Expansion of Lgr5+ cells and hair cells can be achieved in situ in the cochlea

Hair cells can be generated from cells of the adult mouse and primate cochlea

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.

The limited ability of cochlear cells to regenerate is unusual compared to other epithelia, but, despite different capacities for regeneration, Lgr5+ cochlear supporting cells have several characteristics in common with Lgr5+ cells from the intestine. Activation of both the Wnt and Notch signaling pathways has been independently demonstrated to be required for the establishment of Lgr5+ cells in the cochlea during development (). Wnt signaling is required for hair cell differentiation (), which is increased by concurrent inhibition of Notch (). This is strikingly similar to intestinal epithelia, where Wnt and Notch signaling are required for stem cell expansion, and lack of Notch signaling with active Wnt leads to differentiation to mature epithelial cell types (). Some expansion of Lgr5+ cells from the cochlea could be achieved by propagation as cochlear spheres (), but heterogeneous cell populations are obtained and the yield of hair cells upon differentiation is low. Changes in gene expression of progenitors results in a loss of sphere-forming capacity in the adult mouse cochlea (). Studies on progenitor cells have thus been limited by the small number of Lgr5+ cells, which comprise a subset of the few cells in the cochlear epithelium, and inefficient protocols for their expansion. Here, by employing a cocktail of drugs and growth factors to modulate multiple pathways, we demonstrate mechanisms to clonally expand Lgr5+ cells from both newborn and normally unresponsive, adult tissue and to efficiently differentiate these colonies into nearly pure populations of hair cells in high yield. We show, furthermore, that the same drug cocktail drives the generation of hair cells and supporting cells in both healthy and damaged neonatal organ of Corti.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

Regulation of cell fate in the sensory epithelia of the inner ear.

Lgr5, an epithelial cell protein first discovered as a marker for intestinal stem cells and then shown to be critical for their function (), was recently shown to be expressed in cochlear supporting cells that surround the hair cells (). These Lgr5-expressing cells could be induced to undergo limited proliferation when stimulated by Wnt in the normally post-mitotic cochlear sensory epithelium (). Indeed, consistent with a progenitor role, supporting cells that expressed Lgr5 gave rise to new Lgr5+ cells by propagation and to hair cells that were Lgr5−, whereas supporting cells that did not express this receptor did not give rise to hair cells (). Consistent with its role in upstream regulation of the transcription factor Atoh1 (), which is a master regulator of hair cell differentiation (), upregulation of Wnt also increased hair cell differentiation. This combination of the ability to divide in response to Wnt signaling and the potency to differentiate into hair cells suggested that Lgr5+ cells were acting as progenitor cells of the cochlear epithelium (). Indeed, in the newborn cochlea, Lgr5+ cells showed the capacity to regenerate spontaneously after damage (). These data supported a role for the Lgr5+ cells as cochlear progenitor cells, but spontaneous regeneration capacity was lost after the first postnatal week, and, indeed, no cell division or cell replacement occurs in the sensory epithelium of the adult cochlea (). Supporting cell transdifferentiation can lead to some hair cell replacement (), but regenerating a functional cochlea would require both stimulating these cells to divide and differentiating them to hair cells, an approach that would benefit greatly from the isolation of Lgr5+ progenitor cells to develop protocols for their expansion and differentiation to hair cells.

Regulation of cell fate in the sensory epithelia of the inner ear.

Hearing impairment is a major health challenge estimated by the World Health Organization to affect over 5% of the world’s population (360 million people, including 32 million children). The sensory hair cells that detect sound and transmit their signal to the brain via the auditory nerve are susceptible to damage. After loss, the hair cells are never replaced (), and thus, the number of cells, which is low (15,000 per ear in humans and 3,000 in mice) at the start of postnatal life, only decreases with age, and the absence of cell replacement leads to a high prevalence of acquired forms of deafness. Indeed, hair cell and auditory nerve damage, typically caused by noise exposure, ototoxic drugs, viral or bacterial infections, and aging, accounts for more than 80% of all cases of hearing loss ().

Hearing disorders in the population: first phase findings of the MRC National Study of Hearing.

