Neuromuscular junction formation between human stem-cell-derived motoneurons and rat skeletal muscle in a defined system

Abstract

To date, the coculture of motoneurons (MNs) and skeletal muscle in a defined in vitro system has only been described in one study and that was between rat MNs and rat skeletal muscle. No in vitro studies have demonstrated human MN to rat muscle synapse formation, although numerous studies have attempted to implant human stem cells into rat models to determine if they could be of therapeutic use in disease or spinal injury models, although with little evidence of neuromuscular junction (NMJ) formation. In this report, MNs differentiated from human spinal cord stem cells, together with rat skeletal myotubes, were used to build a coculture system to demonstrate that NMJ formation between human MNs and rat skeletal muscles is possible. The culture was characterized by morphology, immunocytochemistry, and electrophysiology, while NMJ formation was demonstrated by immunocytochemistry and videography. This defined system provides a highly controlled reproducible model for studying the formation, regulation, maintenance, and repair of NMJs. The in vitro coculture system developed here will be an important model system to study NMJ development, the physiological and functional mechanism of synaptic transmission, and NMJ- or synapse-related disorders such as amyotrophic lateral sclerosis, as well as for drug screening and therapy design.

FIG. 1.

(A–D) Phase pictures of human stem-cell-derived MNs and rat eSKM coculture. (A) Day 3 after plating in the coculture. Myocytes are shown to be proliferating actively at this time period and a number of them have begun to line up to initiate myotube formation. (B) Day 11 after plating. The myotubes and nerve cells are distinguishable easily by morphology. (C) A sample picture for a large myotube with striations (arrows) from day 10 in culture. (D) A micrograph demonstrating coculture of the human stem-cell-derived MNs and rat myotubes from day 10 in culture. The myotube striations are indicated by the green arrows. A nearby MN (indicated by the yellow arrow) sends out processes that approach the myotube (indicated by the blue arrow). (E) Triple staining of the coculture with antibodies against β-III Tubulin, myosin heavy chain, and α-bungarotoxin, Alexa Fluor^® 488 conjugate (BTX)-488 to demonstrate the presence of neurons, myotubes, and the distribution of AchRs in a day 9 coculture. MNs, motoneurons; eSKM, embryonic skeletal muscle; AchRs, acetylcholine receptors. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Electrophysiological recordings from MNs and eSKM in the coculture. (A, B) Sample traces of a voltage-clamp recording (A) and a current-clamp recording (B) from an MN at day 13 in the coculture. The inset picture indicates the recorded MN. (C, D) Sample traces of a voltage-clamp recording (C) and a current-clamp recording (D) from a myotube at day 26 in the coculture. The inset picture indicates the recorded myotube. The scales are the same in (A) and (B). Color images available online at www.liebertonline.com/ten.

FIG. 3.

Coimmunostaining of β-III Tubulin for neurons (green) and BTX-488 for AchRs (red) in a 13-day-old coculture. (A) A sample micrograph demonstrates that a neurite terminal branched upon contact with a myotube (left arrow). Moreover, this contact is surrounded by a cluster of AchRs. Another neurite–myotube contact site (right arrow), indicated by the neurite indentation and slight enlargement, is also in proximity to an AchR cluster. (B) An optical section from a confocal image indicates the close apposition of the neurite terminals and the AchR clusters as illustrated by β-III Tubulin and BTX-488 costaining (indicated by arrows). These nerve terminals presented a varicosity morphology, resembling typical presynaptic terminals. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Close apposition of a neurite and AchR clusters on the myotube demonstrated by synaptophysin (red) and BTX-488 (green) costaining in a 26-day-old coculture (indicated by arrows). Color images available online at www.liebertonline.com/ten.

FIG. 5.

Glut can specifically induce muscle contractions in the coculture, which can be stopped by curare. (A) Coimmunostaining of β-III Tubulin (blue), choline acetyl transferase (ChAT) (red), and GlutR (green) in a day 31 culture. As indicated, while all the neurons are positive for β-III Tubulin, those that are positive for ChAT also show high level expression of GlutRs. (B) Specific muscle contractions were induced in the MN-eSKM cocultures (Supplemental Videos S1–S4; available online at www.liebertonline.com/ten). GlutRs, glutamate receptors. Color images available online at www.liebertonline.com/ten.