culture treated 10cm plates in differentiation medium, consisting of a 1:1 mix of advanced-DMEM/F12 and neurobasal media, 10% knockout serum replacement (KO-SR), 1x L-glutamine/Penicillin/Streptomycin and 0.1mM 2-mercaptoethanol (hereafter, termed ADNFK media; all components source from Gibco). For generation of medial motor column (MMC) identity motor neurons, mESCs (20,000 cell/ml) were incubated for 2 days at 37°C, 5% CO₂ as floating embryoid bodies (EBs) in ADFNK, before being split 1:2 in ADFNK media supplemented with 1μM retinoic acid (RA; Sigma) and 0.5μM smoothened agonist (Sag; Sigma) between day 3-5 of differentiation. For generation of lateral motor column (LMC) identity motor neurons the modified “caudalized-ventralized” (C-V) protocol was used³⁰: briefly, mESCs (10,000 cell/ml) were incubated for 2 days at 37°C, 5% CO₂ as floating embryoid bodies (EBs) in ADFNK, before being split 1:2 in unsupplemented ADFNK media between day 3-5 of differentiation and then transferred to ADFNK supplemented with 1μM RA between differentiation day 5-7.

On differentiation day 5 or 7, differentiated EBs were pre-treated with 1μg/ml of mitomycin-C for 2hrs, washed in L15 media and dissociated in Accumax [ThermoFisher], washed in L15 media, passed through a 20μm cell strainer and collected in PBS at a final concentration of 50,000 cells/μl; differentiated motor neurons were kept on ice prior to in vivo engraftment.

Single nucleotide polymorphism (SNP) analysis of mESC lines:

mESCs derived from clone #C9G, along with a C57BL/6J mESC line and the commonly used HBG3 (Hb9::GFP) mESC line, as controls, were grown to confluence in the absence of feeder cells, in mESC media and cells were collected and frozen as dry cell pellets, before being sent for commercial SNP analysis (Charles River Laboratories).

Production of implantable optical stimulation devices:

Optical stimulation devices were largely produced as described by Montgomery et al. (2014)³¹, with the following modifications: the design of individual printed circuit boards (PCBs) was modified to enlarge the central pads and the PCB was then fabricated on 0.2-mm thick FR4 board, which greatly facilitated assembly and mounting of individual components (Fig. 5a,b and Supplementary CAD files); an alternative 470nm (145mcd) LED (Wurth Elektronik, Manufacturer Part No: 150120BS75000) was attached to 1.5cm paired trailing wire connected to the power-receiving rectifier circuit, again to facilitate assembly; a 3-turn power-receiving coil with an internal diameter of 1.7mm was used. Following positioning of Schottky diodes and capacitors on the PCB, and after coating with lead-free solder paste (Chip Quick), brief exposure to a flame from a gas soldering iron was used in lieu of reflow soldering. Fully assembled devices were cleaned in an ultrasonic water bath, rinsed in 100% ethanol, dried and then encapsulated by insertion into a silicone-grease coated 0.2ml PCR tube that was then partially filled with optically clear epoxy (Opti-tec 5001) to cover the device. The epoxy was cured for 1hr at 65°C and remaining uncured epoxy was then applied to the inverted trailing LED to form a droplet, before additional curing at 65°C. After encapsulation, devices were removed from 0.2ml PCR tubes, cleaned again in an ultrasonic water bath (to remove residual silicone grease), before being immersed in 100% ethanol for sterilization. Modification of the encapsulation method was necessary to ensure longevity of the device following in vivo implantation. Prior to implantation, devices were transferred to sterile PBS and tested in the RF-resonance chamber (see below) to ensure they were fully functional.

Radio Frequency (RF) resonance cavity power transmission system:

As above, the power transmission system used to activate the implanted LED devices was largely as described by Montgomery et al. (2014)³¹, with the following modifications: a WindFreak SynthNV RF Signal Generator was used to generate a constant 1497MHz RF output (1-2bBm), that was fed into an absorptive SPDT, Solid State Switch (MiniCircuits; ZX80-DR230-S+), powered by a 1.5V CR2032 coin battery, which was controlled by a USB-TTL Interface (Prizmatix), to generate specific optical stimulation patterns (Supplementary Fig. 4).