Hair cell regeneration was achieved in cochlear explants treated with gentamicin, which causes hair cell death in the basal portion of the organ of Corti, where transduction channels are active in the neonate ( Figures 7 G–7I). Gentamicin caused extensive hair cell death in the base of the cochlea, but after 3 days of treatment with CVP, new Atoh1-nGFP hair cells appeared ( Figures 7 G and 7H). The number of hair cells was close to normal after treatment and 7-fold greater than that observed for control-treated cochlea ( Figure 7 H). Supporting cells were EdU+, indicating that supporting cell division was a part of the mechanism for hair cell replacement ( Figure 7 I). Thus, the treatment with CVP that expanded Lgr5+ cells from the cochlea after their isolation and placement into a 3D culture was also able to expand supporting cells in situ and force the generation of new hair cells.

The drugs that were critical for expansion and differentiation of Lgr5+ cells (HDAC inhibitors, GSK3β inhibitors, and γ-secretase inhibitors) have all been used clinically for other indications and could potentially be candidates for clinical development. To investigate their effects in a more clinically relevant tissue, we applied the drugs to cochlear explants. Supporting cells play a key role in cochlear function and homeostasis. Therefore, we treated intact and hair cell-damaged explants from postnatal day 2 mice with small molecules from the expansion conditions (CVP) rather than the differentiation conditions in an attempt to maintain a supporting cell population and permit spontaneous differentiation ( Figure S4 ). We performed these cultures without growth factors in the presence of the surrounding tissue. These tests resulted in extensive proliferation of supporting cells and differentiation to hair cells. Whereas Lgr5-GFP was absent in a control cochlea in the region between the third Deiters cell and inner pillar cells (i.e., outer pillar cells, first and second Deiters cells), treatment with CVP for 3 days caused upregulation of Lgr5-GFP in all supporting cells ( Figures 7 A, 7C, and 7D ). There was a highly significant (p < 0.001) ∼2-fold increase in myosin VIIa+ inner and outer hair cells after 3 days of drug treatment ( Figures 7 B and 7D) as compared to control cochlea ( Figure 7 C). Addition of 616452 to VPA and CHIR did not increase the generation of hair cells. The new hair cells had morphology similar to the intact cochlea, with phalloidin+ stereociliary bundles and hair cells that were separated by intact supporting cells, suggesting that the treatment caused proliferation and subsequent differentiation ( Figure 7 E). Supporting cells had incorporated EdU, suggesting that they had divided ( Figure 7 F), and some of the hair cells, identified as “new” based on their expression of Sox2 (), had transdifferentiated from supporting cells that had taken up EdU, similar to the division of supporting cells stimulated by Wnt signaling ().

(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.

(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.

(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.

(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.

(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.

(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.

(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.

We further tested the conditions using one sample of healthy human inner ear tissue isolated from a 40-year-old male patient undergoing a labyrinthectomy to access a tumor on the brain. The inner ear tissue was microdissected to remove bone, debris, and nerve tissue. The tissue was then treated identically to the mouse tissue to isolate single cells for culture. The single cells formed clonal colonies after 12 days under EFICVP6 conditions, although expansion was not as robust as that seen for neonatal cells ( Figure 6 F). The colonies stained for Sox2, a known marker of inner ear progenitor cells ( Figure 6 F). After 12 days of expansion, the cultures were treated with LY411575 and CHIR for 10 days to differentiate the colonies. The colonies stained positively for the hair cell marker myosin VIIa ( Figure 6 G), suggesting that sensory epithelium from adult human inner ear can also give rise to hair cell progenitors.

We next tested whether the expansion and differentiation condition could be applied to non-human primates. Inner ear epithelial cells were isolated from adult rhesus macaques and cultured with EFICVP6. These preliminary results indicated that the cells formed clonal colonies ( Figure 6 E). However, differentiation to hair cells was not achieved due to repeated contamination likely caused by non-sterile conditions encountered during the temporal bone isolation.

