Method for producing inner ear cells

Abstract

To provide a method for efficiently producing an inner ear cell having connexin 26 gap junction from a pluripotent stem cell.SOLUTION: A method for producing an inner ear cell having connexin 26 gap junction includes culturing an embryoid body, which is a pluripotent stem cell cultured in the presence of insulin, in the presence of a cochlea-derived feeder cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a new method for producing inner ear cells that have connexin 26 gap junctions. [Background technology]

[0002] Hearing loss is the most common congenital sensory disability, with approximately 1 in 1000 people experiencing severe hearing loss at birth or during infancy, defined as prelingual deafness, about half of which is genetically determined. There are more than 100 known forms of nonsyndromic hearing loss associated with identified genetic loci. Mutations in the gap junction beta 2 gene (Gjb2), which encodes connexin 26 (CX26), account for 50% of nonsyndromic sensorineural hearing loss. CX26 and CX30, encoded by Gjb2 and Gjb6, respectively, assemble and participate in the formation of intercellular gap junctions. These connexins are two of the most abundant gap junction-forming proteins in the cochlea. Gap junctions facilitate the rapid removal of K+ ions from cochlear hair cells and return them to the endolymph, thereby maintaining cochlear homeostasis. CX26 and CX30 form heterogeneous and heterotypic channels in many cochlear gap junction plaques (GJPs) and in vitro experiments. Our recent research has shown that disruption of CX GJPs is associated with the development of GJB2-related hearing loss, and that the assembly of cochlear GJPs is dependent on CX26 (Non-Patent Document 1). We have also reported that cochlear gene transfer of GJB2 using adeno-associated virus significantly improved GJP formation and hearing function (Non-Patent Document 2). We have also developed a novel strategy for inner ear cell therapy using bone marrow mesenchymal stem cells (Non-Patent Document 3). Furthermore, the present inventors have reported that inner ear cells with CX26 gap junctions can be produced by culturing pluripotent stem cells such as mouse iPSCs in the presence of one or more additives selected from BMP, a TGF-β1 receptor inhibitor, and FGF, and then culturing the resulting embryoid bodies in the presence of cochlear-derived feeder cells (Non-Patent Document 4, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2017 / 146035 Brochure [Non-patent literature]

[0004] [Non-Patent Document 1] J.Clin.Invest.124,1598-1607(2014) [Non-patent document 2] Hum.Mol.Genet.24,3651-3661(2015) [Non-patent document 3] Am.J.Pathol.171,214-226(2007) [Non-patent document 4] Stem Cell Reports(2016)7(6),1023-1036 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the method for producing inner ear cells with CX26 gap junctions from pluripotent stem cells previously reported by the inventors is still insufficient in terms of yield, and it is desirable to provide a more efficient method for producing inner ear cells with CX26 gap junctions. [Means for solving the problem]

[0006] The present inventors therefore investigated additives for inducing differentiation of pluripotent stem cells into embryoid bodies. As a result, they found that inner ear cells with CX26 gap junctions can be obtained with high efficiency by culturing pluripotent stem cells in the presence of insulin instead of the conventionally used growth factors such as BMP, TGF-β1 receptor inhibitors, and FGF to form embryoid bodies, and then culturing these embryoid bodies in the presence of cochlear-derived feeder cells, thereby completing the present invention.

[0007] That is, the present invention provides the following [1] to [4].

[0008] [1] A method for producing inner ear cells having connexin 26 gap junctions, characterized by culturing embryoid bodies obtained by culturing pluripotent stem cells in the presence of insulin, in the presence of cochlear-derived feeder cells. [2] The method for producing inner ear cells according to [1], wherein the cochlea-derived feeder cells are cells formed by trypsinizing cochlear cells and culturing them to form colonies. [3] The method for producing inner ear cells according to [1] or [2], wherein the pluripotent stem cells are iPS cells or ES cells. [4] The method for producing inner ear cells according to any one of [1] to [3], wherein the pluripotent stem cells are human iPS cells or human ES cells. [Effects of the Invention]

[0009] The inner ear cells obtained by the method of the present invention form CX26 and CX26-containing gap junction plaques, similar to those in the cochlea. They also upregulate the otic progenitor cell markers GJB2, GJB6, PAX2, PAX8, and GATA3, and express the cochlear cell markers CX3, SOX2, SPARCL1, KCC3, KIAA1199, MIA, and OTOR. Furthermore, inner ear cells possessing connexin 26 gap junctions derived from iPSCs from hearing-impaired patients were successfully produced, reproducing the pathology of GJB2-related hearing loss. Therefore, the inner ear cells obtained by the present invention can be used for drug screening for hereditary hearing loss caused by GJB2 mutations and for inner ear regenerative medicine. [Brief explanation of the drawings]

