title: Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes.
abstract: Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.
authors.affiliation: Institute of Anatomy and Cell Biology, Ulm University, Germany. Electronic address: maria.demestre@uni-ulm.de.
authors.forename: M
authors.initials: M
authors.lastname: Demestre
authors.affiliation: Department of Neurology, Ulm University, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Orth
authors.affiliation: Clinic for Anaesthesiology, Ulm University, Germany.
authors.forename: K J
authors.initials: KJ
authors.lastname: Föhr
authors.affiliation: Institute of Neuroanatomy, Eberhard Karls University, Tübingen, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Achberger
authors.affiliation: Department of Neurology, Ulm University, Germany.
authors.forename: A C
authors.initials: AC
authors.lastname: Ludolph
authors.affiliation: Institute of Neuroanatomy, Eberhard Karls University, Tübingen, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Liebau
authors.affiliation: Institute of Anatomy and Cell Biology, Ulm University, Germany. Electronic address: tobias.boeckers@uni-ulm.de.
authors.forename: T M
authors.initials: TM
authors.lastname: Boeckers
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D018920:Coculture Techniques; D005260:Female; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology; D008297:Male; D009046:Motor Neurons / Q000166:cytology* / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000502:physiology; D018482:Muscle, Skeletal / Q000166:cytology; D009469:Neuromuscular Junction / Q000378:metabolism*; D051763:PAX7 Transcription Factor / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011950:Receptors, Cholinergic / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D051763:PAX7 Transcription Factor; C497321:PAX7 protein, human; D011950:Receptors, Cholinergic; D014157:Transcription Factors
keywords: Acetylcholine receptor; Amyotrophic lateral sclerosis; Human induced pluripotent stem cells; Motoneuron; Motoneuron diseases; Myotubes; Neuromuscular junction