Previous studies have documented a decline in proliferative capacity and stem cell properties in the inner ear after the early postnatal period (). Since the drug combination applied here enhanced proliferation of neonatal cells compared to previous techniques, we next tested whether the compounds could be used to expand and differentiate otherwise quiescent adult cells into hair cells. Given the low numbers of Lgr5+ cells available from the adult mouse cochlea, we applied the cocktail of agents that we established for passaging neonatal cells, EFICVP6, to generate clonal colonies of adult cells positive for Lgr5 ( Figure 6 A). After the expansion, the cultures were treated with LY411575 and CHIR to differentiate the Lgr5+ cells. The colonies initiated expression of myosin VIIa only after differentiation ( Figures 6 B and 6C), indicating that adult Lgr5+ cells expanded and differentiated into cells that expressed hair cell markers. Myosin VIIa expression varied between colonies, with some colonies expressing the protein more robustly ( Figures 6 B and 6C). Cells isolated from mice at postnatal day 30 (4.67 ± 0.28 cells) and postnatal day 60 (6.75 ± 1.53 cells) formed similar sized colonies (average colony size across both ages of 6.18 ± 0.13 cells; Figure 6 D). Colonies from postnatal day 30 (4.33 ± 0.28 cells) and postnatal day 60 (3.88 ± 1.48 cells) generated a similar number of myosin VIIa+ cells per colony (average across ages of 4.00 ± 1.06 myosin VIIa+ cells per colony; Figure 6 D). The myosin VIIa+ cells were obtained in colonies generated from postnatal day 30 (93.3% ± 5.8% cells) and postnatal day 60 (59.6% ± 13.7% cells); (average across ages of 68.82% ± 0. 28% myosin VIIa cells; Figure 6 D). No significant differences were seen across ages.

(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.

(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.

(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.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

qPCR studies to determine gene expression profiles before and after differentiation using the optimal conditions for Atoh1-nGFP quantification (EFICVP/LYC) revealed that myosin VIIa was upregulated between day 0 and day 10 after expansion, while Lgr5 expression decreased ( Figure 5 G). The differentiated cells expressed the tip-link genes cadherin23 and protocadherin15 (). The transduction adaptation component myosin Ic (), the synapse-associated calcium channel CaV1.3, and the ribbon synapse component ribeye were also upregulated (). The α9 acetylcholine receptor Chrna9 and the transduction channel component transmembrane channel 1 (Tmc1) () both had increased expression. Prestin, the motor protein, and oncomodulin, a calcium modulator, both of which are found in outer hair cells, as well as the inner hair cell calcium modulator vesicular glutamate transporter 3, also showed increased expression. Our differentiation conditions thus generated inner and outer cochlear hair cell types that contained components of synaptic specializations, the transduction apparatus receptors, and ion channels of hair cells that were identified by both staining for specific markers and real-time qPCR.

TMC1 and TMC2 are components of the mechanotransduction channel in hair cells of the mammalian inner ear.

Further analyses showed that differentiating expanded supporting cells with LYC resulted in large colonies that were almost uniformly positive for myosin VIIa and contained actin-rich protrusions within the inner lumen ( Figure 5 A; Movie S1 ). Closer inspection of hair cell colonies revealed that myosin VIIa+ cells contained CtBP2+ ribbon synapse-like puncta in the basal region, where ribbon synapses are found in native hair cells ( Figure 5 B, arrowheads). Hair cell colonies were either negative ( Figure 5 C, left) or positive ( Figure 5 C, right, arrowhead) for prestin, a motor protein located in the membrane of outer hair cells, identifying a subset of the differentiated cells as outer hair cells (). Colonies of new hair cells also expressed vesicular glutamate transporter3 (vGlut3), an inner hair cell marker (), which was only found in colonies that did not express prestin ( Figure 5 D). The staining also revealed that the actin-rich protrusions comprised several individual stereocilia on the cells’ apical surface ( Figure 5 E). The new colonies of hair cells rapidly accumulated the dye FM1-43, which enters hair cells through active transduction channels () ( Figure 5 F).

(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.

(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.

(B) Myosin VIIa+ cells had CtBP2+ puncta at the basal end of the cell near the membrane (arrowheads). n = 4. Scale bar, 15 μm.

(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.

