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Modeling neuromuscular junctions in vitro: A review of the current progress employing human induced pluripotent stem cells

ICAGEN, 2090 E. Innovation Park Dr., Oro Valley, Arizona USA 85755

Special Issues: iPS Cell Technologies in Human Diseases

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disorder of undetermined etiology with no effective treatments. Motor weakness and bulbar dysfunction lead to premature death, usually resulting from respiratory failure. While much of the research has focused on the role of neuronal dysfunction in ALS etiology, evidence from human patients and animal models indicates that the neuromuscular junction (NMJ) shows significant functional and structural abnormalities prior to the onset of motor neuron degeneration and behavioral symptoms. The development of novel experimental approaches will allow the study and manipulation of human NMJs and significantly contribute to our understanding of ALS pathogenesis, leading to advances in pharmacological treatments for the disease. A novel approach that has been employed in recent years is the use of human induced pluripotent stem cells (iPSCs) to generate cell types contributing to synaptic connectivity at the NMJ. The generation and differentiation of cells derived from ALS patient iPSCs is a promising method for investigating disease mechanisms and drug screening at NMJs in vitro. In this review, we cover the theories underlying the mechanisms of ALS pathogenesis at the NMJ, an overview of the recent developments in the generation of functional human neuromuscular connectivity in vitro, and the advances in human iPSC programming and differentiation technology.
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1. Caroscio JT, Mulvihill MN, Sterling R, et al. (1987) Amyotrophic lateral sclerosis. Its natural history. Neurol Clin 5: 1–8.

2. Logroscino G, Traynor BJ, Hardiman O, et al. (2010) Incidence of amyotrophic lateral sclerosis in Europe. J Neurol Neurosurg Psychiatry 81: 385–390.    

3. Ji AL, Zhang X, Chen WW, et al. (2017) Genetics insight into the amyotrophic lateral sclerosis/frontotemporal dementia spectrum. J Med Genet 54: 145–154.    

4. Czaplinski A, Yen AA, Simpson EP, et al. (2006) Slower disease progression and prolonged survival in contemporary patients with amyotrophic lateral sclerosis: is the natural history of amyotrophic lateral sclerosis changing? Arch Neurol 63: 1139–1143.    

5. Sawada H (2017) Clinical efficacy of edaravone for the treatment of amyotrophic lateral sclerosis. Expert Opin Pharmacother 18: 735–738.    

6. Wroe R, Wai-Ling Butler A, Andersen PM, et al. (2008) ALSOD: the amyotrophic lateral sclerosis online database. Amyotroph Lateral Scler 9: 249–250.    

7. Rosen DR, Siddique T, Patterson D, et al. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362: 59–62.    

8. Kabashi E, Valdmanis PN, Dion P, et al. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 40: 572–574.    

9. Sreedharan J, Blair IP, Tripathi VB, et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319: 1668–1672.    

10. Kwiatkowski TJ, Bosco DA, Leclerc AL, et al. (2009) Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323: 1205–1208.    

11. Vance C, Rogelj B, Hortobágyi T, et al. (2009) Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323: 1208–1211.    

12. Chow CY, Landers JE, Bergren SK, et al. (2009) Deleterious variants of FIG4, a phosphoinositide phosphatase, in patients with ALS. Am J Hum Genet 84: 85–88.    

13. Johnson JO, Mandrioli J, Benatar M, et al. (2010) Exome sequencing reveals VCP mutations as a cause of familial ALS. Neuron 68: 857–864.    

14. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, et al. (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72: 245–256.    

15. Renton AE, Majounie E, Waite A, et al. (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72: 257–268.    

16. Majounie E, Renton AE, Mok K, et al. (2012) Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol 11: 323–330.    

17. Arai T, Hasegawa M, Akiyama H, et al. (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351: 602–611.    

18. Neumann M, Sampathu DM, Kwong LK, et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314: 130–133.    

19. Ling SC, Polymenidou M, Cleveland DW (2013) Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79: 416–438.    

20. Mackenzie IR, Bigio EH, Ince PG, et al. (2007) Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol 61: 427–434.    

21. Scotter EL, Chen HJ, Shaw CE (2015) TDP-43 proteinopathy and ALS: insights into disease mechanisms and therapeutic targets. Neurotherapeutics 12: 352–363.    

22. Rothstein JD (1995) Excitotoxicity and neurodegeneration in amyotrophic lateral sclerosis. Clin Neurosci 3: 348–359.

23. Blasco H, Mavel S, Corcia P, et al. (2014) The glutamate hypothesis in ALS: pathophysiology and drug development. Curr Med Chem 21: 3551–3575.    

24. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443: 787–795.    