[0010] [Figure 1]This shows that insulin treatment induces GJB2 / GJB6 genes and otic precursor marker genes in iPSCs. (A) Procedure for differentiation of CX26-expressing cells from human iPSCs in SFEBq culture. (B) Quantitative PCR analysis of undifferentiated human iPSCs (day 0) and iPSC-derived aggregates (day 7) for expression of NANOG, GJB2, GJB6, PAX2, PAX8, and GATA3 (n = 4 from two to four independent experiments). mRNA expression levels were calculated relative to day 7 aggregates without insulin (Ins(-)). SF: StemFit AK02N; Y: Y-27632; Ins: insulin. Statistical significance was determined by Scheffe's multiple comparison test. Mean ± standard error (SE), *p<0.05, **p<0.01. [Figure 2]Insulin treatment induces CX26-expressing cells in iPSCs. (A-D) Immunostaining for CX26 (red) on day 7 aggregates. Arrows indicate CX26+ vesicles. The areas outlined in (A) and (B) are enlarged in (C) and (D), respectively. (E) Number of CX26+ vesicles per mm3 in day 7 aggregates (n = 9 or 15 aggregates from 2 or 3 independent experiments). (F) Average diameter of CX26+ vesicles on day 7 aggregates (n = 8 or 20 CX26+ vesicles from 3 independent experiments). (G) Average number of cells consisting of CX26+ vesicles on day 7 aggregates (n = 12 CX26+ vesicles) from 3 independent experiments. (H) Ratio of CX26-positive cells to CX26-negative cells in day 7 aggregates (n = 9 or 14 from day 7 aggregates) from 2 to 3 independent experiments. White columns represent CX26-negative (CX26-) cells. Black columns represent CX26-positive (CX26+) cells. (I-N) Immunostaining for CX26 (red) and F-ACTIN (green) on CX26+ vesicles at day 7 aggregates. Nuclear DAPI counterstaining (blue in L-N) is shown. Boxed areas in (I), (J), (L), and (M) are enlarged in (J), and (K) and (M), respectively, show the GJPs (N) and (O,P). 3D images were reconstructed from the image in (M). Arrows indicate GJPs. Scale bars: 200 μm (A and B); 50 μm (C and D); 20 μm (I and L); 10 μm (J, M, P), 5 μm (K, N, O). Statistical significance was determined by Student's t-test, mean ± SE; *p < 0.05; **** p < 0.01. [Figure 3]Growth of iCX26GJCs on adherent culture. (A) Procedure for the growth of iCX26GJCs from human iPSC aggregates using adherent culture. (B) Phase-contrast microscopy (PCM) image from a 21-day adherent culture. (C) Enlarged view of the boxed area in (B) for CX26 (red) and PCM (white) staining. (D-G) Staining of CX26 (red) and F-ACTIN (green). Enlarged views of the boxed areas in (D), (E), and (F). (E), (F), (G), and (G), respectively. (H and I) Staining of CX26 (red) and DAPI (blue) (H). (I) and (H) are the same area in (F) and (G). The boxed area (H) is enlarged in (I). (J,K) 3D image showing GJPs reconstructed from the image in (I). Arrowheads indicate GJPs. Scale bars: (B); 100 μm, (D); 50 μm, (C and E); 10 μm, (F and H); 5 μm (G and I-K). [Figure 4] Immunostaining and gene expression of known cochlear markers in adherently grown iCX26GJCs are shown. (A-G) Nuclei of CX26 (red), CX30 (A, green), SOX2 (B, green), SPARCL1 (C, green), SLC12A6 (KCC3; D, green), Pan-cytokeratin (P-CK; E, green), CK8 (F, green), and CK18 (G, green) were stained with DAPI (blue). (H) Quantitative PCR analysis of undifferentiated human iPSCs (hiPSCs) and iCX26GJCs for expression of NANOG, SOX2, KIAA1199, SPARCL1, MIA, and OTOR (n = 4 from two to three independent experiments). mRNA expression levels were calculated relative to undifferentiated human iPSCs (hiPSCs). Scale bars (A–G) 20 μm (A: columns 2, 3, and 4) 10 μm. Statistical significance was determined by Student's t test, mean ± SE; *p < 0.05; **p < 0.01. [Figure 5]CX30 GJP formation and GJP length in normal or patient iPSC-derived iCX26GJCs are shown. (A) Family history of the individuals from whom these iCX26GJCs were derived. Squares indicate male family members; circles indicate female family members; filled shapes indicate diagnosed family members. (B and C) Audiometric phenotypes of patient 1 (B) and patient 2 (C) indicate severe hearing loss. (D) iCX26GJC formation by normal (201B7) or patient (GP05-235delC, GP06-235delC) iPSCs. These samples were colabeled with anti-CX26 (red) and anti-CX30 (green) antibodies. (E) Maximum GJP length along a single cell boundary (mean ± SE, n = 