To determine whether Lgr5+ cells were the source of hair cells, we crossed Lgr5-Cre-ER mice with Rosa26-flox-tdTomato mice and followed tdTomato expression in any new cells. When 4-hydroxytamoxifen was added to the culture at day 0 to activate the Lgr5-Cre, Lgr5+ cells formed colonies in the cocktail of growth factors and drugs, in which all cells within a colony were positive for tdTomato ( Figure 4 B). After differentiation with LY411575 and CHIR, hair cells (marked by hair cell-specific myosin VIIa ()) were also tdTomato-positive ( Figure 4 C), indicating that the myosin VIIa+ cells were derived from Lgr5-expressing cells. Colonies that were tdTomato-negative did not produce myosin VIIa+ cells, indicating that Lgr5-negative cells do not generate hair cells, as also demonstrated previously (). Cultures stained for EdU given 1 day after LY411575 and CHIR administration showed that cells expressing hair cell genes did not proliferate in the differentiation conditions ( Figure 4 D). These data thus suggest that hair cells did not proliferate during colony formation or differentiation.

We quantified hair cell production using flow cytometry to count Atoh1-nGFP cells after expansion (day 0) and differentiation (day 10) phases of cultures originating from isolated epithelial cells from a single Atoh1-nGFP mouse cochlea ( Figure 4 A). We found that the addition of 616452 caused leakage of the enhancer-mediated Atoh1-nGFP (which was confirmed through co-staining with myosin VIIa during expansion). It was therefore removed from hair cell quantification assays using the Atoh1-nGFP reporter. Treatment with growth factors alone or with CHIR, VPA, and pVc produced few Atoh1-nGFP+ cells (0.72% with the growth factors and 1.108% in the presence of growth factors with drugs, p > 0.05) at the end of the 10-day expansion. Our results suggest that CHIR leads to greater differentiation than LY411575 (5.6-fold vs. 2.6-fold) when compared to removal of growth factors alone, and combining LY411575 and CHIR (LYC) further increases hair cell yield ( Figure 4 A). Expansion using EFICVP, followed by differentiation with LYC, produced the maximum number and purity of hair cells when multiple differentiation conditions were analyzed, suggesting that inclusion of growth factors in culture during differentiation reduces hair cell formation ( Figure 4 A). Expansion with EFICVP and differentiation with LYC resulted in ∼26% of all cells in culture expressing Atoh1-nGFP, which corresponds to ∼11,600 Atoh1-nGFP+ cells per cochlea compared to the 0.72% and average of 173 cells generated per cochlea when differentiating with LYC after expanding with EFI (p < 0.0001). Our optimal conditions generated a highly reproducible hair cell yield (26.3% ± 2.5%, n = 5 independent experiments; Figure 4 A) that was 67-fold greater than previous methods using only growth factors to culture supporting cells () and represented a 580-fold increase of viable hair cells during culture (20 Atoh1-nGFP cells).

(E) High-power view of the EdU+ cell in (D). EdU was added at day 1.

(C) Immunocytochemical staining of Lgr5-Cre-tdTomato cells for myosin VIIa following 10 days of culture in LYC. Scale bar, 15 μm; n = 5.

(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.

(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.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

Although Notch inhibition and β-catenin expression were separately shown to promote hair cell differentiation in vitro from inner ear progenitor cells at a higher rate than removal of growth factors, the number of hair cells produced remained low due to the inability to expand progenitor cells and sufficiently convert them into hair cells (). To test whether the expanded Lgr5+ cells were able to generate higher yields of hair cells after simultaneous Notch inhibition and Wnt activation, we treated Lgr5-GFP or Atoh1-nGFP cells, expanded by the above procedures, with LY411575, a γ-secretase inhibitor previously used to differentiate inner ear progenitor cells (), and CHIR, the GSK3β inhibitor. Following 10 days of differentiation, the expression of Lgr5 was diminished ( Figure S3 A), suggesting that they were differentiated cells. Atoh1-nGFP cells were rare during the expansion phase of the culture but increased in prevalence during the differentiation step of the protocol ( Figure S3 B). This suggests that the combination of LY411575 and CHIR induced the differentiation of the expanded Lgr5+ cells and transformed the colonies into high-purity populations of Atoh1-nGFP hair cells ( Figure S3 B).

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

Consistent with previous reports (), Lgr5+ cells expressed the supporting cell marker Sox2, and a single optical slice revealed that Lgr5-GFP colonies cultured in EFICVP6 comprised pure populations of Sox2-expressing supporting cells with nuclear localization in the basal portion of the cell ( Figure 3 A). Exposure of EFICVP6 cultures to ethynyldeoxyuridine (EdU) revealed that Lgr5-GFP colonies were actively proliferating ( Figure 3 B). Tracking of single Lgr5-GFP cells over time revealed that Lgr5-GFP colonies were formed clonally ( Figure 3 C).

