The drastic disruption of GJP was observed in in vitro disease model of GJB2 mutation

Mutation in GJB2 (CX26) is the most frequent cause of hereditary deafness worldwide

Mutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP-forming cells has not been reported. Here, we detail the development of a novel strategy to differentiate induced pluripotent stem cells into functional CX26-GJP-forming cells that exhibit spontaneous ATP- and hemichannel-mediated Ca 2+ transients typical of the developing cochlea. Furthermore, these cells from CX26-deficient mice recapitulated the drastic disruption of GJPs, the primary pathology of GJB2-related hearing loss. These in vitro models should be useful for establishing inner-ear cell therapies and drug screening that target GJB2-related hearing loss.

Here we developed an efficient induction method for the differentiation of mouse iPSCs into CX26-expressing cells that, through subsequent adherent culture on cochlear feeder cells, form CX26-GJPs typical of cochlear tissue.

Induced pluripotent stem cells (iPSCs) can be produced by the reprogramming of somatic cells, and are capable of self-renewal and differentiation into various types of cells such as embryonic stem cells (ESCs) (). Human cochlear cells are not readily accessible for biopsy or direct drug administration because of anatomical limitations. Therefore, ESCs/iPSCs are an important tool for studying the molecular mechanisms underlying inner-ear pathology as well as for generating cells for replacement therapies. It was recently reported that ESCs/iPSCs could be differentiated into inner-ear progenitor cells by in vitro differentiation in adherent monolayer culture and/or floating aggregation culture (). For recapitulation of neural tissue formation in a three-dimensional (3D) context, floating aggregation culture is advantageous as it allows more flexible adaptation of cell and tissue shapes compared with 2D culture approaches ().reported in vitro differentiation of ESCs into cortical tissues when the cells were cultured as floating aggregates in a serum-free medium, thereby establishing the technique of serum-free floating culture of embryoid body-like aggregates with quick re-aggregation (SFEBq culture). Koehler and colleagues reported differentiation of ESCs into inner-ear hair cell-like cells using modified SFEBq methods (). For the establishment of strategies for inner-ear cell therapy or the development of a disease model for GJB2-related hearing loss, it is necessary to develop efficient differentiation methods for inducing iPSCs to form cells with CX26-containing GJPs. Although several studies have demonstrated the induced differentiation of ESCs/iPSCs into CX37/40/43/45-expressing cells (), no such protocol has been reported for the differentiation of ESCs/iPSCs into CX26-expressing cells.

In our recent study, it was shown that disruption of the CX26 GJP is associated with the pathogenesis of GJB2-related hearing loss and that the assembly of cochlear GJP is dependent on CX26 (). It was also reported that cochlear gene transfer of GJB2 using an adeno-associated virus significantly improved GJP formation and auditory function (). In our alternative approach, a novel strategy was developed for inner-ear cell therapy with bone marrow mesenchymal stem cells ().

Gap junctions facilitate the rapid removal of Kfrom the base of the cochlear hair cells, resulting in Krecycling back to the endolymph to maintain cochlear homeostasis (). CX26 is also involved in the developmental organization of mammalian cochlea, for example, tunnel of Corti, Nuel's space, or spaces surrounding the outer hair cells (). CX26 and CX30 form heteromeric and heterotypic channels in most of the cochlear gap junction plaques (GJPs) () and in in vitro experiments (). Recently, expression of various transcription factors and other proteins in human developmental fetal cochleae from gestational weeks 9–22 were investigated using immunohistochemistry (), and it has been found that the expression of CX26 and CX30 is detectable in the outer sulcus cells at 18 weeks of gestation ().

Development of the stria vascularis and potassium regulation in the human fetal cochlea: insights into hereditary sensorineural hearing loss.

Hearing loss is the most common congenital sensory deficit (). Approximately 1 child in 1,000 is affected with severe hearing loss at birth or during early childhood, and this is defined as prelingual deafness (), with about half of the cases attributable to genetic causes (). There are more than 100 known forms of non-syndromic deafness associated with identified genetic loci (available at http://hereditaryhearingloss.org ). Mutations in the Gap Junction Beta 2 gene (GJB2), encoding connexin 26 (CX26), account for up to 50% of cases of non-syndromic sensorineural hearing loss in some populations (). CX26 and CX30, which are encoded by GJB6, co-assemble and participate in the formation of gap junctions between cells, and these connexins are the two most abundantly expressed gap junction proteins in the cochlea () ( Figure S1 and Movie S1 ).

Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice.

Autosomal dominant prelingual hearing loss with palmoplantar keratoderma syndrome: variability in clinical expression from mutations of R75W and R75Q in the GJB2 gene.

