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However, these engrafted cells are not connected to the descending inputs within the central nervous system that normally control motor function; therefore, their neural activity must be regulated by an artificial control system. Such engrafted ESC-derived motor neurons can be electrically stimulated 7 , but this approach stimulates endogenous as well as engrafted neurons. It has also been shown that viral expression of ChR2 in motor neurons of adult rats can enable optical stimulation of muscle function We generated genetically modified ESC-derived motor neurons that express both ChR2, to enable optical stimulation, as well as glial -derived neurotrophic factor Gdnf , a neurotrophic factor that promotes long-term motor neuron survival see supplementary materials and methods.

When cultured on an ESC-derived astrocyte feeder layer fig.

Neuromuscular Junction Process in Skeletal Muscle

S1 , ChR2 motor neurons mature electrically over a period of 35 days, until they fire trains of action potentials in response to optical stimulation and closely resemble adult motor neuron activity patterns induced by electrical stimulation 15 Fig. Expression of Gdnf in ChR2 motor neurons enhances survival and enables them to mature electrically in vitro. Error bars indicate SEM. One representative of three separate experiments is shown.

Then, ESC clones that stably express ChR2 and Gdnf transgene were selected, cultured in astrocyte feeder layer, and allowed to differentiate into motor neurons suitable for engraftment and in vivo experiments. These ESCs were subsequently directed to differentiate into motor neurons. Stable expression of Gdnf improves motor-neuron survival. Show that ESC-derived motor neurons stably express light-sensitive ChR2 on the membrane and that the expression of Gdnf improves survival of motor neurons, and determine whether and when ESC-derived ChR2 motor neurons become functionally mature.

These observations would establish the criteria for generating motor neurons suitable for engraftment for other experiments from this point forward. Analysis of ESC-derived embryoid bodies containing ChR2 motor neurons using confocal scanning microscope. Expression of the following proteins is analyzed:. Differentiated motor neurons are isolated using magnetic-activated cell sorting MACS and grown in culture.

ESC-derived motor neurons are immunostained for Gdnf and stained with a dye for DNA, followed by analysis using fluorescent confocal microscopy:. DNA blue is present in both left and right images, which indicates GFP, YFP, and Gdnf are localized with cells and do not appear to be the result of non-specific immunostaining or fluorescence.

ESC-derived motor neurons transfected with Gdnf transgene triangle, red line are grown in culture in parallel with wild-type motor neurons with open circles, dark blue line or without closed circles, light blue line Gdnf supplement.

Surviving cells per well are counted y-axis over 35 days in culture x-axis and plotted here. This supports the hypothesis that stable Gdnf expression in motor neurons improves survival. In order to test functional maturation of ESC-derived motor neurons in culture, motor neurons expressing light-sensitive ChR2-YFP and Gdnf are optically stimulated at days 3, 7, and Neuron action potentials are recorded and presented as spikes on the graph. Scale bar indicates duration of action potentials in milliseconds ms and voltage of action potentials in millivolts mV.

At day 3, ChR2 motor neurons did not generate action potentials following optical stimulation. At day 17, ChR2 motor neurons show action potentials in response to optical stimulation.

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At day 35, mature ChR2 motor neurons generate rapid action potentials following optical stimulation. Supplementary Figure 1A below shows representative action potential recordings of ESC-derived ChR2 motor neurons in response to electrical stimulation top panel and optical stimulation bottom panel , up to 35 days in culture. The rapid action potentials induced by optical stimulation at 35 days in culture are similar to those induced by electrical stimulation. Together, the data indicate functional maturation of ChR2 motor neurons at 35 days. Motor-neuron survival is improved with Gdnf protein expression.

ESC-derived ChR2 motor neurons generate action potentials in response to optical stimulation at 35 days in culture, which indicates functional maturity. Together, these results establish the criteria for generating ESC-derived ChR2 motor neurons suitable to engraftment for the remainder of the study. The authors also concluded that ChR2 motor neurons mature after 35 days, when functional testing should be performed. After developing these ChR2 motor neurons, we next established an in vivo model to assess the feasibility of restoring muscle function with optical control of the engrafted cells, using the sciatic nerve.

Muscle denervation was induced by sciatic nerve ligation in adult mice. This procedure results in a complete initial denervation, followed by limited regeneration of endogenous axons through the ligation site, thereby creating a partially denervated environment resembling the partial muscle denervation of early-stage ALS fig. Three days postligation, embryoid bodies containing ChR2 motor neurons were engrafted distal to the ligation into the tibial and common peroneal branches of the sciatic nerve.

Histological analysis revealed that the engrafted ChR2 motor neurons not only survive for at least 35 days in the peripheral nerve environment Fig. S4 and S5. Additionally, ChR2 motor neurons extended large numbers of axons Fig.