(C) A single Lgr5+ cell tracked over 9 days while cultured in the presence of EFICVP6. n = 7. Scale bar, 15 μm. D, day.

(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.

We further examined the potential function of individual factors. The effects of CHIR in increasing Lgr5-GFP cell number and percentage could be partially replicated with Wnt3a in combination with R-spondin1 ( Figures S2 D–S2G), suggesting a role of CHIR in activating the Wnt pathway. Using an Atoh1-nGFP mouse line, we found that VPA suppressed spontaneous differentiation of supporting cells into hair cells ( Figure S2 H), which is consistent with the role of VPA in maintaining Notch activation in intestinal stem cells ().

To examine the relative importance of individual factors in our culture system (without passaging), we separately removed each factor from the medium and quantified cell proliferation and Lgr5 expression of inner ear epithelial cells following 10 days of culture ( Figures 2 C and 2D). Removal of CHIR or bFGF had the greatest effect on Lgr5-GFP cell number and percentage, while removal of CHIR had the greatest effect on Lgr5 expression. Removing EGF or 616452 caused a significant reduction in Lgr5-GFP cell number, while removing VPA or pVc greatly reduced Lgr5 expression. The presence of IGF-1 had a marginal beneficial effect on Lgr5 cell number and percentage. The treatment with the combined agents (EFICVP6) yielded the highest number of total cells, Lgr5+ cells, and percentage of Lgr5+ cells following 10 days of culture. These results suggest that bFGF and CHIR were most critical to Lgr5+ cell culture, while the other factors promoted maximal Lgr5 cell growth and expression. Similar results were obtained by direct visualization of GFP expression and cell growth ( Figure 2 D).

Without Wnt stimulation (EFI alone), Lgr5-GFP expression diminished, and, unlike the intestinal colonies, the number of Lgr5-GFP cells diminished after passage. Although the Lgr5-GFP cell numbers decreased after passage, further Wnt stimulation allowed Lgr5-GFP cells to be maintained in culture for extended periods of time (out to 45 days, the longest time point tested). We reasoned that other factors were needed to optimize the culture of Lgr5-GFP cells and thus performed screening to identify additional factors to enable the colonies’ prolonged culture and passaging. Addition of 2-phospho-L-ascorbic acid (pVc or P), a stable form of vitamin C, increased Lgr5+ cell expansion by an additional 2- to 3-fold ( Figures S2 A and 2B). Addition of a transforming growth factor β (TGF-β) receptor (Alk5) inhibitor, 616452 (or 6), also increased cell expansion (by 2- to 3-fold) and was required for the passage of colonies ( Figures 2 C, 2D, and S2 C). Collectively, the addition of small molecules (CVP6), compared to growth factors alone, increased Lgr5+ cell numbers by >2,000-fold with high consistency ( Figure 2 B).

(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).

(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.

(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.

Lgr5+ cells represent a subset of supporting cells within the cochlear epithelium ( Figure 1 A). Using an Lgr5-GFP mouse line, we tested the activation or inhibition of multiple pathways to expand single Lgr5+ supporting cells isolated from the neonatal cochlea in a Matrigel-based 3D culture system ( Figure 1 B). Initially, we aimed to adapt conditions we previously developed for culture of intestinal stem cells to the inner ear progenitor cells (). We added the glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 (CHIR or C) and the histone deacetylase (HDAC) inhibitor valproic acid (VPA or V) to the growth factor cocktail that was previously used for the culture of inner ear spheres, which includes epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or F), and insulin like growth factor 1 (IGF-1 or I) (). In parallel, we tested conditions used for the culture of intestinal stem cells, which includes EGF (E), R-Spondin1 (R), and Noggin (N) (). The addition of CHIR and VPA to EFI (EGF, bFGF, IGF-1) significantly increased the total number and percentage of Lgr5-GFP cells in culture, which outperformed the conditions previously used to expand intestinal Lgr5-GFP cells (ENR) ( Figure S1 A). The addition of CV to previously used factors led to the formation of large GFP+ colonies ( Figure S1 B), consistent with our previous finding in the intestine ().

(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.

Discussion