As shown in Figure 6 P, GJP lengths in CX26P0-Cre-iCx26GJC (1.63 ± 0.07 μm) are significantly shorter (p < 0.01) than those of the WT (5.34 ± 0.28 μm), as in the cochlea ( Figure 6 O; WT, 4.72 ± 0.17 μm; CX26P0-Cre, 1.76 ± 0.06 μm).

To investigate whether the induction strategy can apply to disease model for CX26-associated deafness, we generated an iPSC line from a CX26-deficient deafness mouse model, CX26P0-Cre (), and tested the potential of this iPSC line to differentiate into GJP-forming cells. WT-iCx26GJC showed large, planar GJPs ( Figures 6 G , 6I, 6K, and 6M) at the cell border that formed an orderly pentagonal structure of hexagonal outlines, as in the wild-type (WT) cochlea. In contrast, CX26P0-Cre-iCx26GJC showed drastically fragmented, small vesicle-like GJPs ( Figures 6 H, 6J, 6L, and 6N), as in the CX26P0-Cre-cochlea ( Figures 6 B, 6D, and 6F).

(O and P) Length of the largest GJPs (brackets in E, F, M, and N) along a single cell border (mean ± SE, n = 25, 38, 41, 48 cell borders from four independent experiments). The statistical difference was determined by Student's t test; ∗∗ p < 0.01.

(A–N) GJP formation in 16-week-old adult mouse cochleae (A–F) and iCx26GJC at day 15 (G–N). These samples were co-labeled with anti-CX26 (red) and anti-CX30 (green) antibodies and were counterstained with DAPI (blue). WT-iCx26GJC showed large, planar GJPs (G, I, K, and M) as WT mouse cochlea (A, C, and E). In contrast, CX26 f/f P0-Cre-iCx26GJC showed drastically fragmented, small vesicle-like GJPs (H, J, L, and N) as CX26 f/f P0-Cre-mouse cochlea (B, D, and F). (I and J) Image of each boxed region in (G) and (H), respectively. (E, F, M, and N) Image of each boxed region in (C), (D), (K), and (L), respectively.

The iCx26GJCs from CX26-Deficient Mouse Recapitulated the Drastic Disruption of GJPs as in Cochlea

Figure 6 The iCx26GJCs from CX26-Deficient Mouse Recapitulated the Drastic Disruption of GJPs as in Cochlea

To investigate whether this spontaneous Caactivity required ATP and hemichannels, as reported for this activity in developing cochlea (), we pharmacologically inhibited Caactivity with the p2x receptor antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2′,4'-disulphonic acid) () and connexin hemichannel blocker FFA (flufenamic acid) (). The spontaneous Casignaling activity was clearly inhibited by PPADS and FFA, and the activity was restored after removal of the inhibitors ( Figure 5 C and Movie S5 ). In these analysis, subset of the cells showed continuous slow oscillation at random regions of the cell culture ( Movie S5 ) and some of them showed clear propagated waves as in developing cochlea ( Figures 5 A, 5B, and 5D–5F; Movies S4 and S6 ).

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca 2+ signals across the inner ear.

To test whether iCx26GJCs propagate spontaneous Catransients via gap junctions in the developing auditory system (), we performed Caimaging with the Caindicator fluo-4. Spontaneous Catransients and their propagation were detected in 2D cultures under the B/S condition ( Figures 5 A, 5B , and 5D–5F; Movies S4 and S6 ). The BMP condition also showed the same Catransients and propagation (data not shown), although the frequency was much less than under the B/S condition.

(E) Time-dependent changes in fluorescence intensity in four cells (labeled 1–4). Cells were imaged for 40 s at one frame per 260 ms. Arrowheads indicate time points corresponding to those in (F).

(C) Spontaneous Ca 2+ signaling activity is reversibly inhibited by the p2x receptor antagonist PPADS and the connexin hemichannel blocker FFA (flufenamic acid). The cells were imaged for 14 min at one frame per 500 ms.

(B) Pseudocolor images indicating the range from low (black) to high (white) signal intensity of the images in (A). The cells were imaged for 50 s at one frame per 500 ms.

(A) The propagation of spontaneous Ca 2+ transients at each time point (0, 4, 7, 10, 24, and 50 s) at near-physiological temperature (32°–35°C). The propagation region is encircled by a dashed red line.

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca 2+ signals across the inner ear.

