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

Figure 4

Stem cell-derived neurons innervated the centre of CN slices within 14 days. Cochlear nucleus (CN) slices were cultured with (right, a) or without (left, a) stem cell- (SC-) derived neurons for 14 days. After 14 days, both the CN-only (control) cultures and CN-SC cocultures were immunostained for neuronal (NFH, blue) (c, e, g), human neuronal (hNFM, red) (d, f, h), and the synaptic markers synapsin1 and VGLUT 1 (synapsin I: green (e, f) and VGLUT1: green (c, d, g, h)). In the CN-SC slice cocultures (b, red square), SC-derived neurites were observed innervating CN neurons within the centre of the slice (red, e–h). The stem cell-derived neurites were positive for both synapsin I (e, f) and VGLUT1 (g, h), similar to stem cell-derived neurites on the edge of CN slices. CN-only slice control (c, d) showed some VGLUT1 immunoreactivity but no hNFM immunoreactivity (g, h). Scale bar in (b) = 500 μm; scale bar in (h) = 20 μm, applies to all panels (c)–(h).