25. Barber SC, Shaw PJ (2010) Oxidative stress in ALS: key role in motor neuron injury and therapeutic target. Free Radical Bio Med 48: 629–641.    

26. Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10: 524–530.    

27. Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat Med 10: S10–S17.    

28. Collard JF, Côté F, Julien JP (1995) Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. Nature 375: 61–64.    

29. Williamson TL, Cleveland DW (1999) Slowing of axonal transport is a very early event in the toxicity of ALS–linked SOD1 mutants to motor neurons. Nat Neurosci 2: 50–56.    

30. Bilsland LG, Sahai E, Kelly G, et al. (2010) Deficits in axonal transport precede ALS symptoms in vivo. Proc Natl Acad Sci USA 107: 20523–20528.    

31. Strong MJ (2010) The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS). J Neurol Sci 288: 1–12.    

32. Pieri M, Albo F, Gaetti C, et al. (2003) Altered excitability of motor neurons in a transgenic mouse model of familial amyotrophic lateral sclerosis. Neurosci Lett 351: 153–156.    

33. Wainger BJ, Kiskinis E, Mellin C, et al. (2014) Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep 7: 1–11.    

34. Menon P, Kiernan MC, Vucic S (2015) Cortical hyperexcitability precedes lower motor neuron dysfunction in ALS. Clin Neurophysiol 126: 803–809.    

35. Navone F, Genevini P, Borgese N (2015) Autophagy and neurodegeneration: insights from a cultured cell model of ALS. Cells 4: 354–386.    

36. Tang BL (2016) C9orf72's interaction with Rab GTPases-modulation of membrane traffic and autophagy. Front Cell Neurosci 10: 228.

37. Freibaum BD, Lu Y, Lopez-Gonzalez R, et al. (2015) GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525: 129–133.    

38. Zhang K, Donnelly CJ, Haeusler AR, et al. (2015) The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525: 56–61.    

39. Murray LM, Talbot K, Gillingwater TH (2010) Neuromuscular synaptic vulnerability in motor neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy. Neuropathol Appl Neurobiol 36: 133–156.    

40. Moloney EB, de Winter F, Verhaagen J (2014) ALS as a distal axonopathy: molecular mechanisms affecting neuromuscular junction stability in the presymptomatic stages of the disease. Front Neurosci 8: 252.

41. Sanes JR, Lichtman JW (2001) Development: Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nat Rev Neurosci 2: 791–805.

42. Slater CR (2008) Structural factors influencing the efficacy of neuromuscular transmission. Ann Ny Acad Sci 1132: 1–12.    

43. Wu H, Xiong WC, Mei L (2010) To build a synapse: signaling pathways in neuromuscular junction assembly. Development 137: 1017–1033.    

44. Fertuck HC, Salpeter MM (1974) Localization of acetylcholine receptor by 125I-labeled α-bungarotoxin binding at mouse motor endplates. Proc Natl Acad Sci USA 71: 1376–1378.    

45. Feng Z, Ko CP (2008) The role of glial cells in the formation and maintenance of the neuromuscular junction. Ann NY Acad Sci 1132: 19–28.    

46. Griffin JW, Thompson WJ (2008) Biology and pathology of nonmyelinating Schwann cells. Glia 56:1518–1531.    

47. Barik A, Li L, Sathyamurthy A, et al. (2016) Schwann cells in neuromuscular junction formation and maintenance. J Neurosci 36: 9770–9781.    

48. Hanyu N, Oguchi K, Yanagisawa N, et al. (1982) Degeneration and regeneration of ventral root motor fibers in amyotrophic lateral sclerosis: morphometric studies of cervical ventral roots. J Neurol Sci 55: 99–115.    

49. Bjornskov EK, Norris FH, Mower-Kuby J (1984) Quantitative axon terminal and end-plate morphology in amyotrophic lateral sclerosis. Arch Neurol 41: 527–530.    

50. Sasaki S, Maruyama S (1994) Synapse loss in anterior horn neurons in amyotrophic lateral sclerosis. Acta Neuropathol 88: 222–227.    

51. Siklós L, Engelhardt J, Harati Y, et al. (1996) Ultrastructural evidence for altered calcium in motor nerve terminals in amyotrophc lateral sclerosis. Ann Neurol 39: 203–216.    

52. Nagao M, Misawa H, Kato S, et al. (1998) Loss of cholinergic synapses on the spinal motor neurons of amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 57: 329–333.    

53. Maselli RA, Wollman RL, Leung C, et al. (1993) Neuromuscular transmission in amyotrophic lateral sclerosis. Muscle Nerve 16: 1193–1203.    