To investigate whether the gap junction structures observed in proliferated iCx26GJCs ( Figures 2 and 3 ) were functional in the formation of gap junction intercellular communication (GJIC) networks, we performed scrape-loading assays with Lucifer yellow (LY) () on proliferated iCx26GJCs, with undifferentiated iPSCs and TRIC feeder cell as controls. We used two different iPSC strains and repeated the experiments at least three times, with similar results. We quantified the extent of dye transfer by measuring the distance from the scrape line to the point where the fluorescence intensity dropped to 1.5× the background fluorescence intensity. In the iCx26GJC culture, we observed that LY diffused beyond the wounded parental cells ( Figures 4 C and 4F ). In contrast, such extent of dye transfer was not observed in undifferentiated iPSCs or cochlear feeder cells ( Figures 4 A, 4B, 4D, and 4E). As shown in the Figure 4 J, quantitative distance of dye transfer in iCx26GJCs (128.2 ± 4.24 μm) was significantly longer (p < 0.01) than in undifferentiated iPSCs (14.8 ± 0.96 μm) or TRIC feeder cells (14.3 ± 0.74 μm).

(J) Quantitative analysis of intercellular LY transfer after scrape loading. Columns represent the mean distance of LY transfer from the scrape line to the point where the fluorescence intensity dropped to 1.5× the background intensity (TRIC feeder cells and undifferentiated iPSCs: n = 20 from four independent experiments; iCx26GJC: n = 68 from six independent experiments). The statistical difference was determined by Scheffe's multiple comparison test, mean ± SE; ∗∗ p < 0.01.

(D–F) Pseudocolor images indicating the range from low (black) to high (red) signal intensity of the images in (A)–(C), respectively.

Digital fluorescence images of cultured cells after scrape loading. The undifferentiated iPSCs (A, D, and G), TRIC feeder cells (B, E, and H) and BMP/SB-treated iPSC-derived 2D culture containing iCx26GJC at day 15 (C, F, and I) were incubated in 0.1% LY and imaged 15 min after wounding with a scalpel blade. Wounded cells integrated dye in all cases, but there was no transfer of dye from primary wounded cells to neighboring cells.

We also examined the ultrastructure of the cell surfaces and borders using scanning EM of 2D cultures at day 15 ( Figures 3 J and 3K). The cell surfaces of 2D culture showed distinct, straight cell borders that formed hexagonal or pentagonal shapes with microvilli distributed along the borders as in normal cochlea ( Figures 3 H and 3I). In contrast, TRIC feeder cells showed unstructured cell borders without microvilli as in conventional mesenchymal cells ( Figures 3 L and 3M).

We also analyzed the ultrastructure at the cell junction sites in transmission electron micrographs (TEM) of iPSC-derived aggregates. We found ultrastructure typical of gap junctions, i.e., an intercellular layer between the two distinct plasma membranes with a uniform intermembrane space of ∼2–4 nm ( Figures 3 A–3G ). Figures 3 C–3E show three distinct gap junctions around a tricellular junction site among three iPS-derived cells. Such characteristic ultrastructures of gap junctions were observed in two medium conditions, BMP ( Figures 3 A–3E) and B/S ( Figures 3 F and 3G), but not in the others (data not shown).

(K) Magnification of boxed region in (J). The surface and borders of cells from 2D culture were quite similar to those of cochlear cells. The individual cells are colored.

(I) Scanning EM of cell surfaces and borders in cochlea from a P6 mouse. The individual cells are colored. OSC, outer sulcus cell; OHC, outer hair cell.

(H) Scanning EM of cell surfaces and borders in cochlea from a 7-week-old mouse. The individual cells are colored. ISC, inner-sulcus cell; IHC, inner hair cell.

(G) Magnification of boxed region in (F). Ultrastructure is typical of gap junctions, with clear distinct intermembrane layer between the plasma membranes.

(B) Magnification of boxed region in (A). Ultrastructure corresponding to three gap junctions was observed at the tricellular junction.

Ultrastructure of Gap Junctions in iPSC-Derived Aggregates at Day 7, Cell Surfaces in Cochlea, 2D Cultures at Day 15, and TRIC Feeder Cells

Figure 3 Ultrastructure of Gap Junctions in iPSC-Derived Aggregates at Day 7, Cell Surfaces in Cochlea, 2D Cultures at Day 15, and TRIC Feeder Cells