27, 30, and 37 cell boundaries from three and four independent experiments). Statistical significance was determined by Scheffe's multiple comparison test. Mean ± standard error, *p < 0.05, ** p < 0.01. [Figure 6]Dye transfer after cell scrape-loading and subsequent quantitative analysis is shown. (A-O) Digital fluorescence images of cultured cells after scrape-loading. iPSC-derived adherent cultures include healthy human iPSCs (201B7) (A-C), feeder cells (TRICs) (D-F), iCX26GJCs derived from healthy human iPSCs (201B7-iCX26GJCs) (G-I), and iCX26GJCs derived from iPSCs from patient 1 (GP5-235delC-iCX26GJCs) (J-L) and patient 2 (GP6-235delC-iCX26GJCs) (M-O) as healthy controls. (A, D, G, J, M) Dye transfer using Lucifer Yellow (B, E, H, K, N). Pseudocolor images show: Images of the transfer range from low (black) to high (red) signal intensity in the same area as shown above. (C, F, I, L, O) Phase-contrast microscopy images of the same area. (P) Quantitative analysis of intercellular dye transfer after scraping. Columns represent mean values. Dye migration distance from the scrape line (TRICs and random 201B7: n = 40 from five independent experiments; 201B7-iCX26JCs, GP5-235delC-iCX26JCs, GP6-235delC-iCX26JCs: n = 40 from two or three independent experiments). Statistical significance was determined by Scheffe's multiple comparison test. Mean ± SE; different letters (a–c) indicate significant differences, p < 0.01. Scale bar represents 50 m. [Figure 7] This figure shows the procedure for differentiation of human iPSC cultures into CX26-expressing cells by treatment with BMP and / or SB. On day 7, BMP4 (10 ng / ml) and / or SB (10 μM) were added to gfCDM. [Figure 8]Treatment with BMP and / or SB does not promote the induction of CX26-expressing cells in SFEBq / gfCDM cultures. (B and C) qPCR analysis of GJB2 (B) and GJB6 (C) expression (n = 3 from three independent experiments) at day 7. (D→I) Immunostaining for CX26 (red: D→F) and F-ACTIN (green: G→I) aggregates at day 7. Arrows indicate CX26-positive vesicles. (J) Average CX26-positive vesicle aggregates at day 7 (n = 8-16 aggregates from three independent experiments). BMP, BMP4, SB, SB431542. Each bar represents the mean ± SE. Differences between samples were assessed by one-way ANOVA and Scheffe's multiple comparison test; *p < 0.05; **p < 0.01. [Figure 9] The morphology of aggregates on day 7 of culture is shown. (A) The morphology of aggregates on day 0 or day 7. Differentiation of human iPSC aggregates in gfCDM without insulin (-) produced more cell debris than gfCDM with insulin (+). (B) The diameter of aggregates on day 7 with or without insulin (+). (n = 30 aggregates from three independent experiments). Statistical significance was determined by Student's t-test, mean ± SE. *p < 0.05; **p < 0.01. Scale bar: 200 μm (A). [Figure 10] This shows the procedure for differentiation of CX26-expressing cells from human iPSC culture. On day 7, BMP4 (10 ng / ml) and / or SB (10 μM) were added to gfCDM. [Figure 11]Treatment with BMPs and / or SB does not enhance insulin-induced induction of CX26-expressing cells in SFEBq / gfCDM cultures. (B and C) qPCR analysis of GJB2 (B) and GJB6 (C) expression (n = 4 from four independent experiments) at day 7. (D → I) Immunostaining for CX26 (red: D → F) and F-ACTIN (green: G → I) aggregates at day 7. Arrows indicate CX26-positive vesicles. (J) Average CX26-positive vesicle aggregates at day 7 (n = 8-16 aggregates from three independent experiments). Insulin (Insulin), BMPs, BMP4, SB, SB431542. Each bar represents the mean ± SE. Significant differences between samples were assessed by one-way ANOVA and Scheffe's multiple comparison test; *p < 0.05; **p < 0.01. [Figure 12] Other cytokeratins expressed in the skin are not observed in human iCX26GJC. Immunostaining of CX26 (red) and CK5, 10, and 14 (green) in adherent cultures with nuclear counterstaining by DAPI (blue) is shown. Scale bar: 10 μm. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides a method for producing inner ear cells having CX26 gap junctions, which is characterized by culturing embryoid bodies obtained by culturing pluripotent stem cells in the presence of insulin, in the presence of cochlear-derived feeder cells.

