Machado Carolina Barcellos, Pluchon Perrine, Harley Peter, Rigby Mark, Gonzalez Sabater Victoria, Stevenson Danielle C, Hynes Stephanie, Lowe Andrew, Burrone Juan, Viasnoff Virgile, Lieberam Ivo
Centre for Stem Cells and Regenerative Medicine, King's College London, London SE1 9RT, UK; Centre for Developmental Neurobiology/MRC Centre for Neurodevelopmental Disorders, King's College London, London SE1 1UL, UK.
Centre for Stem Cells and Regenerative Medicine, King's College London, London SE1 9RT, UK; Centre for Developmental Neurobiology/MRC Centre for Neurodevelopmental Disorders, King's College London, London SE1 1UL, UK; Mechanobiology Institute, National University of Singapore, Singapore 117411.
Adv Biosyst. 2019 Jul;3(7). doi: 10.1002/adbi.201800307. Epub 2019 May 8.
Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Studies of neuromuscular junction formation and homeostasis have been largely confined to models. In this study we have merged three powerful tools - pluripotent stem cells, optogenetics and microfabrication - and designed an open microdevice in which motor axons grow from a neural compartment containing embryonic stem cell-derived motor neurons and astrocytes through microchannels to form functional neuromuscular junctions with contractile myofibers in a separate compartment. Optogenetic entrainment of motor neurons in this reductionist neuromuscular circuit enhanced neuromuscular junction formation more than two-fold, mirroring the activity-dependence of synapse development We incorporated an established motor neuron disease model into our system and found that coculture of motor neurons with astrocytes resulted in denervation of the central compartment and diminished myofiber contractions, a phenotype which was rescued by the Receptor Interacting Serine/Threonine Kinase 1 (RIPK1) inhibitor Necrostatin. This coculture system replicates key aspects of nerve-muscle connectivity and represents a rapid and scalable alternative to animal models of neuromuscular function and disease.
运动神经元将轴突从后脑和脊髓投射到肌肉，在那里它们通过在神经肌肉接头处释放神经递质来诱导肌纤维收缩。对神经肌肉接头形成和稳态的研究在很大程度上局限于模型。在本研究中，我们融合了三种强大的工具——多能干细胞、光遗传学和微加工技术，并设计了一种开放式微器件，其中运动轴突从包含胚胎干细胞衍生的运动神经元和星形胶质细胞的神经隔室通过微通道生长，以在单独的隔室中与收缩性肌纤维形成功能性神经肌肉接头。在这个简化的神经肌肉回路中，对运动神经元进行光遗传学诱导，使神经肌肉接头的形成增加了两倍多，这反映了突触发育对活动的依赖性。我们将一个已建立的运动神经元疾病模型纳入我们的系统，发现运动神经元与星形胶质细胞共培养导致中央隔室失神经支配，并减少了肌纤维收缩，这种表型可通过受体相互作用丝氨酸/苏氨酸激酶1（RIPK1）抑制剂Necrostatin挽救。这种共培养系统复制了神经 - 肌肉连接的关键方面，代表了一种快速且可扩展的替代神经肌肉功能和疾病动物模型的方法。
