Optimal conditions for graft survival and reinnervation of denervated muscles after embryonic motoneuron transplantation into peripheral nerves undergoing Wallerian degeneration.

Motoneuron transplantation into peripheral nerves undergoing Wallerian degeneration may have applications in treating diseases causing muscle paralysis. We investigated whether functional reinnervation of denervated muscle could be achieved by early or delayed transplantation after denervation. Adult rats were assigned to six groups with increasing denervation periods (0, 1, 4, 8, 12, and 24 weeks) before inoculation with culture medium containing (transplantation group) or lacking (surgical control group) dissociated embryonic motoneurons into the peroneal nerve. Electrophysiological and tissue analyses were performed 3 months after transplantation. Reinnervation of denervated muscles significantly increased relative muscle weight in the transplantation group compared with the surgical control group for denervation periods of 1 week (0.042% ± 0.0031% vs. 0.032% ± 0.0020%, respectively; p = 0.009), 4 weeks (0.044% ± 0.0069% vs. 0.026% ± 0.0045%, respectively; p = 0.0023), and 8 weeks (0.044% ± 0.0029% vs. 0.026% ± 0.0008%, respectively; p = 0.0023). The ratios of reinnervated muscle contractile forces to naïve muscle in the 0, 1, 4, 8, and 12 weeks transplantation groups were 3.79%, 18.99%, 8.05%, 6.30%, and 5.80%, respectively, indicating that these forces were sufficient for walking. The optimal implantation time for transplantation of motoneurons into the peripheral nerve was 1 week after nerve transection. However, the neurons transplanted 24 weeks after denervation survived and regenerated axons. These results indicated that there is time for preparing cells for transplantation in regenerative medicine and suggested that our method may be useful for paralysed muscles that are not expected to recover with current treatment.

BDNF-overexpressing human umbilical cord mesenchymal stem cell-derived motor neurons improve motor function and prolong survival in amyotrophic lateral sclerosis mice.

OBJECTIVE: To investigate the beneficial effect of brain-derived neurotrophic factor (BDNF) -overexpressing human umbilical cord mesenchymal stem cell (hUC-MSC)-derived motor neurons in the human Cu, Zn-superoxide dismutase1 (hSOD1)G93A amyotrophic lateral sclerosis (ALS) mice. METHODS: The BDNF gene was transfected into hUC-MSC-derived motor neurons by the lentivirus-mediated method. hSOD1G93A mice were assigned to the ALS, ALS/MN, and ALS/MN-BDNF groups, and intrathecally administrated phosphate-buffered saline (PBS), motor neurons, or motor neurons overexpressing BDNF, respectively. The control group included non-transgenic wild-type littermates administrated PBS. One month after transplantation, the motor function of the mice was assessed by the rotarod test, and the lumbar enlargements were then isolated to detect the expression of hSOD1 and BDNF by western blotting, and the expression of choline acetyltransferase (ChAT), homeobox protein 9 (HB9), major histocompatibility complex I (MHCI) and microtubule-associated protein-2 (MAP-2) by immunofluorescence assay. RESULTS: After transplantation, mice in the ALS/MN-BDNF and ALS/MN groups both exhibited longer latency to fall and longer survival than those in the ALS group (P < 0.01 vs. P < 0.05), and the improvement was more significant in the former than in the latter. However, cell transplantation did not delay disease onset. In the lumbar enlargements of the ALS/MN-BDNF and ALS/MN groups, the expression of hSOD1 was slightly reduced without statistical significance (P > 0.05), but the expression of BDNF, ChAT and HB9, and the co-expression of MHCI and MAP-2 were significantly greater than in the ALS group (P < 0.01), with the differences also being more prominent in the former group than in the latter. CONCLUSIONS: Transplantation of BDNF-overexpressing hUC-MSC-derived motor neurons can improve motor performance and prolong the survival of hSOD1G93A mice. Combining stem cell-derived motor neurons with BDNF might provide a new therapeutic strategy for ALS.

Sequentially induced motor neurons from human fibroblasts facilitate locomotor recovery in a rodent spinal cord injury model.

Generation of autologous human motor neurons holds great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows generation of target cells from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic that are not scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequentially inducing POU5F1(OCT4) and LHX3. Our strategy enables scalable production of pure iMNs because of the transient acquisition of proliferative iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited hallmarks of spinal motor neurons including transcriptional profiles, electrophysiological property, synaptic activity, and neuromuscular junction formation. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in rodent SCI model. Together, our advanced strategy will provide tools to acquire sufficient human iMNs that may represent a promising cell source for personalized cell therapy.

