Modeling motor axon degeneration using hiPSC-derived caudalized organoids

Abstract

Motor axons in the spinal cord connect to muscles and control body movement. Motor axon degeneration is a crucial pathological feature of neurodegenerative disease such as amyotrophic lateral sclerosis (ALS). Oxidative stress is a major factor of degeneration of motor axons by reactive oxygen species (ROS), which leads to cellular damage including apoptosis. Regeneration of damaged axons is inhibited by environmental factors in injured CNS, which makes a model of human motor axons necessary to study neuronal damage and recovery. To develop a model to mimic motor axon degeneration, I differentiated motor neurons using caudalized spinal organoids derived from human induced pluripotent stem cells (hiPSCs). To induce motor neurons in organoids, I generated caudal neural stem cells that contribute to spinal cord. With SHH-inducer treatment, OLIG2-expressing motor neuron precursors arose followed by subsequent differentiation of motor neurons. Inhibiting NOTCH signals resulted in a great expression of ISL1 motor neuron marker in cell body and robust axon outgrowth from the organoids. To better monitor dynamic branches of motor axons and the behavior of growth cones on the axon tips, I then attached the motor nerve organoids on coverslips. Robust axon growth and growth cones with lamellipodial protrusions were observed, labeled with motor axon and cytoskeleton markers. Furthermore, the extent of motor axon degeneration following oxidative stress by H2O2 treatment was measured in three parameters: axon length, axon continuity, and growth cone morphology. In the H2O2-treated group, decreased axon extension and density, fragmented projections, and changes in growth cone morphology to blunt structure and decreased growth cone area were detected. Taken together, these results suggest that the hiPSC-derived motor axon model could be useful for studying axon degeneration by visualizing individual axon projections and growth cones.