To examine the similarities to cochlear cells, we analyzed the expression of known cochlear proteins using immunohistochemistry. CX30 protein, another causative gene product frequently encountered in hereditary deafness, co-localized with CX26 in most of the CX26-GJPs of the differentiated cells ( Figures 2 K, 2L, and S4 E–S4H). Therefore, we inferred that CX26 and CX30 were the two main components of these GJPs, as was the case in the cochlear supporting cells—specifically the outer- and inner-sulcus cells ( Figures S4 A–S4D). Furthermore, the iCx26GJCs on TRIC feeder cells co-expressed P27 Figures 2 M and S5 C–S5I), which is a cyclin-dependent kinase inhibitor expressed in cochlear supporting cells such as the Deiters' cells and outer- and inner-sulcus cells ( Figures S5 A and S5B). The P27signals were observed after day 15, corresponding to the stage at which the colony expansion ( Movie S2 ) had almost stopped (∼days 14–16). We observed both heterogeneous ( Figures 2 M and S5 C–S5E) and homogeneous ( Figures S5 F–S5I) P27-positive regions including iCx26GJCs under the same condition. Furthermore, SOX2, which is a transcript factor essential for maintaining self-renewal, or pluripotency, of undifferentiated or neural stem cells, was co-expressed in part of cochlear supporting cells ( Figures S6 A–S6C). SOX2 signals were partly observed in iCx26GJCs at day 15. We observed both CX26/SOX2cells and CX26/SOX2cells in the same cell population ( Figures 2 N and S6 D–S6I).

The regions with iCx26GJC-containing small vesicles were separated from day 7–11 aggregates and subcultured in growth medium on TRIC feeder cells ( Figure 1 B). With other feeder cells, for example, feeder cells from chicken embryonic inner ear, or in non-feeder conditions on non-coated or gelatin-coated dishes, the separated outer epithelia and small vesicles did not proliferate and were effectively dead. Although the iPSC aggregates that had been subcultured on Matrigel-coated dishes proliferated, CX26 was not observed by immunohistochemistry (data not shown). We observed migration and proliferation of subcultured small vesicles in 2D culture ( Movie S2 ). The subcultured small vesicles indeed colonized on TRIC feeder cells, and the colonies contained iCx26GJC. In TRIC feeder cells, iCx26GJCs proliferated significantly in adherent culture conditions, and the CX26-containing GJPs were preserved ( Figures 2 E–2H and Movie S3 ) as in the cochlear supporting cells—specifically the outer- and inner-sulcus cells ( Figures 2 A–2C). Although the proportion of iCx26GJCs was only 1.8% ± 1.0% and 1.8% ± 1.0% in the aggregates of BMP and B/S, respectively, they increased to 32.2% ± 7.4% and 45.1% ± 9.6% in the 2D culture on TRIC feeder cells ( Figure 2 I). Lengths of the largest GJPs along a single cell border were 2.46 ± 0.45 μm and 1.86 ± 0.15 μm in the BMP- or B/S-treated aggregates, respectively, and were significantly increased to 4.02 ± 0.2 μm and 5.76 ± 0.35 μm in the 2D culture on TRIC feeder cells ( Figure 2 J).

(N) Staining for SOX2 (red), CX26, and DAPI (blue) in 2D culture at day 15. Heterogeneous SOX2-positive regions including iCx26GJCs. Magnification of boxed region in Figures S6 D–S6F.

(M) Staining for CX26 (red), P27(green), and DAPI (blue) in 2D culture at day 15. Heterogeneous P27-positive regions including iCx26GJCs. Magnification of boxed region in Figure S5 E. Arrowheads point to GJPs.

(L) Staining for CX30 (green) and DAPI (blue) in 2D culture at day 14. Magnification of boxed region in Figure S4 E. Arrowheads point to GJPs.

(K) Staining for CX26 (red) and DAPI (blue) in 2D culture at day 14. Magnification of boxed region in Figure S4 E. Arrowheads point to GJPs.

(J) Lengths of the largest GJPs along a single cell border in 3D culture at day 7 and 2D culture at day 15 (3D culture: n = 5 from three independent experiments; 2D culture: n = 25 from five independent experiments). The statistical difference was determined by Student's t test, mean ± SE; ∗∗ p < 0.01.

(I) Percentage of iCx26GJC in 3D culture at day 7 and 2D culture on TRIC at day 15 (3D culture: n = 15 from three independent experiments; 2D culture: n = 20 from four independent experiments). The statistical difference was determined by Student's t test, mean ± SE; ∗∗ p < 0.01.

To analyze the localization of CX26 in iPSC aggregates, we performed immunohistochemistry with day-7 aggregates for which BMP and B/S showed the highest CX26/CX30 mRNA increases ( Figure 1 A). These aggregates formed a distinct outer epithelium that enclosed small vesicles ( Figures 1 C and 1D). In a number of cells and particularly within these vesicles, we distinguished large planar CX26-containing GJPs, which, as we reported previously (), are characteristic of normal mouse cochlea ( Figures 1 E–1H, S3 D, and S3E). These cells, termed iPSC-derived CX26-expressing GJP-forming cells (iCx26GJCs), were disseminated throughout the small vesicles of the aggregates. In contrast, undifferentiated (Nanog-positive) iPSCs and TRIC feeder cells did not show immunolabeling for CX26 ( Figures S3 A–S3C). We also used scanning electron microscopy (EM) to examine the ultrastructure of cell surfaces and borders of the small vesicles from BMP/SB-treated aggregates. The surfaces of the small vesicles showed distinct cell borders with associated microvilli ( Figures 1 I–1K).