Upper Extremity Axon Counts and Clinical Implications for Motor Nerve Transfer.

BACKGROUND: Nerve transfers are planned based on the following parameters: location, number of branches, and axon count matching of the donor and recipient nerves. The authors have previously defined the former two in upper limb muscles. In the literature, axon counts are obtained from various sources, using different methods of histomorphometry. This study describes the axon counts of the same primary motor nerve branches from the authors' previous study using a uniform method of manual histomorphometry and completes the authors' blueprint of upper limb neuromuscular anatomy for reconstructive surgery. METHODS: The distal ends of the primary nerve branches of 23 upper limb muscles were harvested from 10 fresh frozen cadaveric upper limbs. Manual quantitative histomorphometry was performed by two independent investigators, and the average was reported. RESULTS: The primary nerve branches of the arm muscles had higher average axon counts (range, 882 to 1835) compared with those of the forearm muscles (range, 267 to 883). In the forearm, wrist flexor (range, 659 to 746) and extensor (range, 543 to 745) nerve branches had axons counts that were similar to those of potential donors (e.g., supinator, n = 602; pronator teres, n = 625; flexor digitorum superficialis, n = 883; and flexor digitorum profundus, n = 832). CONCLUSIONS: Apart from describing the axon counts of the upper limb, the authors have found that the forearm axon counts are very comparable. This insight, when combined with information on the location and number of primary nerve branches, will empower surgeons to tailor bespoke nerve transfers for every clinical situation.

Differentiation of human adipose-derived stem cells into neuron/motoneuron-like cells for cell replacement therapy of spinal cord injury.

Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression.

Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons.

BACKGROUND: Transplantation of human pluripotent stem cell (hPSC)-derived neurons into chick embryos is an established preliminary assay to evaluate engraftment potential. Yet, with recent advances in deriving diverse human neuronal subtypes, optimizing and standardizing such transplantation methodology for specific subtypes at their correlated anatomical sites is still required. NEW METHOD: We determined the optimal stage of hPSC-derived motor neuron (hMN) differentiation for ex ovo transplantation, and developed a single injection protocol that implants hMNs throughout the spinal cord enabling broad regional engraftment possibilities. RESULTS: A single injection into the neural tube lumen yielded a 100% chick embryo survival and successful transplantation rate with MN engraftment observed from the rostral cervical through caudal lumbar spinal cord. Transplantation of HB9+/ChAT- hMN precursors yielded the greatest amount of engraftment compared to Pax6+/Nkx6.1+/Olig2+ progenitors or mature HB9+/ChAT+ hMNs. COMPARISON WITH EXISTING METHOD(S): Our single injection hMN transplant method is the first to standardize the optimal hMN phenotype for chick embryo transplantation, provide a rubric for engraftment quantification, and enable broad engraftment throughout the spinal cord with a single surgical intervention. CONCLUSION: Transplantation of HB9+/ChAT- hMN precursors into chick embryos of Hamburger Hamilton (HH) stages 15-18 using a single luminal injection confers a high probability of embryo survival and cell engraftment in diverse regions throughout the spinal cord.

Upper Extremity Innervation Patterns and Clinical Implications for Nerve and Tendon Transfer.

BACKGROUND: The authors previously studied the intramuscular innervation of 150 upper limb muscles and demonstrated that certain patterns of intramuscular innervation allowed muscles to be split into compartments with independent function. This study aims to determine the location, extramuscular course, and number of motor nerve branches of upper limb peripheral nerves. The authors want to combine this information with their previous work to create a blueprint of upper limb neuromuscular anatomy that would be useful in reconstructive surgery. METHODS: Ten fresh frozen cadaveric upper limbs were dissected. The origin of branches from the peripheral nerve trunk, their course, and the number of motor nerves per muscle were determined. The authors reviewed all the images of the Sihler-stained muscles from their earlier study. RESULTS: Motor nerve branches arise at the intersection of nerve trunk and muscle belly and are clustered near the origin of muscle groups. Two patterns of extramuscular innervation were noted, with one group having a single motor nerve and another group with consistently more than one motor nerve. A modified classification of muscles was proposed based on the orientation of muscle fibers to the long axis of the limb, the number of muscle compartments, and the number of heads of origin or the tendons of insertion. CONCLUSIONS: Motor nerve clusters can be located based on fixed anatomical landmarks. Muscles with multiple motor nerves have morphology that allows them to be split into individual compartments. The authors created a muscle and nerve blueprint that helps in planning nerve and split muscle transfers.