54. Ikemoto A, Nakamura S, Akiguchi I, et al. (2002) Differential expression between synaptic vesicle proteins and presynaptic plasma membrane proteins in the anterior horn of amyotrophic lateral sclerosis. Acta Neuropathol 103: 179–187.    

55. Liu JX, Brännström T, Andersen PM, et al. (2013) Distinct changes in synaptic protein composition at neuromuscular junctions of extraocular muscles versus limb muscles of ALS donors. PLoS One 8: e57473.    

56. Sasaki S, Iwata M (1996) Ultrastructural study of synapses in the anterior horn neurons of patients with amyotrophic lateral sclerosis. Neurosci Lett 204: 53–56.    

57. Kato T (1989) Choline acetyltransferase activities in single spinal motor neurons from patients with amyotrophic lateral sclerosis. J Neurochem 52: 636–640.    

58. Hirano A, Nakano I, Kurland LT, et al. (1984) Fine structural study of neurofibrillary changes in a family with amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 43: 471–480.    

59. Sasaki S, Iwata M (1996) Impairment of fast axonal transport in the proximal axons of anterior horn neurons in amyotrophic lateral sclerosis. Neurology 47: 535–540.    

60. Chen H, Qian K, Du Z, et al. (2014) Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons. Cell stem cell 14: 796–809.    

61. Tang-Schomer MD, Johnson VE, Baas PW, et al. (2012) Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury. Exp Neurol 233. 364–372.

62. Clark JA, Yeaman EJ, Blizzard CA, et al. (2016) A case for microtubule vulnerability in amyotrophic lateral sclerosis: altered dynamics during disease. Front Cell Neurosci 10: 204.

63. Lavado A, Guo X, Smith AST, et al. (2017) Evaluation of a holistic treatment for ALS reveals possible mechanism and therapeutic potential. Int J Pharm Pharm Res 11: 348–374.

64. Fournier C, Bedlack R, Hardiman O, et al. (2013) ALS untangled no. 20: The Deanna protocol. Amyotroph Lateral Scler Frontotemporal Degener 14: 319–323.    

65. Gurney ME, Pu H, Chiu AY, et al. (1994) Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science 264: 1772–1775.    

66. Chiu AY, Zhai P, Dal Canto MC, et al. (1995) Age-dependent penetrance of disease in a transgenic mouse model of familial amyotrophic lateral sclerosis. Mol Cell Neurosci 6: 349–362.    

67. Frey D, Schneider C, Xu L, et al. (2000) Early and selective loss of neuromuscular synapse subtypes with low sprouting competence in motoneuron diseases. J Neurosci 20: 2534–2542.    

68. Fischer LR, Culver DG, Tennant P, et al. (2004) Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol 185: 232–240.    

69. Schaefer AM, Sanes JR, Lichtman JW (2005) A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis. J Comp Neurol 490: 209–219.    

70. Gould TW, Buss RR, Vinsant S, et al. (2006) Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS. J Neurosci 26: 8774–8786.    

71. Feiguin F, Godena VK, Romano G, et al. (2009) Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior. FEBS Lett 583: 1586–1592.    

72. Wang J, Farr GW, Hall DH, et al. (2009) An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans. PLoS Genet 5: e1000350.    

73. Ramesh T, Lyon AN, Pineda RH, et al. (2010) A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease. Dis Model Mech 3: 652–662.    

74. Cappello V, Vezzoli E, Righi M, et al. (2012) Analysis of neuromuscular junctions and effects of anabolic steroid administration in the SOD1G93A mouse model of ALS. Mol Cell Neurosci 51: 12–21.    

75. Sakowski SA, Lunn JS, Busta AS, et al. (2012) Neuromuscular effects of G93A-SOD1 expression in zebrafish. Mol Neurodegener 7: 44.    

76. Rocha MC, Pousinha PA, Correia AM, et al. (2013) Early changes of neuromuscular transmission in the SOD1 (G93A) mice model of ALS start long before motor symptoms onset. PLoS One 8: e73846.    

77. Coyne AN, Siddegowda BB, Estes PS, et al. (2014) Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis. J Neurosci 34: 15962–15974.    

78. Da Costa MM, Allen CE, Higginbottom A, et al. (2014) A new zebrafish model of SOD1 ALS produced by TILLING replicates key features of the disease and represents a tool for in vivo therapeutic screening. Dis Model Mech 7: 73–81.    

79. Chew J, Gendron TF, Prudencio M, et al. (2015) C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science 348: 1151–1154.    

80. Coyne AN, Lorenzini I, Chou CC, et al. (2017) Post-transcriptional inhibition of Hsc70-4/HSPA8 expression leads to synaptic vesicle cycling defects in multiple models of ALS. Cell Rep 21: 110–125.    