[0012] Pluripotent stem cells used in the present invention include iPS cells and ES cells. Pluripotent stem cells derived from non-human animals such as mice can also be used, but it is preferable to use human-derived iPS cells or human ES cells. Herein, iPS cells may be abbreviated as iPSCs. ES cells may be abbreviated as ESCs.

[0013] In the present invention, embryoid bodies can be formed from pluripotent stem cells by culturing the pluripotent stem cells in the presence of insulin. There is no need to use BMP, TGF-β1 receptor inhibitors, FGF, and the like, which are used in the methods described in Patent Document 1 and Non-Patent Document 4. The concentration of insulin added to the medium is preferably 1 to 20 μg / mL, more preferably 1 to 15 μg / mL.

[0014] To form embryoid bodies derived from pluripotent stem cells, pluripotent stem cells are preferably cultured in a known medium for culturing pluripotent stem cells, such as gfCDM medium, DFNB medium, N2β27 medium, or GEM medium, supplemented with insulin at 30-40°C for 7-10 days. Embryoid body formation can be confirmed by the formation of cell aggregates and differentiation into sensory epithelial cells. Differentiation into sensory epithelial cells can also be confirmed by an increase in the mRNA levels of CX26 and CX30.

[0015] The resulting pluripotent stem cell-derived embryoid bodies can be cultured on cochlear-derived feeder cells to obtain inner ear cells with CX26 gap junctions. The cochlear inner ear feeder cells are preferably cells formed by trypsinizing cochlear cells and culturing them to form colonies, preferably by adding 0.25% trypsin to the cochlear labyrinth tissue including the lateral cochlear wall and the organ of Corti, culturing the tissue, and obtaining the colony-forming cells. The seeding density of cochlear-derived feeder cells used to culture the disaggregated embryoid bodies was 5 × 10 3 cells / cm 2 ~5×10 4 cells / cm 2 The medium used is gfCDM medium, DFNB medium, or DMEM GlutaMAX+10% fetal bovine serum. The culture is preferably carried out at 30 to 40°C for 3 to 4 days.

[0016] By this culture, inner ear cells having CX26 gap junctions can be efficiently obtained. Here, CX26 expression can be confirmed by an increase in the concentration of Cx26 mRNA. The inner ear cells obtained by the present invention form CX26 and CX26-containing gap junction plaques, similar to those in the cochlea, and upregulate the otic progenitor cell markers GJB2, GJB6, PAX2, PAX8, and GATA3. They also express the cochlear cell markers CX3, SOX2, SPARCL1, KCC3, KIAA1199, MIA, and OTOR. Furthermore, inner ear cells possessing connexin 26 gap junctions derived from iPSCs of hearing-impaired patients were successfully produced, reproducing the pathology of GJB2-related hearing loss. Therefore, the inner ear cells obtained by the present invention can be used for screening drugs for hereditary hearing loss caused by GJB2 mutations and for inner ear regenerative medicine. [Example]

[0017] The present invention will now be described in more detail with reference to examples.

[0018] Example 1 (method) (1) Cultivation of human iPSCs A healthy human iPSC line (201B7) was provided by the RIKEN BioResource Center Cell Bank. Two phenotypic iPSC lines (JUFMDOi005-A and JUFMDOi006, specific cell line names, respectively) generated from PBMCs from sibling patients are known, namely, GPSC-235delC / 235delC and GP6-235delC / 235delC (i.e., GP5-235delC and GP6-235delC; family history and audiograms are shown in Figure 5, A–C ). These three human iPSC lines were maintained in a feeder-free culture system on iMatrix-511 (Nippi)-coated plates with StemFit AK02N (AjinomotoWako).

[0019] (2) Differentiation of human iPSCs Human iPSCs were dissociated with 0.5% TrypLE Select and suspended in maintenance medium (Stem Fit) supplemented with Y-27632 (20 μM), then plated at 100 μL / well (9000 cells) into a 96-well low cell attachment V-bottom plate (Thermo Fisher Scientific). After 2 days of incubation at 37°C and 3% CO , aggregates were transferred to 96-well low cell attachment V-bottom plates (Sumitomo Bakelite) in 100 μL of gfCDM (Table 1 below) containing 2% Matrigel (Corning). On days 7-11, the aggregates were hemi-dissected using forceps. The aggregates were transferred to adherent cultures containing cochlear-derived feeder cells (TRIC, see below) in DFNB medium (Table 1 below). After 7 days of incubation, the medium was changed to growth medium (DMEM GlutaMAX supplemented with 10% FBS; Table 1 below).