To develop disease model cells targeting GJB2-related hearing loss, we induced CX26-expressing GJP-forming cells from iPSCs using methods modified from previous studies for the differentiation of inner-ear sensory epithelia (). On day 7 of differentiation, aggregates showed similar morphology composed of differentiated outer regions and undifferentiated core regions (NANOG-GFP-positive cells, Figure S2 ) as previously reported (). In this study, we used the NANOG-GFP reporter system to monitor the differentiation level. NANOG is a transcription factor used to identify undifferentiated cells. To screen the conditions to induce high CX26/CX30 expression, we compared mRNA levels in day-7 aggregates, including addition of bone morphogenetic protein 4 (BMP-4: BMP), inhibitor of activating receptor-like kinase (ALK) receptors (SB-431542: SB), BMP/SB (B/S), B/S + fibroblast growth factor 2 (FGF-2: B/S + FGF), B/S + inhibitor of ALK receptors (LDN-193189: B/S + LDN), and B/S + FGF/LDN (B/S + F/L) ( Figure 1 A ). CX26/CX30 levels were significantly higher especially in BMP and B/S. In contrast to B/S + F/L, a condition for hair cell differentiation (), BMP and B/S showed high mRNA levels both for CX26 and CX30. Therefore, these two conditions were selected for further isolation of CX26/CX30-expressing cells. On days 7–11 of differentiation, BMP- and B/S-treated aggregate were transferred to adherence culture (2D) with trypsin-resistant inner-ear cells (TRIC), which we generated as feeder cells (see Experimental Procedures ) ( Figure 1 B).

(K) Magnification of boxed region in (J). Surface of the small vesicle. The individual cells, which form the surface of the small vesicle, are colored.

(E) Merge of CX26 (red) and phase contrast microscopy (PCM; white) images in the cryosection. A small vesicle is encircled by a dashed line.

(D) Magnification of boxed region in (C). The small vesicle is encircled by a dashed line.

(A) qPCR analysis of mRNA to assay effects of growth factor/inhibitor addition on day-0 (undifferentiated iPSCs) and day-7 aggregates. mRNA expression levels were calculated relative to untreated aggregates (control). BMP, human bone morphogenetic protein 4; SB, SB-431542, inhibitor of activin receptor-like kinase (ALK) receptors; FGF, human fibroblast growth factor 2; LDN, LDN-193189, inhibitor of ALK receptors; F/L, the combination of FGF and LDN. Both CX26 and CX30 were significantly upregulated in BMP, BMP/SB, B/S+FGF, B/S+LDN, and B/S/+F/L samples compared with controls. Statistical differences were determined by Student's t test. n = 4 independent experiments, mean ± SE; ∗∗ p < 0.01.

Discussion

Koehler and Hashino, 2014 Koehler K.R.

Hashino E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Koehler et al., 2013 Koehler K.R.

Mikosz A.M.

Molosh A.I.

Patel D.

Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. We report the development of a method to generate and proliferate iCx26GJCs from mouse iPSCs for use as a disease model and in inner-ear cell therapies targeting GJB2-related hearing loss, the most frequent type of hereditary deafness worldwide. Our induction method until day 7 was based on previous studies ().

Koehler and Hashino, 2014 Koehler K.R.

Hashino E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Koehler et al., 2013 Koehler K.R.

Mikosz A.M.

Molosh A.I.

Patel D.

Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. However, after the day-7 procedures, which screen for a CX26/CX30 highly expressing condition, isolation of CX26-positive small vesicles, and transferring CX26-positive small vesicles onto TRIC feeder cells, differ from previous studies ().

Chen et al., 2012 Chen W.

Jongkamonwiwat N.

Abbas L.

Eshtan S.J.

Johnson S.L.

Kuhn S.

Milo M.

Thurlow J.K.

Andrews P.W.

Marcotti W.

et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Koehler and Hashino, 2014 Koehler K.R.

Hashino E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Koehler et al., 2013 Koehler K.R.

Mikosz A.M.

Molosh A.I.

Patel D.

Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Oshima et al., 2010 Oshima K.

Shin K.

Diensthuber M.

Peng A.W.

Ricci A.J.

