Method Profile
Assemble human motor-neuron spheroids with skeletal-muscle bundles, validate coupled activity, induce intermittent-hypoxia injury and compare distinct NAD⁺-metabolism interventions.
Explore neuromuscular injury mechanisms and distinguish biochemical from functional treatment effects.
CAS-cell-bank human induced pluripotent stem cells differentiated into motor-neuron spheroids plus purchased human skeletal-muscle cells; three to four spheroids per bundle. Mouse C2C12 cells were confined to a separate formation test.
Patterned open PDMS device; four-day intermittent hypoxia, cycling 40 minutes at 1% oxygen and 20 minutes at 21%; separate electrical, movement and structural endpoints, plus NMN, NAT and 78c NAD/ATP comparisons.
Human tissue components
Motor-neuron spheroids from CAS DYR0100 iPSCs and Lifeline FC-0091 skeletal-muscle cells; normoxic and component-only comparisons.
Measurements and interventions
Optogenetic/curare functional controls, microelectrode-array activity, MUSCLEMOTION image-based contraction analysis and NAD⁺/NADH/ATP assays; nicotinamide mononucleotide (NMN) precursor, NAT NAMPT activator and 78c CD38 inhibitor.
Compare component and assembled tissues, functional and structural responses, and individual metabolic drug endpoints. MUSCLEMOTION software quantifies contraction from changes in image pixels; electrode activity has limited coverage in three dimensions.
Validate coupled neuronal–muscle responses, induce intermittent-hypoxia injury and compare three metabolic interventions.
Contraction/assembled activity n = 4 assembloids; whole-construct RNA sequencing: 5 hypoxia and 4 control constructs; NAD⁺/NADH/ATP drug readouts n = 3 independent experiments.
Normoxia, component-only constructs, neuronal stimulation with or without curare, and hypoxia with the three NAD-pathway agents.
Hypoxia impaired neural/contractile outputs despite some preserved axon measures. 78c corrected NAD⁺/NADH balance, NMN mainly restored ATP, and NAT had negligible effects. Post-treatment recovery of contraction/network function was not demonstrated.
A human neuron–muscle construct developed functional neuromuscular connections and hypoxia-related deficits. In a proof-of-concept drug comparison, 78c and NMN improved different NAD⁺/ATP measurements, while the NAMPT activator NAT had little effect; restoration of motor function was not demonstrated by those biochemical results.
