FIG 2.

Human iPSC motor neurons cultured in microfluidic chambers. (A) Human induced pluripotent stem cell (iPSC)-derived motor neurons expressed the neuron axon marker neurofilament (NF), the motor neuron protein choline acetyltransferase (ChAT), and neuron dendrite microtubule marker microtubule-associated protein 2 (MAP2). (B) A microfluidic chamber, with a penny used for scale. (C) Schematic of a microfluidic chamber showing each component of the chamber. iPSC motor neurons, drawn as magenta cells with blue nuclei, are shown growing within the main channel of the somal side and extending their axons through 450-μm-long microgrooves to the axonal side main channel. A hydrostatic gradient can be applied to one side of the channel to prevent the flow of fluid from one side to the other. Here the gradient is depicted as 180 μl on the somal side and 130 μl on the axonal side. (D) An extended focal imaging (EFI) scan of the somal side of a microfluidic chamber containing iPSC motor neurons stained for NF and nuclei. The location of the microgrooves is indicated by the dashed line. The location of the back of the main channel is indicated by the solid line. The width of the somal main channel is ∼2,000 μm. Neurons clustered at regular intervals from the microgrooves. Neurons in clusters closest to the microgrooves extended their axons across the microgrooves (examples are shown with white arrows). (E) Zooming in on the iPSC motor neurons growing across the microgrooves from the somal side to the axonal side shows that only NF-positive axons cross the microgrooves, while MAP2⁺ dendrites surround the nuclei. Bars = 200 μm for ×100-magnification images and 20 μm for ×600-magnification images.