In vivo intraneural engraftment of mESC-derived ChR2⁺ motor neurons:

All procedures and experiments involving animals were carried out under License from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 (Amended Regulations 2012) and following ethical approval from the UCL Queen Square Institute of Neurology Animal Welfare Ethical Review Body (AWERB).

B6.Cg-Tg(SOD1*G93A)1Gur/J mice (JAX) were bred specifically for this study by mating presymptomatic male transgene carriers with congenic C57BL/6J females (Charles River Laboratories); progeny were genotyped by standard PCR analysis and heterozygous transgenic mice were housed under a 12 hr dark/light cycle in IVC cages with behavioural enrichment and ad libitum access to food and water, until they reached the appropriate age. A total of 47 mice were directly used in this study and an additional 129 were used to develop the methodology.

Mice received prophylactic analgesia (buprenorphine, 0.1mg/kg), during induction of anaesthesia and preparation of the surgical site. The immunosuppressant monoclonal antibody (H57-597 mAb) was also administered at the start of the surgical procedure (day 0) by intraperitoneal (i.p.) injection (1mg/kg, diluted in 100μl of sterile saline), which was repeated on days 1, 3, 7 and 14 post-engraftment. Alternatively, in a subset of mice (n = 15), immunosuppression was achieved by daily i.p. administration of FK506 (tacrolimus; 5mg/kg/d, note: due to insolubility in aqueous solution, FK506 (16.67mg/ml) was dissolved in DMSO and stored as frozen 100μl aliquots that were freshly thawed and prepared immediately prior to injection by dilution in 30μl of 100% ethanol (15% final concentration) and 70μl sterile PBS containing 2% Tween-20 (8.33mg/ml final concentration of FK506; 0.6μl/g of body mass, mixed with saline to a final volume of 125μl immediately prior to i.p. injection). Mice were closely monitored and weighed at the time of immunosuppressant administration. As previously reported, H57-597 induced acute loss of body mass, which fully recovered in all cases within 7days from the initial dose; this adverse effect can be blocked by pre-treatment with cyclosporin A⁴².

Intraneural engraftment of ChR2⁺ motor neurons was performed under deep anaesthesia, using aseptic surgical techniques and body temperature was maintained throughout the surgical procedure using a 37°C heat mat, as previously described ¹⁴. Briefly, under a stereoscopic operating microscope (Zeiss), a 3mm skin incision was made in the lower posterior thigh and superficial muscles were separated and retracted to expose the sciatic nerve, immediately proximal to the trifurcation point in the popliteal fossa. The sciatic nerve was elevated from underlying muscles using a curved non-cutting suture needle, placing it under slight tension, and a 27G needle was used to make a small incision in the epineurium of the tibial and common peroneal nerve branches. Immediately prior to intraneural engraftment ChR2⁺ motor neurons (50,000 cells/μl in PBS containing 0.0004% Nile Blue dye, maintained on ice) were carefully resuspended by pipetting and loaded into a 5μl Hamilton syringe, equipped with a customized 33G needle. The needle was then carefully inserted through the incision made in the epineurium and guided along the length of the nerve (approximately 3-4mm), to prevent back-pressure induced leakage of cells; 1μl of cell suspension was slowly injected into the tibial nerve and 0.5μl into the common peroneal nerve and visualization of the Nile Blue labelled cells was used to verify intraneural cell delivery. In procedures during later stages of this study, a fine-tipped (approximately 100μm outer diameter) pulled glass microinjection needle, with a beveled tip and customized positive displacement plunger (Alpha laboratories) was used for intraneural injection of cells, to minimize damage to the nerve. After injection, the needle was carefully withdrawn and the sciatic nerve was returned to its normal position. A subset of mice (n = 7; excluding animals in which the device failed during the stimulation period) underwent implantation of an optical stimulation device (described above). Briefly, fine round-tipped tissue forceps were inserted through the same skin incision, along the length of the thigh bone and rostrally along the back, to create a subcutaneous cavity. The main body of the sterilized device was inserted into the cavity and positioned close to, and parallel with, the lower thoracic spine (to assist alignment of the power receiving coils with the RF-resonance frequency power transmitter). The trailing wire and encapsulated LED was secured in place immediately superficial to the graft site in the sciatic nerve by 8-0 non-absorbable sutures that were also used to return the overlying muscles to their normal position. Finally, the skin incision was closed using 8-0 absorbable sutures and post-operative mice were transferred to a heated chamber and allowed to fully recover before being returned to their home-cage. Mice were closely observed for 10 days following surgery.

