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

Figure 6

Stem cell-derived neurons and early postnatal ANs innervate both inner ear HCs and CN slice within 14 days. Both AN explants (a) and hPSC-derived neurons (SC, e) were cocultured with both HC-only explant and CN slice for 14 days. HC-AN-CN cocultures were stained for neurofilament (pan-NFH, blue), hair cells (parvalbumin, magenta), and VGLUT1 (green). Numerous processes of ANs extended towards both CN slice and HC explant (b). At higher magnification, ANs were observed to innervate the CN slice (c, arrows) and the HCs (d, arrows) with VGLUT1-positive neurites. HC-SC-CN cocultures were stained for neurofilament (pan-NFH, blue), hair cells (parvalbumin, magenta), human neurofilament (hNFM, red), and VGLUT1 (green). Fasciculating bundles of SC-derived neurons extended towards both CN slice and HC explant (f). At higher magnification, SC-derived neurites were observed to innervate the CN slice (g, arrows) and HCs (h, arrows) with VGLUT1-positive neurites. Scale bar in (b) and (f) = 200 μm; scale bar in (d) and (h) = 20 μm, applies to panels (c) and (g), respectively.

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