Heller S. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. 2+ imaging. Previous studies have targeted the generation of inner-ear hair cell-like cells from ESCs/iPSCs (). In contrast, we focused on CX26-GJP-forming cells derived from iPSCs, as the cochlear supporting cell-like cells. It has not been reported that ESCs/iPSCs differentiated into the cells with clear CX26-GJPs characterized by immunolabeling and gene expression for CX26/30, dye transfer assay, and Caimaging.

Koehler et al., 2013 Koehler K.R.

Mikosz A.M.

Molosh A.I.

Patel D.

Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. In the modified SFEBq method, we confirmed that the morphology and outer epithelium thickness of day 7–11 aggregates ( Figure S2 ) were similar to those previously reported (). As these thin outer epithelia did not include the NANOG-GFP cells and were clearly demarcated, they could be separated by dissection for further adherent culture on cochlear feeder cells. In this process ( Figure 1 B), the medium conditions and the timing for optimal treatments were selected according to mRNA expression and gap junction formation as determined in subsequent experiments.

Oshima et al., 2010 Oshima K.

Shin K.

Diensthuber M.

Peng A.W.

Ricci A.J.

Heller S. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. The use of cochlear feeder cells was critical to the proliferation of the iCx26GJCs ( Figure 2 I) and the increase in CX26 plaque length ( Figure 2 J) in the dissected small vesicle of the aggregates. In contrast to the cochlear (TRIC) feeder system, a feeder system with chicken embryonic inner ear used for hair cell differentiation () and feeder-free culture did not promote the cell proliferation or GJP formation of iCx26GJCs. Therefore, it is suggested that TRIC feeder cells may express some molecules to promote the proliferation and GJP formation of iCx26GJCs. Our working hypothesis is that a small number of cells that differentiated from iPSCs into CX26-expressing cells in modified SFEBq culture were induced to proliferate by the growth factors that were secreted from the cochlear feeder cells.

Our observation that iCx26GJCs co-expressed CX26 and CX30 ( Figures 2 K, 2L, and S4 E–S4H) suggested that a number of cells in the aggregates had differentiated into cochlear non-sensory cells ( Figures S4 A–S4D) and generated CX26/CX30 GJPs; moreover, the data suggested that these cells had proliferated on the cochlear feeder cells after dissection of the small vesicles that were attached to the outer epithelium ( Movie S2 ).

Kip1 localization in iCx26GJCs. P27Kip1 is a cyclin-dependent kinase inhibitor that is thought to play a role in maintaining the postmitotic state of cochlear supporting cells such as the Deiters' cells and outer- and inner-sulcus cells. The P27Kip1 signals were observed at the stage at which colony expansion (Kip1-positive regions including iCx26GJCs. This suggests that a portion of the iCx26GJCs gradually matures concomitantly with P27Kip1 expression and that proliferation ceases when the cells stop expressing P27Kip1 to maintain the postmitotic state, i.e., serving as cochlear supporting cells ( White et al. (2006) White P.M.

Doetzlhofer A.

Lee Y.S.

Groves A.K.

Segil N. Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. kip1-positive mouse cochlear supporting cells retained the ability to divide when cultured with periotic mesenchymal cells, which support the growth and differentiation of cochlear sensory progenitors. In the present study, we also used inner-ear tissue-derived feeder cells for the differentiation and proliferation of iPS-derived cells, and we generated CX26+/P27Kip1+ supporting cell-like cells. These results suggest that cochlear feeder cells support the proliferation and further differentiation of iCx26GJCs. As a marker of cochlear supporting cells, we assessed P27localization in iCx26GJCs. P27is a cyclin-dependent kinase inhibitor that is thought to play a role in maintaining the postmitotic state of cochlear supporting cells such as the Deiters' cells and outer- and inner-sulcus cells. The P27signals were observed at the stage at which colony expansion ( Movie S2 ) had almost stopped (∼days 14–16). In the 2D cultures, we observed both heterogeneous ( Figures 2 M and S5 C–S5E) and homogeneous ( Figures S5 F–S5I) P27-positive regions including iCx26GJCs. This suggests that a portion of the iCx26GJCs gradually matures concomitantly with P27expression and that proliferation ceases when the cells stop expressing P27to maintain the postmitotic state, i.e., serving as cochlear supporting cells ( Figures S5 A and S5B).reported that isolated P27-positive mouse cochlear supporting cells retained the ability to divide when cultured with periotic mesenchymal cells, which support the growth and differentiation of cochlear sensory progenitors. In the present study, we also used inner-ear tissue-derived feeder cells for the differentiation and proliferation of iPS-derived cells, and we generated CX26/P27supporting cell-like cells. These results suggest that cochlear feeder cells support the proliferation and further differentiation of iCx26GJCs.