Nerve and Muscle (Studies in Biology)

Engrafted ChR2 motor neuron axons, which grow alongside regenerating endogenous YFP-negative motor axons, are mostly myelinated Fig. Histological analysis also revealed robust reinnervation of muscle fibers by ChR2 motor neurons, although the neuromuscular junctions exhibited hallmarks of inactivity, including poly-innervation as well as collateral and terminal axonal sprouting 16 Fig.

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Nevertheless, quantification of all end plates within a whole TS muscle revealed that Moreover, we observed YFP-positive neuromuscular junctions in both fast-twitch and slow-twitch regions of the TS, indicating that ChR2 motor neurons can innervate different muscle types. Therefore, this time point 35 days was used in subsequent experiments to establish whether the transplanted ChR2 motor neurons were indeed functional and responsive to optical stimuli in vivo.

Robust axonal growth and reinnervation of distal muscles after engraftment of ChR2 motor neurons. A Image montage of a whole nerve and muscle section showing ChR2 motor neuron cell bodies at the graft site and axon projection dashed lines indicate approximate trajectory. C Confocal image of longitudinal and transverse common peroneal nerve sections showing both ChR2 motor neurons and endogenous axons.

D Confocal images of engrafted ChR2 motor neuron axons showing myelination. E Confocal z-stack of ChR2 motor neuron axon terminals innervating multiple neuromuscular junctions within the TS muscle. Arrows indicate preterminal collateral sprouting, arrowheads denote terminal sprouting, and the asterisk indicates an endogenous motor axon. Quantification is shown below. Present data to show that engrafted ESC-derived ChR2 motor neurons can survive and grow in the peripheral environment.

The authors test the hypothesis that engrafted motor neurons resemble normal motor neurons in appearance, extend axons to distal muscles, and form neuromuscular junctions with muscle fibers.

The authors used sciatic nerve ligation to induce peripheral nerve injury, a mouse model of muscle paralysis resulting from denervation. A suture is tied around the sciatic nerve of anesthetized mice, and animals were allowed to recover from the surgery.

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Sciatic nerve ligation induced nerve damage, axon degeneration and muscle denervation, followed by regeneration of a small population of endogenous motor neurons. This partial denervation resembles certain aspects of early-stage ALS. Three days after the ligation surgery, ESC-derived ChR2 motor-neuron embryoid bodies were engrafted into the injured nerve below the ligation site i.

Supplementary Figure 3 shows a schematic illustration of sciatic nerve ligation below : The ligation site and ChR2 motor-neuron graft sites are indicated. Engrafted ChR2 motor neurons are predicted to extend axons along the indicated nerves in the lower leg green. At 35 days after engraftment, immunohistochemistry and confocal microscopy were used to analyze motor neuron growth.

The following motor -neuron markers are examined in Figure 2A:. Image shows dissected nerve and muscle after engraftment of ChR2 motor neurons. The tissue is immunostained for ChAT red , which identifies both endogenous and engrafted motor neurons. The tissue is stained with a dye for DNA blue , which helps visualize the outline of the tissue. Motor-neuron axon projections green and red overlay extend from the graft site toward target muscle.

Center for Neuromuscular Disease | University of Maryland School of Medicine

Together, the data suggest robust axon growth of engrafted ESC-derived ChR2 motor neurons from the graft site into tibial and common peroneal nerves. These images further provide evidence of ESC-derived ChR2 motor-neuron survival and axon extensions along the sciatic nerve and common peroneal nerve in the leg. Myelin sheath is a characteristic of peripheral motor axons. These data implicate healthy ESC-derived ChR2 motor neurons resembling normal motor neurons in the peripheral nerve environment.

The image shows dissected posterior triceps surae TS muscle tissue. Notably, neuromuscular junctions show collateral sprouting and terminal sprouting, indicated by arrows and arrowheads, respectively, which are hallmarks of inactivity. The number of terminals innervated by engrafted ChR2 motor neurons GFP, green is counted and plotted bottom panel , relative to the total number of neuromuscular junctions end-plates innervated by any motor neuron red.

The whole TS muscle shows After never injury in otherwise healthy adult mice, engraftment of ESC-derived ChR2 motor neurons can survive in the peripheral nerve environment. At 35 days after engraftment, ChR2 motor neurons are shown to extend axons along the sciatic nerve and common peroneal nerve to target muscles in the lower leg, where neuromuscular junctions are observed. The data suggest that engrafted ESC-derived ChR2 motor neurons appear to reennervate target muscles in the lower leg after sciatic nerve injury. In anesthetized animals, we used isometric muscle tension physiology to examine the contractile responses elicited from TA, EDL, and TS data summarized in table S1 muscles after optical stimulation of the exposed sciatic nerve using finely controlled pulses of nm blue light generated by a light-emitting diode LED unit and delivered via a light-guide to the graft site Fig.

Short-duration ms light pulses were able to induce submaximal twitch contractions in muscles innervated by transplanted ChR2 motor neurons Fig.