Axonal transport is a critical energy
demanding cellular process describing the movement of organelles, vesicles, and macromolecules toward (retrograde) or away from (anterograde) the soma. Neurons, by virtue of their extraordinarily elongated polarization, are especially dependent on long-range cytoskeletal transport, and defective axonal transport has been implicated in the pathogenesis of a myriad age-related neurodegenerative diseases, including amyotrophic lateral sclerosis .
Here, we describe a simple method to fluorescently label mitochondrial cargo, a surrogate for fast axonal transport, in human induced pluripotent stem cell–derived motor neurons . This method allows for sparse labeling of axons to track directionality of movement and can be adapted to assess not only the cell autonomous effects of a genetic mutation on axonal transport but also the cell non-autonomous effects, through the use of conditioned medium and/or co-culture systems.
We first describe a method for the generation of spinal (lower) motor neurons (MNs) from human induced pluripotent stem cells (iPSCs
) using an established protocol  with minor modifications, yielding a highly enriched and electrophysiologically active neuronal culture, devoid of glia, with circa 60% of cells being positive for islet-1 and islet-2 homeobox MN markers 1 week post platedown . We then describe a method for sparse labeling of neuronal mitochondria , allowing for the quantification of various parameters such as pausing, and speed, but also directionality of movement. It is thus advantageous over dye-based methods, which label all cells, such as MitoTracker .