Mak et al. (2009) Mak A.C.

Szeto I.Y.

Fritzsch B.

Cheah K.S. Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development. We assessed SOX2 localization in iCx26GJCs and observed heterogeneous ( Figures 2 N and S6 D–S6I) SOX2 expressions as in the adult mouse cochlea ( Figures S6 A–S6C).reported that the SOX2 signals were observed in the developing cochlear supporting cells, such as the Deiters' cells, Hensen cells, inner phalangeal cells, pillar cells, and part of inner-sulcus cells.

These results, whereby iCx26GJCs and mouse cochleae were stained with CX26, CX30, P27kip1, and SOX2 ( Table S1 ), suggested that iCx26GJCs are a cell type similar to the cochlear supporting cells.

TEM of iPSC-derived aggregates detected an ultrastructure typical of gap junctions ( Figures 3 A–3G). These characteristic structures were observed in the two medium conditions, BMP ( Figures 3 A–3E) and B/S ( Figures 3 F and 3G), but not in others (data not shown). These results suggest that the upregulated CX26 and CX30 proteins ( Figure 1 A) formed gap junctions within the iPSC aggregates. Prior to this study, little or no such ultrastructural formations typical of gap junctions had been reported as having been reproducibly induced from ESCs/iPSCs. Scanning EM of the cellular ultrastructure from 2D cultures at day 15 showed distinct straight cell borders that formed hexagonal or pentagonal shapes with associated microvilli dispersed along the borders ( Figures 3 J and 3K). This suggested that the differentiated iPSCs formed the surface structures typical of the inner and outer sulcus cells that surround cochlear hair cells ( Figures 3 H and 3I).

Yum et al., 2007 Yum S.W.

Zhang J.

Valiunas V.

Kanaporis G.

Brink P.R.

White T.W.

Scherer S.S. Human connexin26 and connexin30 form functional heteromeric and heterotypic channels. Yum et al., 2010 Yum S.W.

Zhang J.

Scherer S.S. Dominant connexin26 mutants associated with human hearing loss have trans-dominant effects on connexin30. The scrape-loading assay of iCx26GJCs showed gradients and distance of dye transfers typical of those formed by functional GJIC networks ( Figures 4 C, 4F, and 4J) as previously reported (). In contrast, such intercellular transfer of LY was not observed in undifferentiated iPSCs or TRIC used as feeder cells ( Figures 4 A, 4B, 4D, 4E, and 4J). These results suggest that CX26 and CX30 formed functional GJIC networks among iCx26GJCs, as is the case in cochlear tissue.

2+ transients have been reported in postnatal mouse cochlear supporting cells ( Tritsch et al., 2007 Tritsch N.X.

Yi E.

Gale J.E.

Glowatzki E.

Bergles D.E. The origin of spontaneous activity in the developing auditory system. 2+ signals were suggested to be propagated via gap junctions and hemichannels containing CX26 and CX30 ( Anselmi et al., 2008 Anselmi F.

Hernandez V.H.

Crispino G.

Seydel A.

Ortolano S.

Roper S.D.

Kessaris N.

Richardson W.

Rickheit G.

Filippov M.A.

et al. ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear. Schutz et al., 2010 Schutz M.

Scimemi P.

Majumder P.

De Siati R.D.

Crispino G.

Rodriguez L.

Bortolozzi M.

Santarelli R.

Seydel A.

Sonntag S.

et al. The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice. 2+ signals in supporting cells may play a crucial role in generating the periodic, high-frequency burst of activity observed in the auditory center of the brain ( Wang et al., 2015 Wang H.C.

Lin C.C.

Cheung R.

Zhang-Hooks Y.

Agarwal A.

Ellis-Davies G.

Rock J.

Bergles D.E. Spontaneous activity of cochlear hair cells triggered by fluid secretion mechanism in adjacent support cells. Such spontaneous Catransients have been reported in postnatal mouse cochlear supporting cells (), and the Casignals were suggested to be propagated via gap junctions and hemichannels containing CX26 and CX30 (). It was also suggested that such spontaneous Casignals in supporting cells may play a crucial role in generating the periodic, high-frequency burst of activity observed in the auditory center of the brain (). Therefore, iCx26GJCs in the 2D cultures may differentiate into postnatal cochlear supporting cells before the onset of hearing.

f/f P0-Cre), GJP formations showed visible drastic disruption ( Kamiya et al., 2014 Kamiya K.

Yum S.W.

Kurebayashi N.

Muraki M.

Ogawa K.

