Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells

Abstract

Death of cochlear hair cells, which do not regenerate, is a cause of hearing loss in a high percentage of the population. Currently, no approach exists to obtain large numbers of cochlear hair cells. Here, using a small-molecule approach, we show significant expansion (>2,000-fold) of cochlear supporting cells expressing and maintaining Lgr5, an epithelial stem cell marker, in response to stimulation of Wnt signaling by a GSK3β inhibitor and transcriptional activation by a histone deacetylase inhibitor. The Lgr5-expressing cells differentiate into hair cells in high yield. From a single mouse cochlea, we obtained over 11,500 hair cells, compared to less than 200 in the absence of induction. The newly generated hair cells have bundles and molecular machinery for transduction, synapse formation, and specialized hair cell activity. Targeting supporting cells capable of proliferation and cochlear hair cell replacement could lead to the discovery of hearing loss treatments.

Figure 1. Diagram of Cochlear Lgr5+ Cell Culture

(A) Lgr5 is expressed in a subset of supporting cells surrounding the cochlear hair cells, including the greater epithelial ridge (GER), inner border cells (IBCs), inner pillar cells (IPCs), and third Deiters cells (3^rd DCs). IHC, inner hair cell; OHC, outer hair cell. (B) Diagram of cochlear epithelial cell isolation and culture as single cells in a 3D system for 10 days.

Figure 2. Characterization of Culture Conditions for Inner Ear Lgr5+ Cells

(A) GFP fluorescence and bright-field images of Lgr5-GFP colonies obtained from inner ear epithelial cells cultured for 10 days in the presence of EGF, bFGF, IGF-1 (EFI); EFI and CHIR, VPA, pVc, 616452 (EFICVP6). Scale bars, 200 μm. (B) Number of live cells and percentage and number of Lgr5-GFP cells from inner ear epithelia cultured for 10 days. n = 3. Error bars represent mean ± SD. (C) Number of live cells and number and percentage of Lgr5-GFP cells from inner ear epithelia cultured for 10 days. Lgr5+ cell number and percentage were highest in cultures containing EGF, bFGF, IGF-1, CHIR, VPA, pVc, and 616452 (EFICVP6) compared to cultures from which individual factors were removed. Each condition was compared to EFICVP6. n = 3. Error bars represent mean ± SD; ***p < 0.001; *p < 0.05; ns, not significant (p > 0.05). (D) GFP fluorescence and bright-field images of cultures as shown in (C). Scale bars, 400 μm.

Figure 3. Lgr5+ Supporting Cells Actively Proliferate and Form Clonal Colonies

(A) Lgr5+ colonies generated in EFICVP6 expressed the supporting cell marker Sox2 in nuclei located in the basal region of the cell. The dashed line indicates the border of a single cell with the apical surface (hollow arrowhead) facing the lumen. Image is a single optical slice. n = 4. Scale bar, 15 μm. (B) EdU staining of an Lgr5-GFP colony. Image is a maximum projection of a z stack. n = 6. Scale bar, 15 μm. (C) A single Lgr5+ cell tracked over 9 days while cultured in the presence of EFICVP6. n = 7. Scale bar, 15 μm. D, day.

Figure 4. Hair Cell Colonies Are Generated from Lgr5+ Colonies

(A) Flow cytometry was performed for quantification of Atoh1+ cells in multiple expansion (blue bars) and differentiation (red bars) conditions in cultures originating from Atoh1-nGFP mice. Inner ear cell culture in growth factors (EFI) or growth factors with VPA and CHIR (EFICVP) did not change the percentage of Atoh1-nGFP cells after 10 days of expansion (shown as day 0 of the experiment; p > 0.05). A combination of LY411575 and CHIR (LYC) was the most effective for differentiation of Atoh1-nGFP cells from EFICVP-expanded cells and was therefore compared to each condition. n = 5. Error bars represent mean ± SD. **p < 0.0001; *p < 0.05; medium without growth factors or drugs (Med). D, day. (B) Lgr5-Cre-tdTomato cells were cultured with EFICVP for 10 days. 4-Hydroxytamoxifen was added to the culture at day 0 of expansion. Expression of Lgr5-GFP and tdTomato is shown. Scale bar, 15 μm; n = 9. (C) Immunocytochemical staining of Lgr5-Cre-tdTomato cells for myosin VIIa following 10 days of culture in LYC. Scale bar, 15 μm; n = 5. (D) EdU and myosin VIIa cells following 10 days of differentiation. Scale bar, 15 μm; n = 5. (E) High-power view of the EdU+ cell in (D). EdU was added at day 1. The arrowheads in (D) and (E) refer to EdU-positive cells.