[0020] [Table 1]

[0021] (3) Preparation of cochlear-derived feeder cells (trypsin-resistant inner ear cells: TRIC) To prepare TRIC, cochlear tissue, including the organ of Corti, basilar membrane, and lateral wall, was obtained from 10-week-old mice (CLEA Japan, Inc.), consisting mainly of supporting cells of the basilar membrane, hair cells, cochlear fibrocytes, and other cells. TRIC was generated by exposing cochlear tissue to trypsin and screening for trypsin-resistant cells, which were then maintained in growth medium. This cell line was used as an inner ear-derived feeder cell for expanding otic progenitor cells. The feeder cell layer consisted of 3 × 10 cells after 3 hours of mitomycin C (10 mg / mL) treatment. 5 TRICs / cm 2 were seeded into gelatin-coated wells of a 24-well culture plate.

[0022] (4) Quantitative reverse transcription PCR of GJB2 and GJB6 mRNA expression The aggregates were harvested on day 7, washed with DPBS, and then total RNA was isolated using reagents from the Neasy Plus Mini Kit (Qiagen) and reverse transcribed into cDNA using reagents from the PrimeScript II First-Strand cDNA Synthesis Kit (Takara). Real-time PCR was performed using the reverse transcription products, TaqMan Fast Advanced Master Mix reagent (Applied Biosystems), and gene-specific TaqMan probes (see below; Applied Biosystems) on a StepOne Real-Time PCR system (Applied Biosystems). Each sample was run in triplicate. The Ct values ​​of each mRNA were analyzed using StepOne software (Applied Biosystems), and their expression was normalized to that of the endogenous control, actin β mRNA. TaqMan probes (assay ID; Applied Biosystems) were used to detect human GJB2 (Hs00269615_S1), GJB6 (Hs00922742_S1), NANOG (Hs02387400_G1), PAX2 (Hs01057416_M1), PAX8 (Hs00247586_M1), GATA3 (Hs00231122_M1), OTOR (Hs00375304_M1), MIA (Hs00197954_M1), SOX2 (Hs01053049_S1), SPARCL1 (Hs00949886_M1), ACTB (Hs99999903_M1), and 18S (Hs99999901_S1). The expression of mRNA was detected.

[0023] (5) Immunostaining and image acquisition Aggregates were fixed with 4% (w / v) paraformaldehyde in 0.01M PBS at room temperature for 1 hour. For whole mounts, aggregates were permeabilized with 0.5% (w / v) Triton X-100 (Sigma-Aldrich) in 0.01M PBS for 30 minutes. Samples were then washed twice with 0.01M PBS and blocked with 2% (w / v) BSA in 0.01M PBS for 30 minutes. Cells from adherent cultures were fixed with 4% (w / v) paraformaldehyde in 0.01M PBS for 15 minutes at room temperature, followed by permeabilization with 0.5% (w / v) Triton X-100 in 0.01M PBS for 5 minutes. Samples were washed twice with 0.01M PBS and blocked with 2% (w / v) BSA in 0.01M PBS for 30 minutes. For immunofluorescence staining, primary and secondary antibody solutions were diluted in 1% (w / v) BSA in 0.01 M PBS. The primary antibodies used were CX26 (rabbit IgG, 71-500; mouse IgG, 33-5800, Life Technologies), CX30 (rabbit IgG, 71-2200, Life Technologies), pan-cytokeratin (mouse IgG, C2562, Sigma-Aldrich), cytokeratin 8 (mouse IgG, MA5-14428, Invitrogen), cytokeratin 18 (mouse IgG, MA5-12104, Invitrogen), SOX2 (goat IgG, SC-17320, Santa Cruz), SPARC-like1 (mouse IgG, AF2728, R&D Systems), and SLC12A6 (rabbit IgG, KCC3). Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse IgG or anti-goat IgG, Cy3-conjugated anti-rabbit IgG (Invitrogen, A11070), and phalloidin FITC staining for F-actin (Invitrogen, 12379). The specimens were washed twice with 0.01 M PBS and mounted with a mounting medium (Vector, DAPI-added VECTASHIELD Mounting Medium). Fluorescence confocal images were obtained using an LSM780 confocal microscope (Zeiss). Images were collected at 0.5 μm intervals (z-stacks), and single image stacks were constructed using the LSM Image Browser (Zeiss). Three-dimensional images were constructed from z-stacked confocal images using IMARIS (Bitplane).

[0024] (6) Scrape-loading / dye-transfer (SL / DT) assay The SL / DT assay was performed according to Mol. Med., 17, 550-556 (2011) and J. Hum. Genet., 50, 76-83 (2005). iCX26GJC-containing proliferated cells were grown for 7-14 days after transfer onto TRICs (feeder cells). Undifferentiated iPSCs and TRICs were grown confluently on dishes as controls. The medium was changed to HBSS + 0.1% Lucifer Yellow CH (L453, Invitrogen). Many parallel lines were cut into the dish with a razor blade. 15 minutes after Lucifer Yellow loading, cells were washed three times with HBSS and imaged. Scrape loading was quantified by measuring the distance from the scrape line to the point where the fluorescence intensity dropped to background intensity. Images were processed and analyzed with NIH ImageJ software, and the average distance was calculated using MicroWeb Excel software.

