A Novel Functional In Vitro 3D iPSC-Derived Neuromuscular Junction Model for Investigating Botulinum Neurotoxin Activity and Neuromuscular Disease


Translational Research

Poster Number: M221


Greg Luerman, PhD, Curi Bio, Jacob Fleming, PhD, Curi Bio, Kevin Gray, Curi Bio, David Nash, Curi Bio, Vincent Leung, Curi Bio, Cam Gelber, Curi Bio, Christos Michas, PhD, Curi Bio, Shawn Luttrell, PhD, Curi Bio, Alec Smith, PhD, Institute for Stem Cell and Regenerative Medicine, University of Washington, David Mack, PhD, University of Washington Medicine, Nick Geisse, PhD, Curi Bio

Diseases of the neuromuscular junction (NMJ) have devastating impacts on individuals’ quality of life and are often ultimately fatal. The development of effective therapeutics necessitates disease models that more closely mimic in vivo conditions, to date few models exist. Here, we present a novel in vitro functional neuromuscular junction (NMJ) model derived from human iPSCs, comprising motor neurons and skeletal muscle 3D tissue constructs. Furthermore, we validate the utility of this model by demonstrating Botulinum toxin (Botox) potency for blocking neuron-mediated muscle contractions.
Human iPSCs were differentiated (separately) into motor neurons bearing a blue light-activated channel (ChR2) and skeletal muscle cells. Functional engineered skeletal muscles were generated in the commercially available MantarrayTM two-post tissue platform, allowing across time label free functional measures. Following 10-days of differentiation these skeletal tissues were combined with 100 neuronal spheroids using a specialized consumable removing the requirement to directly handle spheroids. Following 1-week of co-culture 488mn light application to co-culture tissues produced synchronized functional responses from the underlying muscle at 5, 2, and 1 second intervals.
Application of 10nM botulinum toxin A to co-culture tissues ablated blue light responses within 240 minutes, reducing both force magnitude and fidelity of synchronized force production in response to blue light. This demonstrates the specificity of the motor neuron – skeletal muscle interactions within these tissues, assuring that force generation is dependent on motor neuron derived acetylcholine.
We present this model of the NMJ as a platform for preclinical development of drugs targeting currently intractable diseases of the NMJ, to improve clinical translation of novel therapeutics. Through the application of iPSC derived motor neurons containing disease associated mutations, diseases such as ALS can now be studied in a dish. Additionally, healthy NMJs present a platform for toxicity and potency testing on known and unknown neurotoxins in a human relevant system.