Centre for Gene Therapy & Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, London SE1 9RT, United Kingdom.
Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London SE1 1UL, United Kingdom.
Biofabrication. 2023 Sep 5;15(4):045020. doi: 10.1088/1758-5090/acf39e.
Many devastating neuromuscular diseases currently lack effective treatments. This is in part due to a lack of drug discovery platforms capable of assessing complex human neuromuscular disease phenotypes in a scalable manner. A major obstacle has been generating scaffolds to stabilise mature contractile myofibers in a multi-well assay format amenable to high content image (HCI) analysis. This study describes the development of a scalable human induced pluripotent stem cell (iPSC)-neuromuscular disease model, whereby suspended elastomer nanofibers support long-term stability, alignment, maturation, and repeated contractions of iPSC-myofibers, innervated by iPSC-motor neurons in 96-well assay plates. In this platform, optogenetic stimulation of the motor neurons elicits robust myofiber-contractions, providing a functional readout of neuromuscular transmission. Additionally, HCI analysis provides rapid and automated quantification of axonal outgrowth, myofiber morphology, and neuromuscular synapse number and morphology. By incorporating amyotrophic lateral sclerosis (ALS)-related TDP-43mutant motor neurons and CRISPR-corrected controls, key neuromuscular disease phenotypes are recapitulated, including weaker myofiber contractions, reduced axonal outgrowth, and reduced number of neuromuscular synapses. Treatment with a candidate ALS drug, the receptor-interacting protein kinase-1 (RIPK1)-inhibitor necrostatin-1, rescues these phenotypes in a dose-dependent manner, highlighting the potential of this platform to screen novel treatments for neuromuscular diseases.
目前，许多破坏性的神经肌肉疾病缺乏有效的治疗方法。这在一定程度上是由于缺乏能够以可扩展的方式评估复杂的人类神经肌肉疾病表型的药物发现平台。一个主要的障碍是生成支架以稳定多孔板中成熟的收缩肌纤维，使其适用于高内涵图像（HCI）分析。本研究描述了一种可扩展的人类诱导多能干细胞（iPSC）-神经肌肉疾病模型的开发，其中悬浮弹性体纳米纤维支持 iPSC 肌纤维的长期稳定性、对齐、成熟和重复收缩，由 iPSC 运动神经元在 96 孔板中支配。在该平台中，运动神经元的光遗传学刺激引发了强大的肌纤维收缩，提供了神经肌肉传递的功能读数。此外，HCI 分析提供了轴突生长、肌纤维形态以及神经肌肉突触数量和形态的快速自动定量。通过整合肌萎缩侧索硬化症（ALS）相关的 TDP-43 突变运动神经元和 CRISPR 校正对照，重现了关键的神经肌肉疾病表型，包括肌纤维收缩减弱、轴突生长减少和神经肌肉突触数量减少。用候选 ALS 药物 RIPK1 抑制剂 necrostatin-1 治疗，以剂量依赖的方式挽救了这些表型，突出了该平台筛选神经肌肉疾病新疗法的潜力。