[0025] (7) Statistics The data were analyzed using MicroWeb Excel software and presented as mean ± standard error (SE). Comparisons of mRNA levels, dye migration distance, and GJP length were performed using one-way analysis of variance and Scheffe's multiple comparison test or two-tailed Student's t-test, with p < 0.05 as the significance criterion.

[0026] (result) (1) Differentiation of human iPSCs into CX26-expressing cells The method for inducing iCX26GJCs from human iPSCs, which is a combination of SFEBq culture and adherent culture techniques, was modified from the inventors' previous method for mouse iPSCs (Patent Document 1), and the conditions required for differentiation were then evaluated. For SFEBq culture, human iPSCs were reaggregated (9,000 cells / well) and cultured in maintenance medium (StemFit AK02N) for 2 days (day 2 to day 0). The aggregates were then transferred to growth factor-free chemically defined medium (gfCDM; Table 1). On day 3 of SFEBq culture, BMP4 (BMP) and / or an activin / Nodal / TGF-β pathway inhibitor (SB431542: SB) were added to gfCDM according to the mouse iCX26GJC induction method (Figure 7). However, the addition of these supplements did not increase GJB2 / GJB6 mRNA expression or CX26-positive cell mass (CX26+ vesicles) production. Conversely, addition of SB induced a decrease in GJB2 mRNA expression and CX26+ vesicle production (Fig. 8). Based on these results, in the following experiments, insulin (7 μg / mL) was added to gfCDM from day 0. The modified SFEBq culture for differentiation of human iPSCs into CX26-expressing cells is shown in Figures 1A (SFEBq culture) and 3A (adherent culture).

[0027] (2) Screening for high CX26 expression in SFEBq cultures In the improved SFEBq culture, aggregates were collected on day 7 and mRNA (GJB2, GJB6, PAX2, PAX8, GATA3) levels were measured in each culture group. The GJB6 gene encodes the CX30 protein, which is co-expressed with CX26 in cochlear supporting cells (J. Comp. Neurol., 467, 207-231 (2003), J. Comp. Neurol., 499, 506-518 (2006)). The PAX2, PAX8, and GATA3 gene set has been used as an otic progenitor cell marker in several studies aimed at differentiating ESCs / iPSCs into inner ear cells (Cell 141, 704-716 (2010), Nature, 490, 278-282 (2012), etc.). In the day 7 aggregates, the GJB2, GJB6, PAX2, PAX8, and GATA3 genes were upregulated compared to those in undifferentiated iPSCs (day 0). Furthermore, the expression of PAX2, PAX8, and GATA3 genes was significantly increased compared to undifferentiated iPSCs (Figure 1B). iPSCs cultured in gfCDM supplemented with insulin showed significantly higher expression of these mRNAs compared to cultures without insulin (GJB2: 20.3-fold increase, GJB6: 13.7-fold increase, PAX8: 1.7-fold increase, GATA3: 304.0-fold increase) (Figure 1B). In day 7 aggregates without insulin, cellular debris was observed around the aggregates. In contrast, no debris was observed when insulin was added to the aggregates (Figure 9A). Furthermore, the diameter of the aggregates on day 7 was significantly larger with insulin (mean = 876.6 ± 6.90 μm) than without insulin (mean = 644.8 ± 24.58 μm) (Figure 9B).

[0028] To analyze the localization of CX26 in iPSC aggregates, we performed immunohistochemistry on day 7 aggregates. CX26-expressing cell masses (CX26+ vesicles) were observed in day 7 aggregates, regardless of the presence or absence of insulin (Figure 2A-D). Cells treated with insulin had significantly more CX26 + vesicles (mean = 2.7 ± 0.21) compared with cells cultured without insulin (mean = 1.7 ± 0.37) ( Figure 2E ). On the other hand, addition of SB and / or BMP to insulin-supplemented gfCDM did not increase mRNA expression or CX26+ vesicle production (Figs. 10 and 11). In terms of diameter of CX26+ vesicles, cells treated with insulin (mean = 161.3 ± 10.83 μm) were significantly larger than cells without insulin (mean = 129.2 ± 4.70 μm) ( Figure 2F ). Furthermore, these CX26+ vesicles consisted of 281.9 ± 41.4 cells (with insulin) or 184.2 ± 12.07 cells (without insulin), respectively (Figure 2G). The percentages of CX26-positive cells (CX26(+)) and CX26-negative cells (CX26(-)) in the aggregates were 4.18% and 95.8%, respectively, for insulin(-) aggregates, and 8.86% and 9.13%, respectively, for insulin(+) aggregates (Figure 2H). Confocal analysis of the 7-day insulin-treated aggregates revealed that CX26-expressing cells were seeded throughout the CX26+ vesicles (Figure 2, I-N). These cells formed CX26+ GJs at cell-cell boundaries (Figure 2, J-K, M-N). Three-dimensional reconstruction of the confocal images revealed planar CX26-containing GJPs (Figure 2, O-P).

