Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.

Katja A Puttonen,Nikolay Naumenko,Pasi Tavi,Marika Ruponen,Jari Koistinaho,Outi H Hovatta

doi:10.3389/fncel.2015.00473

Abstract

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

Highlights

Disorders of the neuromuscular junctions (NMJs), the cholinergic synapses between lower motoneurons and skeletal muscle fibers, include myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS; Liang and Han, 2013)
To investigate the differentiation fate of the non-neural cells, we seeded cells from all sphere types derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines onto polyornithine-laminin coated wells as monolayers, and withdrew basic fibroblast factor from the growth medium
Having shown the co-maturation of neurons and myotubes in our cell culture model of human pluripotent stem cells (hPSC)-derived cells, we investigated whether the two cell types are synaptically connected to each other

Summary

Introduction

Disorders of the neuromuscular junctions (NMJs), the cholinergic synapses between lower motoneurons and skeletal muscle fibers, include myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS; Liang and Han, 2013). 2000; Das et al, 2010; Wu et al, 2010; Naumenko et al, 2011; Palma et al, 2011; Thomson et al, 2012) and spinal muscular atrophy (SMA; Russman, 2007), involve early pathological changes at NMJ. While all these disorders belong to the class of orphan (rare) diseases, their prevalence altogether is 100,000—130,000 in the US, 200,000—260,000 in Europe, and approximately 1.5–1.8 million worldwide For modeling diseases of genetic etiology an NMJ model where both neuron and muscle components are derived from the same individual is of great interest

Methods

Results

Conclusion