81. Herranz-Martin S, Chandran J, Lewis K, et al. (2017) Viral delivery of C9orf72 hexanucleotide repeat expansions in mice leads to repeat-length-dependent neuropathology and behavioural deficits. Dis Model Mech 10: 859–868.    

82. Perry S, Han Y, Das A, et al. (2017) Homeostatic plasticity can be induced and expressed to restore synaptic strength at neuromuscular junctions undergoing ALS-related degeneration. Hum Mol Genet 26: 4153–4167.    

83. Rogers RS, Tungtur S, Tanaka T, et al. (2017) Impaired Mitophagy Plays a Role in Denervation of Neuromuscular Junctions in ALS Mice. Front Neurosci 11: 473.    

84. Kennel PF, Finiels F, Revah F, et al. (1996) Neuromuscular function impairment is not caused by motor neurone loss in FALS mice: an electromyographic study. Neuroreport 7: 1427–1431.    

85. Chand KK, Lee KM, Lee JD, et al. (2018) Defects in synaptic transmission at the neuromuscular junction precedes motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis. FASEB J 32: 2676–2689.    

86. Xie Y, Zhou B, Lin MY, et al. (2015) Progressive endolysosomal deficits impair autophagic clearance beginning at early asymptomatic stages in fALS mice. Autophagy 11: 1934–1936.    

87. Shi Y, Lin S, Staats KA, et al. (2018) Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat Med 24: 313–325.    

88. Shaw PJ, Ince PG, Falkous G, et al. (1995) Oxidative damage to protein in sporadic motor neuron disease spinal cord. Ann Neurol 8: 691–695.

89. Rizzardini M, Mangolini A, Lupi M, et al. (2005) Low levels of ALS-linked Cu/Zn superoxide dismutase increase the production of reactive oxygen species and cause mitochondrial damage and death in motor neuron-like cells. J Neurol Sci 232: 95–103.    

90. Pollari E, Goldsteins G, Bart G, et al. (2014) The role of oxidative stress in degeneration of the neuromuscular junction in amyotrophic lateral sclerosis. Front Cell Neurosci 8: 131.

91. Perlson E, Maday S, Fu MM, et al. (2010) Retrograde axonal transport: pathways to cell death? Trends Neurosci 33: 335–344.    

92. Perlson E, Jeong GB, Ross JL, et al. (2009) A switch in retrograde signaling from survival to stress in rapid-onset neurodegeneration. J Neurosci 29: 9903–9917.    

93. Kostic V, Jackson-Lewis V, de Bilbao F, et al. (1997) Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis. Science 277: 559–563.    

94. Parone PA, Da Cruz S, Han JS, et al. (2013) Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci 33: 4657–4671.    

95. Di Giorgio FP, Carrasco MA, Siao MC, et al. (2007) Non–cell autonomous effect of glia on motor neurons in an embryonic stem cell–based ALS model. Nat Neurosci 10: 608–614.    

96. Nagai M, Re DB, Nagata T, et al. (2007) Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci 10: 615–622.    

97. Wong M, Martin LJ (2010) Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum Mol Genet 19: 2284–2302.    

98. Park KH, Vincent I (2008) Presymptomatic biochemical changes in hindlimb muscle of G93A human Cu/Zn superoxide dismutase 1 transgenic mouse model of amyotrophic lateral sclerosis. Biochim Biophys Acta 1782: 462–468.    

99. Fu AK, Fu WY, Cheung J, et al. (2001) Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction. Nat Neurosci 4: 374–381.    

100. Lazaro JB, Kitzmann M, Poul MA, et al. (1997) Cyclin dependent kinase 5, cdk5, is a positive regulator of myogenesis in mouse C2 cells. J Cell Sci 110: 1251–1260.

101. Narai H, Manabe Y, Nagai M, et al. (2009) Early detachment of neuromuscular junction proteins in ALS mice with SODG93A mutation. Neurol Int 1: e16.    

102. Jokic N, Gonzalez de Aguilar JL, Pradat PF, et al. (2005) Nogo expression in muscle correlates with amyotrophic lateral sclerosis severity. Ann Neurol 57: 553–556.    

103. Pasterkamp RJ, Giger RJ (2009) Semaphorin function in neural plasticity and disease. Curr Opin Neurobiol 19: 263–274.    

104. De Winter F, Vo T, Stam FJ, et al. (2006) The expression of the chemorepellent Semaphorin 3A is selectively induced in terminal Schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease. Mol Cell Neurosci 32: 102–117.    

105. Moloney EB, Hobo B, De Winter F, et al. (2017) Expression of a Mutant SEMA3A Protein with Diminished Signalling Capacity Does Not Alter ALS-Related Motor Decline or Confer Changes in NMJ Plasticity after BotoxA-Induced Paralysis of Male Gastrocnemic Muscle. PLoS One 12: e0170314.    

