An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing
Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity, scalability, throughput, and ethical considerations further limit the suitability of animal models...
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Elsevier
2025-01-01
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| Series: | Current Research in Toxicology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666027X25000040 |
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| author | Jacob W. Fleming Molly C. McCloskey Kevin Gray David R. Nash Vincent Leung Christos Michas Shawn M. Luttrell Christopher Cavanaugh Julie Mathieu Shawn Mcquire Mark Bothwell David L. Mack Nicholas A. Geisse Alec S.T. Smith |
| author_facet | Jacob W. Fleming Molly C. McCloskey Kevin Gray David R. Nash Vincent Leung Christos Michas Shawn M. Luttrell Christopher Cavanaugh Julie Mathieu Shawn Mcquire Mark Bothwell David L. Mack Nicholas A. Geisse Alec S.T. Smith |
| author_sort | Jacob W. Fleming |
| collection | DOAJ |
| description | Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity, scalability, throughput, and ethical considerations further limit the suitability of animal models for preclinical screening. Engineered models have emerged as alternatives for studying NMJ functionality in response to genetic and/or pharmacological challenge. However, these models have faced challenges associated with their poorly scalable creation, sourcing suitable cells, and the extraction of reliable, quantifiable metrics. We present a turnkey iPSC-based model of the NMJ employing channelrhodopsin-2 expression within the motor neuron (MN) population driving muscle contraction in response to blue light. MNs co-cultured with engineered skeletal muscle tissues produced twitch forces of 34.7 ± 22.7 µN in response to blue light, with a response fidelity > 92 %. Histological analysis revealed characteristic punctate acetylcholine receptor staining co-localized with the presynaptic marker synaptic vesicle protein-2. Dose-response studies using botulinum neurotoxin showed loss of function in a dose- and time-dependent manner (EC50 − 0.11 ± 0.015 µg). Variability of the EC50 values between 2 different iPSC differentiations of both cell types and 2 users was less than 2 %. Further testing with the acute neurotoxins acetylcholine mustard and d-tubocurarine validated the biological relevance of the postsynaptic machinery of the model. This model marks a meaningful progression of 3D engineered models of the NMJ, providing engineered tissues at a throughput relevant to potency and screening applications with an abundant iPSC cell source and standardized hardware-software ecosystem allowing technology transfer across laboratories. |
| format | Article |
| id | doaj-art-5932ee53bab841288baac55b863e0279 |
| institution | Kabale University |
| issn | 2666-027X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Current Research in Toxicology |
| spelling | doaj-art-5932ee53bab841288baac55b863e02792025-01-30T05:14:58ZengElsevierCurrent Research in Toxicology2666-027X2025-01-018100218An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testingJacob W. Fleming0Molly C. McCloskey1Kevin Gray2David R. Nash3Vincent Leung4Christos Michas5Shawn M. Luttrell6Christopher Cavanaugh7Julie Mathieu8Shawn Mcquire9Mark Bothwell10David L. Mack11Nicholas A. Geisse12Alec S.T. Smith13Curi Bio Inc., 3000 Western Avenue, Seattle, WA, USADepartment of Neurobiology and Biophysics, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USAComparative Medicine Department, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USAComparative Medicine Department, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USADepartment of Neurobiology and Biophysics, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USADepartment of Neurobiology and Biophysics, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA; Department of Bioengineering, University of Washington, Seattle, WA, USA; Department of Rehabilitation Medicine, University of Washington, Seattle, WA, USACuri Bio Inc., 3000 Western Avenue, Seattle, WA, USA; Corresponding authors at: Curi Bio Inc., 3000 Western Avenue, Seattle, WA, USA (N.A. Geisse), Department of Physiology and Biophysics, University of Washington, 850 Republican Street, South Building, S420, Seattle, WA 98109, USA (A.S.T. Smith)Department of Neurobiology and Biophysics, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA; Corresponding authors at: Curi Bio Inc., 3000 Western Avenue, Seattle, WA, USA (N.A. Geisse), Department of Physiology and Biophysics, University of Washington, 850 Republican Street, South Building, S420, Seattle, WA 98109, USA (A.S.T. Smith)Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity, scalability, throughput, and ethical considerations further limit the suitability of animal models for preclinical screening. Engineered models have emerged as alternatives for studying NMJ functionality in response to genetic and/or pharmacological challenge. However, these models have faced challenges associated with their poorly scalable creation, sourcing suitable cells, and the extraction of reliable, quantifiable metrics. We present a turnkey iPSC-based model of the NMJ employing channelrhodopsin-2 expression within the motor neuron (MN) population driving muscle contraction in response to blue light. MNs co-cultured with engineered skeletal muscle tissues produced twitch forces of 34.7 ± 22.7 µN in response to blue light, with a response fidelity > 92 %. Histological analysis revealed characteristic punctate acetylcholine receptor staining co-localized with the presynaptic marker synaptic vesicle protein-2. Dose-response studies using botulinum neurotoxin showed loss of function in a dose- and time-dependent manner (EC50 − 0.11 ± 0.015 µg). Variability of the EC50 values between 2 different iPSC differentiations of both cell types and 2 users was less than 2 %. Further testing with the acute neurotoxins acetylcholine mustard and d-tubocurarine validated the biological relevance of the postsynaptic machinery of the model. This model marks a meaningful progression of 3D engineered models of the NMJ, providing engineered tissues at a throughput relevant to potency and screening applications with an abundant iPSC cell source and standardized hardware-software ecosystem allowing technology transfer across laboratories.http://www.sciencedirect.com/science/article/pii/S2666027X25000040Neuromuscular junctionEngineered skeletal muscleEngineered tissue modelsPotency assayHigh-throughput model |
| spellingShingle | Jacob W. Fleming Molly C. McCloskey Kevin Gray David R. Nash Vincent Leung Christos Michas Shawn M. Luttrell Christopher Cavanaugh Julie Mathieu Shawn Mcquire Mark Bothwell David L. Mack Nicholas A. Geisse Alec S.T. Smith An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing Current Research in Toxicology Neuromuscular junction Engineered skeletal muscle Engineered tissue models Potency assay High-throughput model |
| title | An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing |
| title_full | An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing |
| title_fullStr | An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing |
| title_full_unstemmed | An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing |
| title_short | An automated platform for simultaneous, longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing |
| title_sort | automated platform for simultaneous longitudinal analysis of engineered neuromuscular tissues for applications in neurotoxin potency testing |
| topic | Neuromuscular junction Engineered skeletal muscle Engineered tissue models Potency assay High-throughput model |
| url | http://www.sciencedirect.com/science/article/pii/S2666027X25000040 |
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