Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction.
Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction.
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DOI：
10.3791/63759
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发表时间：
2022-04-14
期刊：
影响因子：
--
通讯作者：
Vunjak-Novakovic G
中科院分区：
文献类型：
--
作者：
Liberman M;Chavez M;Nash TR;Vila OF;Vunjak-Novakovic G
Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs) that are differentiated into motoneurons, we generate neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Here, we describe an automated, unbiased method to characterize NMJ function by combining tissue engineering, optogenetics, microfabrication and video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates the human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform's ability to show functional improvement of neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function. We describe a reproducible, automated and unbiased imaging system to characterize neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.
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影响因子：
14
作者：
Vila OF;Chavez M;Ma SP;Yeager K;Zholudeva LV;Colón-Mercado JM;Qu Y;Nash TR;Lai C;Feliciano CM;Carter M;Kamm RD;Judge LM;Conklin BR;Ward ME;McDevitt TC;Vunjak-Novakovic G
通讯作者：
Vunjak-Novakovic G
影响因子：
13.6
作者：
Osaki T;Uzel SGM;Kamm RD
通讯作者：
Kamm RD
影响因子：
46.9
作者：
Maury, Yves;Come, Julien;Nedelec, Stephane
通讯作者：
Nedelec, Stephane
影响因子：
5.5
作者：
Powell, CA;Smiley, BL;Vandenburgh, HH
通讯作者：
Vandenburgh, HH
影响因子：
8.8
作者：
Jones RA;Harrison C;Eaton SL;Llavero Hurtado M;Graham LC;Alkhammash L;Oladiran OA;Gale A;Lamont DJ;Simpson H;Simmen MW;Soeller C;Wishart TM;Gillingwater TH
通讯作者：
Gillingwater TH