106. Yumoto N, Kim N, Burden SJ (2012) Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses. Nature 489: 438–442.    

107. Cantor S, Zhang W, Delestrée N, et al. (2018) Preserving neuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonist antibody. ELife 7: e34375.    

108. Dadon-Nachum M, Melamed E, Offen D (2011) The "dying-back" phenomenon of motor neurons in ALS. J Mol Neurosci 43: 470–477.    

109. Verstreken P, Ly CV, Venken KJ, et al. (2005) Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47: 365–378.    

110. Vos M, Lauwers E, Verstreken P (2010) Synaptic mitochondria in synaptic transmission and organization of vesicle pools in health and disease. Front Synaptic Neurosci 2: 139.

111. Szepsenwol J (1946) A comparison of growth, differentiation, activity and action currents of heart and skeletal muscle in tissue culture. Anat Rec 95: 125–146.    

112. Bornstein MB, Breitbart LM (1964) Anatomical studies of mouse embryo spinal cord-skeletal muscle in long-term tissue culture. Anat Rec 148: 362.

113. Crain SM (1964) Electrophysiological studies of cord-innervated skeletal muscle in long-term tissue cultures of mouse embryo myotomes. Anat Rec 148: 273.

114. Crain SM (1968) Development of functional neuromuscular connections between separate explants of fetal mammalian tissues after maturation in culture. Anat Rec 160: 466.

115. James DW, Tresman RL (1968) De novo formation of neuro-muscular junctions in tissue culture. Nature 220: 384–385.    

116. Shimada Y, Fischman DA, Moscona AA (1969) Formation of neuromuscular junctions in embryonic cell cultures. Proc Natl Acad Sci USA 62: 715–721.

117. Seecof RL, Teplitz RL, Gerson I, et al. (1972) Differentiation of neuromuscular junctions in cultures of embryonic Drosophila cells. Proc Natl Acad Sci USA 69: 566–570.    

118. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156.    

119. Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78: 7634–7638.

120. Wilmut I, Schnieke AE, McWhir J, et al. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385: 810–813.    

121. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147.

122. Wichterle H, Lieberam I, Porter JA, et al. (2002) Directed differentiation of embryonic stem cells into motor neurons. Cell 110: 385–397.    

123. Li XJ, Du ZW, Zarnowska ED, et al. (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23: 215–221.    

124. Singh Roy N, Nakano T, Xuing L, et al. (2005) Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells. Exp Neurol 196: 224–234.    

125. Lee H, Shamy GA, Elkabetz Y, et al. (2007) Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells 25: 1931–1939.    

126. Barberi T, Bradbury M, Dincer Z, et al. (2007) Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med 13: 642–648.    

127. Darabi R, Gehlbach K, Bachoo RM, et al. (2008) Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat Med 14: 134–143.    

128. Lu B, Czernik AJ, Popov S, et al. (1996) Expression of synapsin I correlates with maturation of the neuromuscular synapse. Neuroscience 74: 1087–1097.    

129. Peng HB, Yang JF, Dai Z, et al. (2003) Differential effects of neurotrophins and schwann cell-derived signals on neuronal survival/growth and synaptogenesis. J Neurosci 23: 5050–5060.    

130. Fischbach GD (1972) Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol 28: 407–429.    

131. Fischbach GD, Cohen SA (1973) The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol 31: 147–162.    

132. Frank E, Fischbach GD (1979) Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses. J Cell Biol 83: 143–158.    

133. Harper JM, Krishnan C, Darman JS, et al. (2004) Axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats. Proc Natl Acad Sci USA 101: 7123–7128.    

134. Umbach JA, Adams KL, Gundersen CB, et al. (2012) Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons. PLoS One 7: e36049.    

135. Dutton EK, Uhm CS, Samuelsson SJ, et al. (1995) Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons. J Neurosci 15: 7401–7416.    

136. Daniels MP, Lowe BT, Shah S, et al. (2000) Rodent nerve‐muscle cell culture system for studies of neuromuscular junction development: Refinements and applications. Microsc Res Tech 49: 26–37.    

137. Das M, Rumsey JW, Bhargava N, et al. (2010) A defined long-term in vitro tissue engineered model of neuromuscular junctions. Biomaterials 31: 4880–4888.    

138. Guo X, Gonzalez M, Stancescu M, et al. (2011) Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system. Biomaterials 32: 9602–9611.    

139. Marteyn A, Maury Y, Gauthier MM, et al. (2011) Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy. Cell Stem Cell 8: 434–444.    