Figure 5. Colonies of Inner Ear Progenitor Cells Generate Hair Cells In Vitro

(A) The combination of CHIR and LY411575 (LYC) converted progenitor colonies into high-purity populations of myosin VIIa+ cells (left, surface view) with actin-rich protrusions projecting into the lumen (right, section through the colony). n = 11. Scale bar, 15 μm. (B) Myosin VIIa+ cells had CtBP2+ puncta at the basal end of the cell near the membrane (arrowheads). n = 4. Scale bar, 15 μm. (C) Myosin VIIa+/prestin− colonies, indicative of inner hair cells and myosin VIIa+/prestin+ (arrowhead) colonies, indicative of outer hair cells, were distinct. n = 4. Scale bar, 15 μm. (D) Myosin VIIa+/vGlut3+ cells were also produced, indicative of inner hair cells. n = 5. Scale bar, 15 μm. (E) Myosin VIIa+ cells had actin-rich bundles on the apical surface comprising several individual stereocilia. n = 11. Scale bar, 15 μm. (F) Atoh1-nGFP colonies incorporated FM1-43 dye. Image is a single optical slice. n = 6. Scale bar, 15 μm. (G) Key hair cell genes were compared by real-time qPCR at days 0 and 10 of differentiation. Myosin VIIa expression increased while Lgr5 expression decreased between days 0 and 10. Hair cell genes (CaV1.3, Ribeye, Chrna9, Tmc1, Pcdh15, Cdh23, myosin Ic, prestin, oncomodulin, and vGlut3), which include the markers measured by antibody staining (A–D), were strongly upregulated. n ≥5 independent samples per gene. Error bars represent mean ± SEM; p < 0.05 for all genes presented; **p < 0.005. D, day.

Figure 6. Expansion and Differentiation of Progenitor Cells from Adult Mouse, Rhesus Macaque, and Human Inner Ear

(A) Adult mouse Lgr5-GFP cells formed colonies in EFICVP6. n = 4. Scale bar, 15 μm. (B) Upon differentiation with LYC, subsets of colonies contained high purity populations of cells expressing hair cell gene, myosin VIIa (Myo VIIa). n = 4. Scale bar, 15 μm. (C) Other colonies showed a smaller percentage of myosin VIIa+ cells. n = 4. Scale bar, 15 μm. (D) Left: cells isolated from 30-day and 60-day old (p30 and p60) animals formed similar sized colonies (p > 0.05). Average (Avg) is also shown. Middle: colonies from 30- and 60-day-old animals generated a similar number of myosin VIIa+ cells per colony (p > 0.05). Average (Avg) is also shown. Right: myosin VIIa+ cells were represented in similar proportions in colonies from 30- and 60-day-old animals (p > 0.05). Average (Avg) is also shown. p30 n = 3; p60 n = 8. Error bars represent mean ± SD. P, postnatal day. (E) Cells isolated from adult rhesus macaque inner ear epithelia generated clonal colonies in EFICVP6 for 7 days. n = 4. Scale bar, 50 μm. (F) Cells isolated from human inner ear epithelia from a 40-year-old male generated clonal colonies that stained for Sox2 after a 12-day EFICVP6 treatment. n = 1. Scale bar, 15 μm. DIC, differential interference contrast. (G) LYC treatment of human inner ear colonies generated populations of hair cell-like cells (Myo VIIa) with few myosin VIIa− cells (arrow). Colony size was 7.25 ± 1.74 cells. The number of myosin VIIa+ cells per colony was 5.25 ± 2.21. The proportion of myosin VIIa+ cells was 66.7% ± 18.0%. Scale bar, 15 μm.

Figure 7. Increase in Hair Cell Numbers after Treatment of Cochlear Explants with GSK3β and HDAC Inhibitors

(A) Cells between the third Deiters and inner border cells (arrowhead) had increased Lgr5-GFP expression in a cochlea treated with CHIR, VPA, and pVc (CVP). n = 9. Scale bar, 25 μm. (B) Increased numbers of inner hair cells, outer hair cells, and total hair cells (IHCs, OHCs, and total HCs) were observed in treated as compared to control cochleae by myosin VIIa (Myo VIIa) expression. n = 4 each. Error bars represent mean ± SD; ***p < 0.001. (C) Control cochleae had typical Lgr5-GFP expression, one row of inner hair cells, and three rows of outer hair cells. n = 3. Scale bar, 15 μm. (D) A cochlear epithelium had an increased number of hair cell after CVP treatment. n = 4. Scale bar, 15 μm. (E) Treated cochlear explant (left) had extra hair cells. The new hair cells possessed microvillar bundles in an orthogonal view (right). Supporting cells remained between new hair cells (arrowheads) as outlined by phalloidin staining. n = 5. Scale bars, 15 μm. (F) Treated cochlear explant (top) showed supporting cells (Sox2+, arrowheads), hair cells (myosin VIIa+, asterisks), and EdU. Staining for EdU was visible in both supporting cells and hair cells (orthogonal view; bottom). n = 3. Scale bar, 25 μm. (G) A cochlea damaged by gentamicin following a 3-day treatment with CVP had an increased number of Atoh1+ cells in the inner and outer hair cell regions. n = 3 each. Scale bar, 25 μm. (H) Treatment with CVP increased hair cell numbers after gentamicin exposure. Dashed line represents hair cell counts in a healthy mouse cochlea. n = 3 each. Error bars represent mean ± SD. **p < 0.01. (I) EdU incorporation into a gentamicin-treated cochlear explant (top) compared to a gentamicin and CVP-treated cochlear explant (bottom). EdU and CVP were added at 16 hr. n = 4. Scale bars, 15 μm.