[0029] iCX26GJCs expressed typical cochlear cell markers. Regions containing iCX26GJC-containing CX26+ vesicles were isolated from the aggregates on days 7–11 and transferred to mouse cochlear feeder cells (trypsin-resistant inner ear cells, TRICs) in DMEM / Ham's F12 supplemented with N2 / B27 (DFNB medium) (Figure 3A). The transferred iCX26GJC-containing regions indeed formed colonies on TRIC feeder cells, and the colonies contained iCX26GJCs (Figure 3B and C). These proliferating cells formed CX26-positive GJs at the cell-cell boundaries (Figure 3D). Three-dimensional reconstructions of confocal images revealed large, planar CX26-containing GJPs (Figure 3IK).

[0030] To determine whether iCX26GJC resembles cochlear supporting cells, we examined the expression of the following proteins (CX30, SOX2, SPARCL1, KCC3, and several cytokeratins) and genes (SOX2, SPARCL1, KIAA1199, MIA, and OTOR) observed in cochlear supporting cells and / or fibrocytes. The results of immunostaining and qPCR for iCX26GJC are summarized in Tables 2 and 3.

[0031] [Table 2]

[0032] [Table 3]

[0033] In human iCX26GJCs, CX30 and CX26 expression was observed in the same cells, but these proteins did not necessarily co-assemble in gap junction plaques (Figure 4A). In human cochlear spiral ligament fibroblasts, CX26 / CX30 proteins form separate GJPs and do not necessarily co-assemble in GJPs (Cell Tissue Res., 365, 13-27 (2016)). However, it is unclear whether these proteins form gap junction plaques in cochlear supporting cells as well as in cochlear fibroblasts. On the other hand, in rodents, the expression patterns of CX26 / CX30 proteins differ between cochlear supporting cells (forming hexagons) and lateral wall fibroblasts (not forming polygons) (Cell Tissue Res., 333, 395-403 (2008), J. Clin. Invest., 124, 1598-1607 (2014)). From the above, we cannot conclude that in our target, human cochlear supporting cells, CX26 / CX30 proteins do not co-assemble GJPs as they do in spiral ligament fibrocytes.

[0034] Furthermore, iCX26GJC coexpressed SOX2, SPARCL1, KCC3, p-CK, CK8, and CK18 proteins (Fig. 4, B-G). In contrast, immunolabeling of the skin markers CK5, CK10, and CK14 was not detected in iCX26GJCs (Figure 12). Furthermore, the GJB2, GJB6, KIAA1199, SPARCL1, MIA, and OTOR genes were significantly upregulated compared to undifferentiated iPSCs (Figure 4H). On the other hand, the expression level of the undifferentiated marker NANOG was significantly decreased compared to undifferentiated iPSCs. The expression level of SOX2, another undifferentiated marker, was also decreased, but not as dramatically as NANOG. It is known that non-sensory cells in the cochlea include cells that express SOX2 (inner phalangeal cells, inner pillar cells, outer pillar cells, Deiters cells, and Hensens cells) (Proc. Natl. Acad. Sci. USA, 105, 18396-18401 (2008)) and cells that do not express SOX2 (inner sulcus cells, outer sulcus cells, and fibrocytes). Furthermore, in the mouse iPSC-derived iCX26GJCs reported by the present inventors in Patent Document 1, the presence or absence of SOX2 expression was observed, as in the cochlear supporting cells. Based on the above, it was concluded that no significant decrease in mRNA expression, such as that of NANOG, was observed in human iPSC-derived iCX26GJC.