Daily optical stimulation training:

After 14d post-engraftment (to allow sufficient time for engrafted motor neurons to extend axons far enough to reach target muscles), mice with implanted stimulation devices were transferred to a round acrylic chamber, containing clean bedding and nesting material, positioned on top of the RF-resonance cavity (Fig. 5c and 6a). Where possible, multiply-housed littermates were transferred to the chamber at the same time. A red acrylic dome was placed in the centre of the chamber to encourage mice to spend time in that location, since resonance power transmission is less efficient at the periphery of the chamber. Engrafted SOD1^G93A mice underwent daily optical stimulation (pulse pattern described below; Supplementary Fig. 4 – 6) for 1hr/d from post-engraftment day 14 until termination of the experiment at late-stage disease (132.4 ±6.8 days).

Isometric muscle tension physiological analysis:

At the experimental end-point, engrafted SOD1^G93A mice underwent isometric muscle tension physiology, in order to accurately determine the contractile properties of target muscles in response to acute optical stimulation, largely as previously described¹⁴, with the following modifications: a PowerLab 4/30 stimulation and recording system (AD Instruments) was used to deliver bespoke electrical stimulation signals, either as direct constant voltage pulses applied to the nerve via bipolar electrodes for electrical nerve stimulation, or used as a 5V TTL signal to activate a 470nm LED light-source (CoolLED; pe100), delivered to the exposed sciatic nerve via a liquid lightguide for optical stimulation; stimulation program available on request. As previously reported, percentage light power intensity can be controlled over a range of 0-80 mW/mm²; 50% (40 mW/mm²) supramaximal stimulation was used for most optically-evoked muscle force recordings, however, maximum motor unit activation was achieved using light intensities ≤2.5 mW/mm². For electrical stimulation, 0.2ms supramaximal constant-voltage (5V) pulses were directly applied to the exposed sciatic nerve via a bipolar stimulating electrode, as individual pulses (for twitch contractions) or at 20, 40, 80 and 100 Hz burst (0.5s duration) with a 5 second rest interval to interrogate the full range of maximal tetanic contractile force values.

Distal tendons of individual target muscles were attached to 25g or 55g UF-1 force transducers coupled to a bridge amp (AD Instruments). LabChart software (AD Instruments) was used for automated data analysis of parameters including maximum twitch and tetanic contractile force, time-to-rise, time-to-peak and ½ relaxation time (during twitch contractions). Motor unit number estimation (MUNE) was performed using a bespoke pattern of rising and falling amplitude electrical stimuli (0-5V, 0.2ms pulse width), repeated every 1s (LabChart programme available upon request), or by 1 s interval 5V TTL trigger pulses in combination with manual cycling of the percentage power output from the pe-100 (470nm) light source (Cool LED) between 1-10%.

Nerve and muscle histology and image processing:

Immediately after physiological analysis, animals were euthanized and the ipsilateral (engrafted) sciatic nerve (SN), attached to the triceps surae (TS) muscle group, along with the contralateral SN and tibialis anterior (TA) and extensor digitorum longus (EDL) muscles, were dissected, rinsed in TBS and post-fixed in 4% paraformaldehyde in TBS for a minimum of 2hrs, followed by cryoprotection in TBS containing 20% sucrose for >12 hrs. Tissue was mounted in aluminum-foil molds containing OCT (Tissue-Tek) and rapidly frozen on dry-ice, then stored at -20°C prior to cryosectioning. Serial longitudinal sections from the ipsilateral SN and TS muscle, and TA/EDL muscles were cut at 30μm thickness, encompassing the entire tissue block to enable 3D reconstruction of the extent of reinnervation throughout the whole muscle (only performed in selected cases due to time- and resource-intensive nature of imaging data acquisition and processing). Transverse 10μm-thick sections were cut from the contralateral SN for axonal analysis. For immunostaining, muscle/nerve sections were washed 3 x 5 mins with TBS, blocked for 1hr in TBS containing 0.2% Triton X-100 and 5% donkey serum and sections were double-labelled with the following primary antibodies, raised in either rabbit or goat, applied in combination overnight in TBS containing 0.2% Triton X-100 and 2% donkey serum: goat anti-choline acetyltransferase, 1:100 (Chemicon); goat anti-green fluorescent protein, 1:500 (Abcam); rabbit anti-green fluorescent protein, 1:500 (Invitrogen); rabbit anti-choline acetyltransferase, 1:200 (Abcam); rabbit anti-βIII tubulin; 1:500 (Biolegend). Secondary antibodies, raised in donkey were diluted 1:500, along with α-bungarotoxin-Alexa568/647 (1:500; Invitrogen) and DAPI (1:1000; Sigma) in TBS containing 0.2% Triton X-100 and 2% donkey serum and applied for 2hrs, before washing 3 x 5 mins with TBS and coverslips were then mounted using fluorescent mounting medium (Invitrogen). Image acquisition was performed using a Zeiss LSM780 confocal microscope, to acquire tile-scan images of entire SN/TS sections using a 20x objective (pinhole set to 30μm to obtain fluorescent signal from entire thickness of the section); high-resolution z-stacks from specific regions of interest were acquired using either 40x or 63x oil-immersion objectives. Images reported here were either prepared directly, using Zeiss Zen Blue/Black image processing software or processed and analyzed using Fiji (ImageJ) and MetaMorph (Molecular Devices) software for 3D reconstruction, as follows: serial tile-scan images encompassing the whole SN/TS tissue block were converted into compressed tiff-format files, assembled into an image stack and individual planes were aligned using the StackReg plugin⁴³, aligned stacks were then converted into rendered 3D reconstructions using MetaMorph software and saved as compressed AVI video files for visualization purposes (note: automated NMJ analysis, described below, was performed on individual image planes from each stack). Tile-scans were also acquired from contralateral SN sections, using a 40x or 63x oil-immersion objective, for automated axon cross-sectional area analysis.

Automated endplate and innervation analysis:

Serial tile-scan images, in RGB Color format, from the whole TS muscle from SOD1^G93A mice at the experimental end-point, 135 days, were analyzed as follows, using Fiji (ImageJ) software: endplates, labelled with α-bungartoxin conjugated to Alexa-568 or Alexa-647 were assigned to the red color channel and color thresholding, using consistent parameters (Hue = 0-17 [pass], Saturation = 0-255 [pass], Brightness = 75-255 [pass]) was performed to specifically identify endplates; the number of endplates in each plane was then quantified using the “Analyze Particles” feature of ImageJ (key parameters include: Area = 50-500μm, Circularity 0-1, Show “Overlay masks”). Quantification of innervated endplates was performed in a similar manner, using colour thresholding parameters to identify double-labelled endplates that stained positive for both α-bungarotoxin (assigned to red channel) and GFP/YFP+ motor axons (assigned to the green channel), using the following parameters: Hue = 23-44; Saturation = 0-255; Brightness = 90-255. This automated analysis method is intended to quantify the total number of endplates and provide an indication of their innervation status; determination of full versus partial innervation would require much higher resolution imaging which is not feasible for the whole TS muscle. Additionally, severe muscle atrophy at late-stage disease decreases the TS muscle volume, thus the number and size of tile-scan images would be approximately 3-fold greater for wild-type mice.

Motor and sensory axon nerve analysis:

Individual channels, representing total axons (stained for β-III tubulin) and motor axons (stained for choline-acetyltransferase), from tile-scans of contralateral SNs were used to determine number and cross-sectional area (CSA) of each axonal type, to determine the effect of different immunosuppression regimens. Briefly, images were thresholded in ImageJ, using identical settings to delineate individual axons, individual branches were then manually circled, using the freehand selection tool, and axonal counts and CSA were automatically quantified using the “Analyze Particles” tool.