140. Miles GB, Yohn DC, Wichterle H, et al. (2004) Functional properties of motoneurons derived from mouse embryonic stem cells. J Neurosci 24: 7848–7858.    

141. Soundararajan P, Lindsey BW, Leopold C, et al. (2007) Easy and Rapid Differentiation of Embryonic Stem Cells into Functional Motoneurons Using Sonic Hedgehog-Producing Cells. Stem Cells 25: 1697–1706.    

142. Guo X, Das M, Rumsey J, et al. (2010) Neuromuscular junction formation between human stem-cell-derived motoneurons and rat skeletal muscle in a defined system. Tissue Eng Part C 16: 1347–1355.

143. Kobayashi T, Askanas V, Engel WK (1987) Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation. J Neurosci 7: 3131–3141.    

144. Mars T, Yu KJ, Tang XM, et al. (2001) Differentiation of glial cells and motor neurons during the formation of neuromuscular junctions in cocultures of rat spinal cord explant and human muscle. J Comp Neurol 438: 239–251.    

145. Gajsek N, Jevsek M, Mars T, et al. (2008) Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures. Chem Biol Interact 175: 50–57.    

146. Yohn DC, Miles GB, Rafuse VF, et al. (2008) Transplanted mouse embryonic stem-cell-derived motoneurons form functional motor units and reduce muscle atrophy. J Neurosci 28: 12409–12418.    

147. Puttonen KA, Ruponen M, Naumenko N, et al. (2015) Generation of functional neuromuscular junctions from human pluripotent stem cell lines. Front Cell Neurosci 9: 473.

148. Steinbeck JA, Jaiswal MK, Calder EL, et al. (2016) Functional connectivity under optogenetic control allows modeling of human neuromuscular disease. Cell Stem Cell 18: 134–143.    

149. Smith AST, Long CJ, Pirozzi K, et al. (2013) A functional system for high-content screening of neuromuscular junctions in vitro. Technology 1: 37–48.    

150. Uzel SG, Platt RJ, Subramanian V, et al. (2016) Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units. Sci Adv 2: e1501429.    

151. Cvetkovic C, Rich MH, Raman R, et al. (2017) A 3D-printed platform for modular neuromuscular motor units. Microsyst Nanoeng 3: 17015.    

152. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676.    

153. Takahashi K, Tanabe K, Ohnuki M, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872.    

154. Yu J, Vodyanik MA, Smuga-Otto K, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920.    

155. Byrne SM, Mali P, Church GM (2014) Genome editing in human stem cells. Methods Enzymol 546: 119–138.    

156. Wang L, Yi F, Fu L, et al. (2017) CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs. Protein Cell 8: 365–378.    

157. Corti S, Nizzardo M, Simone C, et al. (2012) Genetic correction of human induced pluripotent stem cells from patients with spinal muscular atrophy. Sci Transl Med 4: 165ra162.

158. Baum C, Von Kalle C, Staal FJ, et al. (2004) Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol Ther 9: 5–13.

159. Fusaki N, Ban H, Nishiyama A, et al. (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85: 348–362.    

160. Nishimura K, Sano M, Ohtaka M, et al. (2011) Development of defective and persistent Sendai virus vector a unique gene delivery/expression system ideal for cell reprogramming. J Biol Chem 286: 4760–4771.    

161. Nishimura K, Ohtaka M, Takada H, et al. (2017) Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302. Stem Cell Res 23: 13–19.    

162. Okita K, Matsumura Y, Sato Y, et al. (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8: 409–412.    

163. Chou BK, Mali P, Huang X, et al. (2011) Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res 21: 518–529.    

164. Kim D, Kim CH, Moon JI, et al. (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4: 472–476.    

165. Warren L, Manos PD, Ahfeldt T, et al. (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618–630.    

166. Li XJ, Hu BY, Jones SA, et al. (2008) Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules. Stem Cells 26: 886–893.    

167. Chambers SM, Fasano CA, Papapetrou EP, et al. (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27: 275–280.    

168. Hu BY, Zhang SC (2009) Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc 4: 1295–1304.    

169. Karumbayaram S, Novitch BG, Patterson M, et al. (2009) Directed differentiation of human‐induced pluripotent stem cells generates active motor neurons. Stem Cells 27: 806–811.    

170. Du ZW, Chen H, Liu H, et al. (2015) Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun 6: 6626.    

171. Son EY, Ichida JK, Wainger BJ, et al. (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9: 205–218.    

172. Dimos JT, Rodolfa KT, Niakan KK, et al. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321: 1218–1221.    

173. Bilican B, Serio A, Barmada SJ, et al. (2012) Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability. Proc Natl Acad Sci USA 109: 5803–5808.    

