Intrinsic regenerative potential of murine cochlear supporting cells

Abstract

The lack of cochlear regenerative potential is the main cause for the permanence of hearing loss. Albeit quiescent in vivo, dissociated non-sensory cells from the neonatal cochlea proliferate and show ability to generate hair cell-like cells in vitro. Only a few non-sensory cell-derived colonies, however, give rise to hair cell-like cells, suggesting that sensory progenitor cells are a subpopulation of proliferating non-sensory cells. Here we purify from the neonatal mouse cochlea four different non-sensory cell populations by fluorescence-activated cell sorting (FACS). All four populations displayed proliferative potential, but only lesser epithelial ridge and supporting cells robustly gave rise to hair cell marker-positive cells. These results suggest that cochlear supporting cells and cells of the lesser epithelial ridge show robust potential to de-differentiate into prosensory cells that proliferate and undergo differentiation in similar fashion to native prosensory cells of the developing inner ear.

Figure 1. Dissection of the neonatal organ of Corti into 5 distinct cell populations.

a, Immunohistological staining with CD271, CD326, and CD146 of middle-turn cryosections of fixed P3 Math1-nGFP cochlea. Primary antibodies were visualized with Cy5-conjugated secondary antibodies (shown in red), the hair cells are nGFP-positive (shown in green). b, Fragment of a typical P3 Math1-nGFP cochlear duct used for cell dissociation and FACS analysis. The lesser (LER) and greater epithelial ridges (GER) are indicated; hair cells are nGFP-positive. c, FACS regimen. 2.3 ±0.2% of the total viable cells were GFP⁺ (green box). c', 2.6 ±0.3% of the GFP⁻ cells were CD271^High (red box), and c”, 7.2 ±0.7% of the GFP⁻/CD271^Low cells were CD326⁺/CD146^Low (light blue box), 21.2 ±2.0% were CD326⁺/CD146^High (dark blue box), and 63.2 ±2.9% were CD326⁻ (yellow box) (n = 9). d, Shown is a heat map representing similarity and divergence in the gene expression levels of the 1,000 most divergent genes among 13 array data sets obtained with independent sorted cochlear cell populations.

Figure 2. Selected genes and their maximum differential expression within the 5 groups of FACS-sorted cells.

Markers known to be specific for hair cells are indicated with light green, markers known to be specific for other cell types or cochlear regions are indicated as follows: monocytes/macrophages in light red, sub-basilar membrane in light yellow, LER and supporting cells in light blue, and GER in purple. In all cases, the maximum expression indicated by x-fold enrichment over the average of all other populations matched the distinct population, which is indicated with saturated colors.

Figure 3. Schematic drawing to visualize the cellular identity of the five FACS-sorted cell populations.

Based on gene array analysis and comparison of marker gene expression levels, we propose a model in which the GFP⁺ cells (shown in green) represent the inner and outer hair cells (IHC, OHC), GFP⁻/CD271^High (shown in red) represent monocytes/macrophages, GFP⁻/CD271^Low/CD326⁺/CD146^Low (light blue) represent LER and organ of Corti supporting cells, GFP⁻/CD271^Low/CD326⁺/CD146^High (dark blue) represent GER cells, and GFP⁻/CD271^Low/CD326⁻ (yellow) represent cells located below the basilar membrane.

Figure 4. Colony formation capacity of cochlear non-sensory cell populations.

a, Sorted cells were cultured with a density of 3 cells/µl in non-adherent conditions and spheres were counted after 5 days (n = 6). _* indicates p<0.05. b, EdU incorporation into spheres after 5 days in culture (n = 3–4). c, 3-day-old colonies on mitotically inactivated chicken utricle stroma-derived feeder cells, immunolabeled with antibodies to E-cadherin (green), visualized EdU incorporation (red) and nuclear DNA staining with 4′,6-diamidino-2-phenylindole (DAPI, blue). d, Number of colonies grown from 5,000 individual cells of each of the different non-sensory populations after 1, 3, 7, and 14 days of culture (n = 3–12). e, Colony cell numbers at the time points indicated. Color codes are indicated in Figs. 1 and 3.

Figure 5. Hair cell-like cells.

a, Upregulation of nGFP in FACS-sorted non-sensory cell populations (n = 3–9). Color codes are indicated in Figs. 1 and 2. _*** indicates p<0.0001. b, Expression of multiple hair cell markers in a subset of nGFP-positive hair cell-like cells. c, Merge of a light microscopic image with a fluorescent image showing 4 nGFP-positive cells in a colony derived from GFP^-/CD271^Low/CD326⁺/CD146^Low cells. d, The same colony as in (c), visualized with SEM. e,f,g, Apical protrusions emanating from the cells labeled in (d) with e, f, and g. The arrow points to the hair bundle-like protrusion in f. In g, the arrow points to a thicker protrusion, a presumptive kinocilium-like structure. h, Higher magnification image of a hair bundle-like protrusion.

Figure 6. Hair cell-like cells originate from proliferating and Sox2-positive cells.

a, EdU incorporation into hair cell-like cells after 10 days in vitro. b, Number of Sox2-expressing cells in FACS-sorted non-sensory cell populations (n = 3–6). Color codes are indicated in Figs. 1 and 2. _* and _*** indicate p<0.05 and p<0.001, respectively. c, Co-labeling for nGFP and Sox2 in colonies derived from GFP^-/CD271^Low/CD326⁺/CD146^Low cells at 7 and 14 days in vitro. d, Fraction of nGFP expression of Sox2-positive cells in GFP⁻/CD271^Low/CD326⁺/CD146^Low colonies at 3, 7, 10, and 14 days in vitro, indicated in percent.