Body proportions as possible predictors for free gracilis one-stage facial reanimation.

BACKGROUND: Gracilis muscle and its motor nerve belongs to most commonly used flap for facial reanimation. However, it is performed in two steps, which is time consuming. One stage technique can be also performed, but the length of the motor nerve cannot be currently determined before surgery. AIM: The present study was conducted in order to evaluate the body composition on the length and suitability of the motor nerve of gracilis muscle for one stage facial reanimation. METHODS: The gracilis flaps along with the motoric nerve were dissected from 20 fresh cadavers (6 females, 14 males). The length of the lower extremity from superior iliac anterior spine to the bottom of the heel and BMI were measured. Regression analysis of lower extremity length and BMI to the actual length of the motor nerve of gracilis flap was performed. RESULTS: The linear regression analysis showed a positive correlation between the length of the lower limb and the size of the motor nerve length (r = 0.5060, p < 0.05), as well as between the BMI and the size of the motor nerve length (r = 0.5073, p < 0.05). Also, the males had longer motor nerve when compared to females by 13 % (p < 0.05). No difference between females and males in BMI was observed. CONCLUSION: The length from the superior iliac anterior spine, BMI and gender seemed to be potential factors that could help to predict the length of the gracilis flap motor nerve for the one stage facial reanimation. However, further studies evaluating other anatomical factors and validating the possible prediction rule for one stage reanimation success are needed (Fig. 3, Ref. 14).

Modelling and treating amyotrophic lateral sclerosis through induced-pluripotent stem cells technology.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease affecting primarily the population of motor neurons, even though a non-cell autonomous component, involving neighbouring non-neuronal cells, is more and more described. Despite 140 years of disease experience, still no efficient treatment exists against ALS. The inability to readily obtain the faulty cell types relevant to ALS has impeded progress in drug discovery for decades. However, the pioneer work of Shinya Yamanaka in 2007 in the stem cell field was a real breakthrough. Recent advances in cell reprogramming now grant access to significant quantities of CNS disease-affected cells. Induced pluripotent stem cells (iPSc) have been recently derived from patients carrying mutations linked to familial forms of ALS as well as from sporadic patients. Precise and mature protocols allow now their differentiation into ALS-relevant cell subtypes; sustainable and renewable sources of human motor neurons or glia are being available for ALS disease modelling, drug screening or for the development of cell therapies. In few years, the proof-of-concept was made that ALS disease-related phenotypes can be reproduced with iPSc and despite some remaining challenges, we are now not so far to provide platforms for the investigation of ALS therapeutics. This paper also reviews the pioneering studies regarding the applicability of iPSc technology in ALS animal models. From modest slowing down of ALS progression to no severe adverse effects, iPSc-based cell therapy resulted in promising premises in ALS preclinical paradigms, although long-term surveys are highly recommended.

Transplantation of embryonic motor neurons into peripheral nerve combined with functional electrical stimulation restores functional muscle activity in the rat sciatic nerve transection model.

Reinnervation of denervated muscle by motor neurons transplanted into the peripheral nerve may provide the potential to excite muscles artificially with functional electrical stimulation (FES). Here we investigated whether transplantation of embryonic motor neurons into peripheral nerve combined with FES restored functional muscle activity in adult Fischer 344 rats after transection of the sciatic nerve. One week after sciatic nerve transection, cell culture medium containing (cell transplantation group, n = 6) or lacking (surgical control group, n = 6) dissociated embryonic spinal neurons was injected into the distal stump of the tibial and peroneal nerves. Electrophysiological and tissue analyses were performed in the cell transplantation and surgical control groups 12 weeks after transplantation, as well as a in naïve control group (n = 6) that received no surgery. In the cell transplantation group, ankle angle was measured during gait, with and without FES of the peroneal nerve. Ankle angle at mid-swing was more flexed during gait with FES (26.6 ± 8.7°) than gait without FES (51.4 ± 12.8°, p = 0.011), indicating that transplantated motor neurons in conjunction with FES restored ankle flexion in gait, even though no neural connection between central nervous system and muscle was present. These results indicate that transplantation of embryonic motor neurons into peripheral nerve combined with FES can provide a novel treatment strategy for paralysed muscles. Copyright © 2013 John Wiley & Sons, Ltd.

