A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts

Stem Cells and Cloning: Advances and Applications Dovepress open access to scientific and medical research Original Research Stem Cells and Cloning: Advances and Applications downloaded from https://www.dovepress.com/ by 37.59.46.207 on 21-Dec-2018 For personal use only. Open Access Full Text Article A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts This article was published in the following Dove Press journal: Stem Cells and Cloning: Advances and Applications Marwah Abd Al Samid 1 Jamie S McPhee 2 Jasdeep Saini 1 Tristan R McKay 1 Lorna M Fitzpatrick 1 Kamel Mamchaoui 3 Anne Bigot 3 Vincent Mouly 3 Gillian Butler-Browne 3 Nasser Al-Shanti 1 1 Healthcare Science Research Institute, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK; 2 Department of Sport and Exercise Science, Manchester Metropolitan University, Manchester, UK; 3Center for Research in Myology, Sorbonne Université-INSERM, Paris, France Background: Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. Objective: We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. Methods: Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. Results: Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. Conclusion: We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy. Keywords: motor unit, neuromuscular junctions, human embryonic stem cells, neuronal progenitor cells, human myoblasts Introduction Correspondence: Nasser Al-Shanti School of Healthcare Science, Faculty of Science and Engineering, Manchester Metropolitan University, John Dalton Building, Chester Street, M1 5GD Manchester, UK Tel +44 161 247 5712 Fax +44 161 247 6831 Email 85 submit your manuscript | www.dovepress.com Stem Cells and Cloning: Advances and Applications 2018:11 85–93 Dovepress © 2018 Abd Al Samid et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). http://dx.doi.org/10.2147/SCCAA.S178562 Powered by TCPDF (www.tcpdf.org) Neuromuscular junctions (NMJs) serve as the interface between nerves and skeletal muscles. Maintenance, structure, and formation of NMJs depend on the bidirectional molecular interaction between the muscle and motor neuron.1 The NMJ consists of a presynaptic motor neuron terminal, a postsynaptic motor end plate and a synaptic cleft. If chemical or molecular communication is disrupted, NMJ deterioration can follow. This involves axon degeneration, synapse disruption, impaired NMJ transmission, and muscle fiber degradation2 which are the features of neuromuscular diseases, myopathies, and age-associated neuromuscular impairments.3 Despite decades of intensive research to characterize the structure and function of NMJs by utilizing animals and ex vivo models,4 effective treatment of neuromuscular Dovepress Stem Cells and Cloning: Advances and Applications downloaded from https://www.dovepress.com/ by 37.59.46.207 on 21-Dec-2018 For personal use only. Abd Al Samid et al and neurodegenerative diseases remains a significant unmet clinical need. This is mainly due to the failure of experimental animal models to reflect complex processes of human aging and disease progression.5 In order to advance this field, novel, alternative, experimental models are needed. There has been recent progress toward the development of in vitro co-culture models using human induced pluripotent stem cells (iPSCs);6,7 mouse,8 rat,9 and human primary myoblasts;10,11 and human embryonic stem cells (hESCs)12–14 and cross-species models.15,16 However, existing in vitro motor neuron and skeletal muscle co-culture systems typically require a complex neural growth medium that contains serum and cocktails of around 15 neural growth factors (some of which are derived from animals).11,12,17 This further complicates drug discovery and toxicology studies due to possible cross-communication of the novel compound with factors contained within the added media, possibly explaining why many promising therapies do not translate to clinics. Another issue with existing models is that muscle contraction is induced by applied electrical or chemical stimulation, which does not replicate the native physiological stimulation required for muscle contractions.8,17–19 Recent innovation in the use of iPSCs offers the potential to derive myoblasts and motor neurons for use with in vitro NMJ models. However, cells derived from iPSCs may exhibit genetic inconsistency and genetic modification, which limit their use.20 Recent human iPSC-based studies have failed to recapitulate the severe neuronal loss observed in human neurodegenerative diseases.21–23 Human skeletal myoblasts which were used in some of the abovementioned models10,11 were obtained from primary cells (eg, muscle biopsy or surgical samples), but their life span is limited to just a few passages which restricts experimentation and necessitates repeated supply of the primary cells.24,25 Furthermore, primary cells have varied cell purity26 and (...truncated)