Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Abstract

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated microcompartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker α-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

FUS-ALS hiPSC-derived astrocytes impair human motor units through both gain-of-toxicity and loss-of-support mechanisms

“…hiPSC-derived motor neurons and astrocytes were cocultured in microfluidic devices with human primary myoblasts using a slightly modified version of a recently published protocol [ 26 ]. On day 10 of the coculture timeline, 250 000 motor neuron-NPCs were plated in a compartment on one side of the microgrooves of the device, while 40 000 myoblasts were plated in the opposite compartment.…”

“…ICC analyses of NMJ formation and neurite outgrowth were performed in XC150 microfluidic devices, while remaining stainings were imaged on 13 mm #1.5 coverslips (VWR, Monroeville, PA, USA, Cat N° 631-0150P) in 24-well plates (Greiner bio-one cellstar, Vilvoorde, Belgium, Cat N° 662160) using a previously described method [ 25 , 26 ]. Briefly, cells were fixed using 4% paraformaldehyde (Cat N° 28908) in DPBS (Cat N° 14190250) for 15–20 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Cat N° T8787) in DPBS for 20 min, and subsequently incubated with 5% normal donkey serum (NDS) (Sigma-Aldrich, Cat N° D9663) in 0.1% Triton X-100/DPBS for 30 min.…”

“…Astrocyte, motor neuron and NMJ morphologies were evaluated with SEM at day 28 of the coculture timeline using a previously published method [ 25 , 26 ]. In brief, cultures were fixed in 2.5% glutaraldehyde (Agar Scientific, Essex, UK, Cat N° R1020) in 0.1 M Na-cacodylate buffer (Sigma-Aldrich, Cat N° C0250) and the SND75 devices were carefully removed from the Aclar sheets.…”

“…Recordings were made using a previously described protocol [ 25 , 26 ]. On day 28 of the coculture timeline (1 week after adding astrocytes), myotubes were labelled with 5 μM Fluo-4 AM.…”

“…In this study, we aimed to unravel the role of hiPSC-derived astrocytes in the context of FUS -ALS and include these cells in our previously established human motor unit microfluidics model [ 25 , 26 ] to evaluate the astrocytic effect in a novel multicellular system. We discovered that FUS -ALS astrocytes showed increased reactivity and secretion of inflammatory cytokines.…”