Karasawa K.

Miwa A.

Guo X.

Gotoh S.

Sugitani Y.

et al. Assembly of the cochlear gap junction macromolecular complex requires connexin 26. f/f P0-Cre mouse have the potential to differentiate into iCx26GJC as an in vitro disease model of GJB2-related hearing loss. By using these cells, it is expected to establish the drug screening and inner-ear cell therapy after in vitro restoration of GJPs by the GJB2 gene transfer ( Iizuka et al., 2015 Iizuka T.

Kamiya K.

Gotoh S.

Sugitani Y.

Suzuki M.

Noda T.

Minowa O.

Ikeda K. Perinatal Gjb2 gene transfer rescues hearing in a mouse model of hereditary deafness. In iCx26GJC from a CX26-deficient deafness mouse model (CX26P0-Cre), GJP formations showed visible drastic disruption ( Figures 6 H, 6J, 6L, and 6N), reported to be a primary pathology of GJB2-related hearing loss (). This suggests that the iPSCs derived from CX26P0-Cre mouse have the potential to differentiate into iCx26GJC as an in vitro disease model of GJB2-related hearing loss. By using these cells, it is expected to establish the drug screening and inner-ear cell therapy after in vitro restoration of GJPs by the GJB2 gene transfer () targeting GJB2-related hearing loss.

Koehler and Hashino, 2014 Koehler K.R.

Hashino E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Koehler et al., 2013 Koehler K.R.

Mikosz A.M.

Molosh A.I.

Patel D.

Hashino E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. 2+ transients typical of the developing cochlea. By using this method, we generated the in vitro disease model cells with GJP disruption for GJB2-related hearing loss ( Figure 7 Schematic of In Vitro iPSC Differentiation into Functional iCx26GJCs and Disease Model Cells for GJB2-Related Hearing Loss Show full caption Undifferentiated iPSCs formed floating aggregate in serum-free medium (SFEBq culture). SFEBq culture generates neuroectoderm tissues such as retinal and neuronal tissues. Non-neural ectoderm such as inner-ear hair cell is also generated by this culture. The culture conditions including BMP (BMP4) and TGF-β inhibitor (SB-431542) treatment were selected to generate high-CX26/CX30 aggregates with high mRNA expressions. Unlike hair cell differentiation, these characteristic iPSC aggregates form distinct epithelia and small vesicles attached to the outer epithelium. After the removal of NANOG(+)-undifferentiated region, these small vesicles, which contain iCx26GJCs, are dissected and added onto cochlear feeder cells. They attach to the cochlear feeder cells (TRIC) and undergo colony expansion. The small vesicle-derived colony exhibits proliferation potency and contains iCx26GJCs. The iCx26GJCs form functional CX26-GJPs that exhibit spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. In GJB2 (CX26) mutant iPSCs, GJP disruption, which has been reported to be a primary pathology of GJB2-related hearing loss, is recapitulated in vitro. In summary, we hypothesize that iPS-derived cells formed floating aggregate in serum-free medium (SFEBq culture), after which the culture conditions including BMP (BMP4) and TGF-β inhibitor (SB-431542) treatment were selected to generate high-CX26/CX30 aggregates. Unlike hair cell differentiation (), these characteristic iPSC aggregates form distinct epithelia and small vesicles attached to the outer epithelium. In 2D culture, these small vesicles colonized on TRIC feeder cells. The small vesicle-derived colony exhibits proliferation potency and contains iCx26GJCs. The iCx26GJCs form functional CX26-GJPs that exhibit spontaneous ATP- and hemichannel-mediated Catransients typical of the developing cochlea. By using this method, we generated the in vitro disease model cells with GJP disruption for GJB2-related hearing loss ( Figure 7 ).

In the present study, we demonstrated that the aggregate formation of iPSCs under several medium conditions followed by adherent culture with cochlear feeder cells induced: (1) the upregulation of mRNAs encoding CX26/CX30; (2) GJP formation composed of CX26/CX30; (3) the ultrastructure typical of gap junctions; (4) functional GJIC networks; and (5) spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of developing cochlea. These are known to be the biological properties of cochlear supporting cells. Cochlear supporting cells are the most CX26-abundant cells that play crucial roles in maintaining proper endocochlear potential via ion transport. Furthermore, the disease model cells with GJB2 mutation showing drastic GJP disruptions in the present study are thought to be the optimum therapeutic target for the treatment of GJB2-related hearing loss, the most typical type of hereditary deafness worldwide. It is expected, then, that these iPS-derived cells, which can be obtained from patients, will be particularly useful for drug screening and inner-ear cell therapies targeting GJB2-related hearing loss.