Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices

Figure 1

Stem cell-derived neurites innervated HCs within 12 days of coculture. Cocultures and explant controls were set up as follows: whole explant where HCs and ANs are kept intact (a), HC-only explant where ANs are dissected from HC rows (b), HC-AN coculture where HC rows and ANs are separated and cocultured (c), HC-SC coculture where HC rows are cocultured with stem cell-derived neurons (d). Whole explant controls were cultured for 1 day in vitro (DIV), whereas HC-only controls, HC-AN cocultures, and HC-SC cocultures were maintained for 12-14 DIV. Control cultures and cocultures were immunostained with antibodies for neurofilament for neurons (blue, (e)–(l)), parvalbumin for hair cells (magenta, (e)–(l)), and synaptic markers synapsin I (green, (e)–(h)) and VGLUT1 (green, (i)–(l)). Synapsin I was observed at the sites of innervation in whole explant control (e), HC-AN coculture (g), and HC-SC coculture (h), whereas it was absent in HC-only control (f). Similarly, VGLUT1 was observed at some sites of innervation in whole explant control (i), HC-AN coculture (k), and HC-SC coculture (l), whereas it was absent in HC-only control (j). Whole explant controls are cultured for 1 DIV and the explant maintains its ultrastructure. HCs in whole explant controls always appear smaller compared to cocultures and HC-only controls, since there is more loss of supporting cells and ultrastructure after weeks in vitro. (e)–(l) were all taken under oil immersion at the same magnification. (m, m, m) reconstructions illustrate HC-AN innervation from three different directions (HC: magenta, parvalbumin; AN: blue, neurofilament; synaptic puncta: green, synapsin 1). (n) The coordinates are reconstructed to illustrate SC-HC innervation and higher magnification images of SC-HC synapses is shown from two directions in (o) and (). Arrowheads in (m) through () inclusive denote putative puncta between hair cells and either AN (m, m, m) or SC (n, o, o). Scale bars (l) = 20 μm, applies to (e)–(l). Scale bars in (m), (m) (applies to (m)), (n), and (o) (applies to (o)) = 10 μm.