Clinical outcome following transfer of the supinator motor branch to the posterior interosseous nerve in patients with C7-T1 brachial plexus palsy.

BACKGROUND: In cases of C7-T1 brachial plexus palsy, a reliable method for the reconstruction of the finger and thumb extension was not established until the transfer of the supinator motor branch to the posterior interosseous nerve was proposed. The long-term outcome of this new technique requires evaluation due to the limited number of cases and the shorter follow-up period of the previous study. OBJECTIVE: This study aims to evaluate the long-term effect of the transfer of the supinator motor branch to the posterior interosseous nerve and to determine the recovery time course for this new technique. METHODS: A retrospective review was conducted in 10 patients with lower brachial plexus injuries who underwent transfer of the supinator motor branch. Patients were followed up postoperatively for a minimum of 24 months, with all patients scheduled to receive a physical examination and electrophysiological testing every 3 months for the first 2 years. RESULTS: Nine patients (90%) recovered to the Medical Research Council (MRC) grade 3 or better for the extensor digitorum communis. The electrophysiologically documented recovery began at an average of 5.7 months after surgery, with the average time required for the first finger extension being 9.1 months (range 5-18 months), and the average time required for achieving MRC grade 3 being 14.3 months (range 9-24 months). Moreover, no complications or loss of supination was observed in any patient. CONCLUSION: The supinator motor branch transfer is a safe procedure that yields recovery of finger extension in C7-T1 brachial plexus palsies with encouraging long-term outcomes.

Electrical stimulation of transplanted motoneurons improves motor unit formation.

Motoneurons die following spinal cord trauma and with neurological disease. Intact axons reinnervate nearby muscle fibers to compensate for the death of motoneurons, but when an entire motoneuron pool dies, there is complete denervation. To reduce denervation atrophy, we have reinnervated muscles in Fisher rats from local transplants of embryonic motoneurons in peripheral nerve. Since growth of axons from embryonic neurons is activity dependent, our aim was to test whether brief electrical stimulation of the neurons immediately after transplantation altered motor unit numbers and muscle properties 10 wk later. All surgical procedures and recordings were done in anesthetized animals. The muscle consequences of motoneuron death were mimicked by unilateral sciatic nerve section. One week later, 200,000 embryonic day 14 and 15 ventral spinal cord cells, purified for motoneurons, were injected into the tibial nerve 10-15 mm from the gastrocnemii muscles as the only neuron source for muscle reinnervation. The cells were stimulated immediately after transplantation for up to 1 h using protocols designed to examine differential effects due to pulse number, stimulation frequency, pattern, and duration. Electrical stimulation that included short rests and lasted for 1 h resulted in higher motor unit counts. Muscles with higher motor unit counts had more reinnervated fibers and were stronger. Denervated muscles had to be stimulated directly to evoke contractions. These results show that brief electrical stimulation of embryonic neurons, in vivo, has long-term effects on motor unit formation and muscle force. This muscle reinnervation provides the opportunity to use patterned electrical stimulation to produce functional movements.

Human motor neurons generated from neural stem cells delay clinical onset and prolong life in ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neuron disease. The etiology and pathogenic mechanisms of the disease remain unknown, and there is no effective treatment. Here we show that intrathecal transplantation of human motor neurons derived from neural stem cells (NSCs) in spinal cord of the SOD1G93A mouse ALS model delayed disease onset and extended life span of the animals. When HB1.F3.Olig2 (F3.Olig2) cells, stable immortalized human NSCs encoding the human Olig2 gene, were treated with sonic hedgehog (Shh) protein for 5-7 days, the cells expressed motor neuron cell type-specific phenotypes Hb9, Isl-1 and choline acetyltransferase (ChAT). These F3.Olig2-Shh human motor neurons were transplanted intrathecally in L5-L6 spinal cord of SOD1G93A mice, and at 4 weeks post-transplantation, transplanted F3.Olig2-Shh motor neurons expressing the neuronal phenotype markers NF, MAP2, Hb9, and ChAT were found in the ventral horn of the spinal cord. Onset of clinical signs in ALS mice with F3.Olig2-Shh motor neuron implants was delayed for 7 days and life span of animals was significantly extended by 20 days. Our results indicate that this treatment modality of intrathecal transplantation of human motor neurons derived from NSCs might be of value in the treatment of ALS patients without significant adverse effects.

Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice.

Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell-derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.

PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons.

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast-iPS-motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.

Electrical stimulation of embryonic neurons for 1 hour improves axon regeneration and the number of reinnervated muscles that function.

Motoneuron death after spinal cord injury or disease results in muscle denervation, atrophy, and paralysis. We have previously transplanted embryonic ventral spinal cord cells into the peripheral nerve to reinnervate denervated muscles and to reduce muscle atrophy, but reinnervation was incomplete. Here, our aim was to determine whether brief electrical stimulation of embryonic neurons in the peripheralnerve changes motoneuron survival, axon regeneration, and muscle reinnervation and function because neural depolarization is crucial for embryonic neuron survival and may promote activity-dependent axon growth. At 1 week after denervation by sciatic nerve section, embryonic day 14 to 15 cells were purified for motoneurons, injected into the tibial nerve of adult Fischer rats, and stimulated immediatelyfor up to 1 hour. More myelinated axons were present in tibial nerves 10 weeks after transplantation when transplants had been stimulated acutely at 1 Hz for 1 hour. More muscles were reinnervated if the stimulation treatment lasted for 1 hour. Reinnervation reduced muscle atrophy, with or without the stimulation treatment. These data suggest that brief stimulation of embryonic neurons promotes axon growth, which has a long-term impact on muscle reinnervation and function. Muscle reinnervation is important because it may enable the use of functional electrical stimulation to restore limb movements.

Retrospective study of the functional recovery of men compared with that of women with long-term facial paralysis.

Sex is likely to play an important part in reanimation of the face after paralysis, with women being superior in terms of resistance to neural injury and regeneration. Our aim was to evaluate the influence of the sex of the patient on the recovery of facial paralysis after surgical reanimation by comparing the degree of restored movement between men and women with long-standing paralysis that was reanimated by transfer of the hypoglossal nerve or cross-face nerve grafting. Between 1999 and 2010 we operated on 174 patients with facial paralysis. Of these we studied 26 cases (19 women and 7 men) with complete long-standing paralysis reanimated with either cross-face nerve grafting (n=14) or transfer of the hemihypoglossal nerve (n=12). The degree of movement restored was recorded in each case. Statistical analysis showed that in cases with long-standing paralysis women had significantly more movement restored than men for both cross-face nerve grafting (p=0.02) and hypoglossal transposition (p=0.04). We conclude that, after a neural injury, women tend to maintain the viability of the facial musculature longer than men, which suggests that they are more resistant to both denervation and the development of muscular atrophy. Whether this phenomenon can be explained by neural or muscular processes, or both, warrants further studies.

Transplanted motoneurons derived from human induced pluripotent stem cells form functional connections with target muscle.

Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency, self-derivation and potential ability to differentiate into motoneurons. However, it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo, which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study, we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system, human iPSCs efficiently differentiated into motoneurons which displayed typical morphology, expressed specific molecules, and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats, they survived robustly, extended axons along the nerve, and formed functional connections with the target muscle (biceps brachii), thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo, and they have potential for stem cell-based therapies for motoneuron diseases.

Acute stimulation of transplanted neurons improves motoneuron survival, axon growth, and muscle reinnervation.

Few options exist for treatment of pervasive motoneuron death after spinal cord injury or in neurodegenerative diseases such as amyotrophic lateral sclerosis. Local transplantation of embryonic motoneurons into an axotomized peripheral nerve is a promising approach to arrest the atrophy of denervated muscles; however, muscle reinnervation is limited by poor motoneuron survival. The aim of the present study was to test whether acute electrical stimulation of transplanted embryonic neurons promotes motoneuron survival, axon growth, and muscle reinnervation. The sciatic nerve of adult Fischer rats was transected to mimic the widespread denervation seen after disease or injury. Acutely dissociated rat embryonic ventral spinal cord cells were transplanted into the distal tibial nerve stump as a neuron source for muscle reinnervation. Immediately post-transplantation, the cells were stimulated at 20 Hz for 1 h. Other groups were used to control for the cell transplantation and stimulation. When neurons were stimulated acutely, there were significantly more neurons, including cholinergic neurons, 10 weeks after transplantation. This led to enhanced numbers of myelinated axons, reinnervation of more muscle fibers, and more medial and lateral gastrocnemius muscles were functionally connected to the transplant. Reinnervation reduced muscle atrophy significantly. These data support the concept that electrical stimulation rescues transplanted motoneurons and facilitates muscle reinnervation.