Digital Cross-sectional area analysis of longitudinal muscle sections (dCALMS):

Briefly, z-stacks from regions of interest incorporating at least 1 GFP/YFP+ motor axon terminal were acquired from the whole 30μm thickness of individual TS muscle sections; the “reslice” feature of ImageJ was then used to obtain digital transverse orientation images from each ROI to interrogate muscle fibre CSA throughout the y-plane of the image stack and correlation with the innervation status; triple labelling with GFP/YFP, α-bungarotoxin and choline-acetyltransferase labelling enabled the innervation status of adjacent fibres within the ROI to be assigned to the following categories: denervated (α-bungarotoxin only), innervated by endogenous motor neurons (α-bungarotoxin and choline-acetyltransferase), innervated by engrafted ChR2⁺ motor neurons (α-bungarotoxin, choline-acetyltransferase and GFP/YFP) or unknown (when no endplate was visible within the ROI. Overexposure of the channel used to acquire choline-acetyltransferase staining enabled visualization of the muscle CSA, which underwent gaussian blurring to aid definition of the sarcolemma (as shown in Fig. 7c, colorized images versus non-blurred images shown in black and white). The edge of each muscle fibre at the level of the endplate (where present) in the y-plane (or the maximum diameter, where no endplate was evident), was manually circled using the freehand selection tool and analysed using the “measure” function of ImageJ. ROIs were analysed from n = 3 mice that underwent ChR2⁺ motor neuron engraftment and optical stimulation training. Only fibres that were entirely within the z-plane were measured at their widest point along the y-plane (Fig. 7c and Supplementary Video 6); partial fibres, were excluded.

Statistics:

All data are presented as mean ± SEM unless otherwise indicated. GraphPad Prism 9 (Prism) was used for statistical analyses. No out-liners or data points were eliminated. Differences between two groups were assessed using multiple two-tailed unpaired t tests. Differences between more than two groups were assessed by using one-way or two-way analysis of variance (ANOVA), with Tukey’s post hoc correction, or mixed model effects analysis. Significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001.

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Acknowledgments: The authors are grateful to Profs Elizabeth Fisher, Jennifer Morgan (UCL) and Victor Tybulewicz (The Crick Institute) for helpful discussions about immuno-compatibility of allogeneic cells in mice and adverse effects of CNIs on muscle, and Dr Henry Lancashire for assistance with modified PCB production. In addition, we are grateful to Prof Ada Poon and Dr Yuji Tanabe (Stanford University) for assistance with establishing their wireless optical stimulation system. JBB was supported by a Motor Neurone Disease Association Lady Edith Wolfson Senior Non-clinical Fellowship (Bryson 965-799), as well as an NIHR BRC UCL Excellence Fellowship award (BRC371/ED/AT/101310), in addition to grant support from the Rosetrees Trust (ref: M643) and an Early Career Researcher Award from the Richard Stravitz Foundation. This study was supported in part by the UK Medical Research Council (MRC) research project grant (MR/R011648/1), awarded to JBB, AD and LG (principal investigator) and a Thiery Latran Foundation project grant (JBB and LG). LG is supported by Brain Research UK and holds The Graham Watts Senior Research Fellowship (LG).

Author contributions: JBB, LG developed original project concept; JBB performed laboratory experiments, data analysis, visualization of results, project supervision and drafted the manuscript; AK contributed to laboratory experiments; JBB, DJ, AD were responsible for optical stimulation device modification and testing; JBB, AD, LG were responsible for funding acquisition; LG contributed to project supervision. All authors reviewed and/or edited the final manuscript.

Competing interests: The authors declare that they have no competing interests.

Correspondence and requests for materials should be addressed to J. Barney Bryson or Linda Greensmith.

There is NO Competing Interest.

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A cell therapy approach to prevent muscle atrophy and paralysis in an aggressive mouse model of ALS

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