Applications of Lgr5-Positive Cochlear Progenitors (LCPs) to the Study of Hair Cell Differentiation

Abstract

The mouse cochlea contains approximately 15,000 hair cells. Its dimensions and location, and the small number of hair cells, make mechanistic, developmental and cellular replacement studies difficult. We recently published a protocol to expand and differentiate murine neonatal cochlear progenitor cells into 3D organoids that recapitulate developmental pathways and can generate large numbers of hair cells with intact stereociliary bundles, molecular markers of the native cells and mechanotransduction channel activity, as indicated by FM1-43 uptake. Here, we elaborate on the method and application of these Lgr5-positive cochlear progenitors, termed LCPs, to the study of inner ear development and differentiation. We demonstrate the use of these cells for testing several drug candidates, gene silencing and overexpression, as well as genomic modification using CRISPR/Cas9. We thus establish LCPs as a valuable in vitro tool for the analysis of progenitor cell manipulation and hair cell differentiation.

Keywords: Lgr5; cochlea; differentiation; epigenetics; hair cells; proliferation; supporting cells.

FIGURE 1

Differentiated LCPs resemble native hair cells. (A) Schematic representation of LCP generation and 3D culture in Matrigel. Sensory epithelial cells are harvested from newborn cochleae, dissociated and plated in 3D Matrigel droplets. When harvested from Lgr5-EGFP-IRES-CreER mice, Lgr5-positive organoids are fluorescent during the expansion phase, while when harvested from Atoh1-nGFP mice, Atoh1-positive organoids are fluorescent during differentiation (modified from McLean et al., 2017). (B–E) Expression analysis of whole-culture samples, using qPCR demonstrates Atoh1 (B) and Myo7a (C) expression patterns that resemble in vivo regulation during development. Lgr5 (D) and Ccnd1 (E) expression is reduced as expected from differentiated cells that are exiting the cell cycle. Results are presented as the average fold change ± SEM, between D0, which reflects the first day on which differentiation medium is applied, and days 1–10 (D1–D10); ^∗p-value ≤ 0.05; ^∗∗p-value ≤ 0.01; ^∗∗∗p-value ≤ 0.005 calculated using one-way ANOVA for D0–D10.

FIGURE 2

Identification of new drugs that induce proliferation and differentiation. (A) Flow cytometry analysis of Lgr5-positive cells after 10 days of proliferation. Addition of WS3, WS6 and 5-azacytidine (AZA) had no affect alone, but significantly increased proliferation yield when combined with CHIR, compared to CHIR alone. (B) Bright-field images of expanded LCPs in basal medium and in combination with CHIR and WS3 and WS6. In the presence of CHIR alone, the organoids appeared larger compared to control basal medium and a portion expressed Lgr5. While the organoids appeared smaller when WS3 or WS6 were added to CHIR, the number of Lgr5-positive colonies increased substantially. Scale bar: 500 μm. (C,D) Treatment with 5-azacytidine in combination with CHIR resulted in the highest increase in differentiation compared to CHIR alone, as evaluated using flow-cytometry analysis of Atoh1-positive cells (C) and validated using qPCR expression analysis of Atoh1 and Myo7a (D). (E) Treatment with BIX01294 in combination with LY-CHIR resulted in the highest increase in differentiation compared to LY-CHIR, while treatment with trichostatin A (TSA) in combination with LY-CHIR resulted in a similar degree of differentiation as LY-CHIR. (F) Expression analysis of Atoh1 and Myo7a using qPCR demonstrated increased expression of Atoh1 and decreased Myo7a after treatment with TSA. Results are presented as average fold change ± SEM; ^∗p-value ≤ 0.05; ^∗∗p-value ≤ 0.01; ^∗∗∗∗p-value ≤ 0.005 calculated using one-way ANOVA. All drug concentrations are in μM. Growth factors (GF); CHIR99021 (CH). Addition of CH at the end of a drug name or initial indicates the treatment combination of the drug and CHIR99021.

FIGURE 3

Lentiviral transduction enables ectopic expression and genome editing in LCPs. (A) Schematic representation of the plasmid used for sgRNA viral transduction. hU6, human U6 promoter. hUbC, human ubiquitin C promoter. (B) Representative bright field images of expanded LCPs after transduction with lentivirus. Intensity of the fluorescence from the transgene (green) varied between experiments. Scale bar: 100 μm. (C) LCPs transduction efficiency varied in proportion to the titer of the lentivirus that was delivered to the cells. Relative number of recovered LCPs after transduction was measured using the expression of GFP from the transgene and correlated to the titer delivered per cell. The titer was measured based on the infection of HEK293T cells prior to LCP transduction. Results are presented as mean ± SEM. (D) Flow cytometry analysis demonstrated overlapping expression of tdTomato and sgRNA-GFP virus after Sox2-driven Cre activation in expanded LCPs. tdTomato was measured as a marker for Cas9 expression after Cre activation with tamoxifen, with similar percentage of positive cells in the presence and absence of the virus. Extensive viral transduction is evident by the 98.9% of cells that were GFP positive. 74.4% of the cells were positive for both tdTomato and GFP, indicating high yield of transduction into Cas9 expressing LCPs, enabling subsequent activity of Cas9 in conjunction with the transduced sgRNA. (E) T7 endonuclease assay was used to validate Cas9/gRNA activation at the target site of Notch1 in Neuro2A and LCPs. While Cas9 is expressed in all cells after Cre activation, two distinct cut bands were detected only in the presence of the Notch1 gRNA denoting the specificity of the gRNA in this locus (red asterisk).