174. Egawa N, Kitaoka S, Tsukita K, et al. (2012) Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci Transl Med 4: 145ra104.

175. Burkhardt MF, Martinez FJ, Wright S, et al. (2013) A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells. Mol Cell Neurosci 56: 355–364.    

176. Devlin AC, Burr K, Borooah S, et al. (2015) Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability. Nat Commun 6: 5999.

177. Kiskinis E, Sandoe J, Williams LA, et al. (2014) Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1. Cell Stem Cell 14: 781–795.    

178. Nizzardo M, Simone C, Rizzo F, et al. (2013) Minimally invasive transplantation of iPSC-derived ALDHhiSSCloVLA4+ neural stem cells effectively improves the phenotype of an amyotrophic lateral sclerosis model. Hum Mol Genet 23: 342–354.

179. Popescu IR, Nicaise C, Liu S, et al. (2013) Neural progenitors derived from human induced pluripotent stem cells survive and differentiate upon transplantation into a rat model of amyotrophic lateral sclerosis. Stem Cells Transl Med 2: 167–174.    

180. Ho R, Sances S, Gowing G, et al. (2016) ALS disrupts spinal motor neuron maturation and aging pathways within gene co-expression networks. Nat Neurosci 19: 1256–1267.    

181. Ambasudhan R, Talantova M, Coleman R, et al. (2011) Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 9: 113–118.    

182. Pang ZP, Yang N, Vierbuchen T, et al. (2011) Induction of human neuronal cells by defined transcription factors. Nature 476: 220–223.    

183. Cornacchia D, Studer L (2017) Back and forth in time: Directing age in iPSC-derived lineages. Brain Res 1656: 14–26.    

184. Hinds S, Bian W, Dennis RG, et al. (2011) The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle. Biomaterials 32: 3575–3583.    

185. Liao IC, Liu JB, Bursac N, et al. (2008) Effect of electromechanical stimulation on the maturation of myotubes on aligned electrospun fibers. Cell Mol Bioeng 1:133–145.    

186. Darabi R, Arpke RW, Irion S, et al. (2012) Human ES-and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice. Cell Stem Cell 10: 610–619.    

187. Tanaka A, Woltjen K, Miyake K, et al. (2013) Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro. PLoS One 8: e61540.    

188. Rao L, Qian Y, Khodabukus A, et al. (2018) Engineering human pluripotent stem cells into a functional skeletal muscle tissue. Nat Commun 9: 126.    

189. Trokovic R, Weltner J, Manninen T, et al. (2012) Small molecule inhibitors promote efficient generation of induced pluripotent stem cells from human skeletal myoblasts. Stem Cells Dev 22: 114–123.

190. Chal J, Al Tanoury Z, Hestin M, et al. (2016) Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nat Protoc 11: 1833–1850.    

191. Goetsch KP, Myburgh KH, Niesler CU (2013) In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair. J Muscle Res Cell Motil 34: 333–347.    

192. Happe CL, Tenerelli KP, Gromova AK, et al. (2017) Mechanically patterned neuromuscular junctions-in-a-dish have improved functional maturation. Mol Biol Cell 28: 1950–1958.    

193. Madden L, Juhas M, Kraus WE, et al. (2015) Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs. Elife 4: e04885.

194. Lenzi J, Pagani F, De Santis R, et al. (2016) Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscular diseases. Stem Cell Res 17: 140–147.    

195. Swartz EW, Baek J, Pribadi M, et al. (2016) A novel protocol for directed differentiation of C9orf72-associated human induced pluripotent stem cells into contractile skeletal myotubes. Stem Cells Transl Med 5: 1461–1472.    

196. Liu Q, Spusta SC, Mi R, et al. (2012) Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells. Stem Cells Transl Med 1: 266–278.    

197. Wang A, Tang Z, Park IH, et al. (2011) Induced pluripotent stem cells for neural tissue engineering. Biomaterials 32: 5023–5032.    

198. Kim HS, Lee J, Lee DY, et al. (2017) Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair. Stem Cell Rep 8: 1714–1726.    

199. Miller JD, Ganat YM, Kishinevsky S, et al. (2013) Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13: 691–705.    

200. Vera E, Studer L (2015) When rejuvenation is a problem: challenges of modeling late-onset neurodegenerative disease. Development 142: 3085–3089.    

201. Li Y, Liu M, Yan Y, et al. (2014) Neural differentiation from pluripotent stem cells: the role of natural and synthetic extracellular matrix. World J Stem Cells 6: 11–23.    

202. Hagbard L, Cameron K, August P, et al. (2018) Developing defined substrates for stem cell culture and differentiation. Philos Trans R Soc Lond B Biol Sci 373: 20170230.

203. Demestre M, Orth M, Föhr KJ, et al. (2015) Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes. Stem Cell Res 15: 328–336.    

204. Toma JS, Shettar BC, Chipman PH, et al. (2015) Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons. J Neurosci 35: 1291–1306.    

205. Lefebvre S, Bürglen L, Reboullet S, et al. (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80: 155–165.    

206. Yoshida M, Kitaoka S, Egawa N, et al. (2015) Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs. Stem Cell Rep 4: 561–568.    

207. Sumner CJ, Huynh TN, Markowitz JA, et al. (2003) Valproic acid increases SMN levels in spinal muscular atrophy patient cells. Ann Neurol 54: 647–654.    

208. Kerr KS, Fuentes-Medel Y, Brewer C, et al. (2014) Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction. J Neurosci 34: 2910–2920.    

209. Fuentes-Medel Y, Ashley J, Barria R, et al. (2012) Integration of a retrograde signal during synapse formation by glia-secreted TGF-β ligand. Curr Biol 22: 1831–1838.    

210. Robitaille R (1998) Modulation of synaptic efficacy and synaptic depression by glial cells at the frog neuromuscular junction. Neuron 21: 847–855.    

211. Smith IW, Mikesh M, Lee Y, et al. (2013) Terminal Schwann cells participate in the competition underlying neuromuscular synapse elimination. J Neurosci 33: 17724–17736.    

212. Son YJ, Thompson WJ (1995) Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells. Neuron 14: 133–141.    

213. O'Malley JP, Waran MT, Balice-Gordon RJ (1999) In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions. J Neurobiol 38: 270–286.    

214. Ullian EM, Harris BT, Wu A, et al. (2004) Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture. Mol Cell Neurosci 25: 241–251.    

215. Gingras M, Beaulieu MM, Gagnon V, et al. (2008) In vitro study of axonal migration and myelination of motor neurons in a three-dimensional tissue-engineered model. Glia 56: 354–364.    

216. Hyung S, Lee BY, Park JC, et al. (2015) Coculture of primary motor neurons and Schwann cells as a model for in vitro myelination. Sci Rep 5: 15122.    

217. Suh JKF, Hyung S (2018) Primary motor neuron culture to promote cellular viability and myelination. In Neurotrophic Factors. Methods Mol Biol 1727: 403–411.    

218. Appel SH, Zhao W, Beers DR, et al. (2011) The microglial-motoneuron dialogue in ALS. Acta Myol 30: 4–8.

219. Douvaras P, Sun B, Wang M, et al. (2017) Directed differentiation of human pluripotent stem cells to microglia. Stem Cell Rep 8: 1516–1524.    

220. Abud EM, Ramirez RN, Martinez ES, et al. (2017) iPSC-derived human microglia-like cells to study neurological diseases. Neuron 94: 278–293.    

221. Zahavi EE, Ionescu A, Gluska S, et al. (2015) A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions. J Cell Sci 128: 1241–1252.    

222. Ionescu A, Zahavi EE, Gradus T, et al. (2016) Compartmental microfluidic system for studying muscle–neuron communication and neuromuscular junction maintenance. Eur J Cell Biol 95: 69–88.    

223. Vilmont V, Cadot B, Ouanounou G, et al. (2016) A system for studying mechanisms of neuromuscular junction development and maintenance. Development 143: 2464–2477.    

224. Black BJ, Atmaramani R, Pancrazio JJ (2017) Spontaneous and Evoked Activity from Murine Ventral Horn Cultures on Microelectrode Arrays. Front Cell Neurosci 11: 304.

225. Kovalchuk MO, Heuberger JA, Sleutjes BT, et al. (2018) Acute effects of riluzole and retigabine on axonal excitability in patients with ALS: A randomized, double-blind, placebo-controlled, cross-over trial. Clin Pharmacol Ther.

226. Hochbaum DR, Zhao Y, Farhi SL, et al. (2014) All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat Methods 11: 825–833.    

227. Kiskinis E, Kralj JM, Zou P, et al. (2018) All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS. Stem Cell Rep 10: 1–14.    

228. Santhanam N, Kumanchik L, Guo X, et al. (2018) Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics. Biomaterials 166: 64–78.    

229. Scannell JW, Blanckley A, Boldon H, et al. (2012) Diagnosing the decline in pharmaceutical R&D efficiency. Nat Rev Drug Discov 11: 191–200.    

230. Van der Worp HB, Howells DW, Sena ES, et al. (2010) Can animal models of disease reliably inform human studies? PLoS Med 7: e1000245.    

231. Van der Schyf CJ (2011) The use of multi-target drugs in the treatment of neurodegenerative diseases. Expert Rev Clin Pharmacol 4: 293–298.    

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