[0035] (3) Formation of CX26 / CX30 gap junction plaques in iCX26GJCs derived from healthy and diseased iPSCs We generated disease-specific iCX26GJC from two iPSC lines generated from sibling patients with a homozygous 235delC mutation in GJB2 ( Fig. 5A ). The 235delC mutation is the most common GJB2 mutation in Asia, including Japan, and results in a profound hearing picture. Two human iPSC lines, GP5-235delC / 235delC and GP6-235delC / 235delC (hereafter referred to as GP5-235delC and GP6-235delC, respectively; audiograms of profoundly hearing-impaired individuals are shown in Figure 5, B and C), have been previously characterized (specialized cell line names: JUFMDOi005-A and JUFMDOi006, respectively). To examine whether disruption of the CX26 / CX30 macromolecular complex (15, 29), a pathological condition of GJB2 hearing loss observed in mouse models, was also observed in patient iPSC-derived iCX26GJCs, we examined CX26 and CX30 by immunostaining. iCX26GJCs derived from healthy iPSCs (201B7) showed large, planar CX30 GJPs at the cell boundaries (Figure 5D, left column). In contrast, iCX26GJCs derived from patient iPSCs (GP5-235delC, GP6-235delC) partially shortened GJPs (Figure 5D, middle, right column). As shown in Figure 5E, the GJP length in diseased iPSC-derived iCX26GJCs (GP5-235delC-iCX26GJC: 6.13 ± 0.35 μm; GP6-235delC-iCX26GJC: 5.91 ± 0.37 μm) was significantly shorter than that in normal iPSC-derived iCX26GJCs (8.62 ± 0.31 μm).

[0036] (4) Functional evaluation of gap junction intercellular communication (GJIC) in iCX26 GJCs derived from healthy and diseased iPSCs To investigate whether there were differences in the function of the GJIC network between healthy and diseased iCX26GJCs, we performed a scrape-loading / dye permeability test (SL / DT) using Lucifer Yellow (Stem Cell Reports, 7, 1023-1036 (2016), Am. J. Physiol. Cell Physiol., 293, C1032-1048 (2007)). SL / DT was performed on iCX26GJC, GP5-235delC-iCX26GJC, and GP6-235delC-iCX26GJC derived from iPSCs from a healthy donor (201B7-iCX26GJC). For comparison, undifferentiated iPSCs (201B7) and feeder cells (TRIC) were included. The extent of dye transfer was quantified by measuring the distance from the scrape line to the point where the fluorescence intensity dropped to background fluorescence intensity. In these iCX26GJC cultures, we observed Lucifer Yellow diffusing beyond the injured parental cells (Figure 6, G, H, J, K, M, and N), indicating the presence of GJIC. In contrast, this degree of dye permeability was not observed in undifferentiated iPSCs or TRIC feeder cells (Figure 6, A, B, D, and E). As shown in Figure 6P, the quantified distances of dye permeability in 201B7-iCX26GJCs (114.3 ± 4.05 μm), GP5-235delC-iCX26GJCs (36.4 ± 1.55 μm), and GP6-235delC-iCX26GJCs (33.3 ± 0.69 μm) were significantly longer than those in undifferentiated iPSCs (22.4 ± 0.65 μm) or TRIC feeder cells (23.8 ± 0.99 μm). Furthermore, the dye penetration distance of both patient-derived iCX26JC (GP5-235delC-iCX26JC and GP06-235delC-iCX26JC) was significantly shorter than that of iCX26JC generated from iPSCs without the GJB2 mutation (201B7-iCX26JC).

[0037] (Consideration) (1) Efficient method for differentiation of human iPS cells into iCX26GJC In this study, CX26 gap junction-forming cells (iCX26GJCs), which have the properties of cochlear supporting cells, were generated from human iPSCs using SFEBq culture and adherent culture as described in Patent Document 1. Furthermore, this induction method was applied to patient-derived iPSCs to reproduce the pathology of GJB2-related hearing loss. The SFEBq culture system is the most suitable method for inducing various ectoderm-derived tissues, such as the forebrain, midbrain, hindbrain, optic cup, and ear cup from ESCs / iPSCs. The use of gfCDM and the addition of insulin in SFEBq culture promotes differentiation into the midbrain and hindbrain regions and increases the number of these regions contained in embryoid bodies. Our target, the inner ear, originates from the otic placode, a superficial part of the non-neural ectoderm (NNE) adjacent to part of the hindbrain. Based on the above, we hypothesized that these conditions for SFEBq culture are suitable for ear induction. Several reports on the differentiation of ESCs / iPSCs into otic progenitor cells (OPCs) have used PAX2, PAX8, and GATA3 as OPC markers. In this study, in addition to these gene sets, we also used GJB2 and GJB6 as indicators of culture conditions. The GJB6 gene encodes the CX30 protein and is co-expressed with CX26 in cochlear supporting cells. We confirmed that these marker genes were upregulated by the addition of insulin in SFEBq cultures. These results suggest that adding insulin to gfCDM in SFEBq culture is the most suitable method for inducing iCX26GJCs. Several reports have characterized cochlear-like cells derived from ESCs / iPSCs using multiple markers. Using immunostaining (CX30, SOX2, SPARCL1, KCC3, and some cytokeratins) and qPCR (SOX2, KIAA1199, SPARCL1, MIA, and OTOR), we confirmed that these markers were expressed in human iCX26GJCs expanded in adherent culture after SFEBq culture. Several studies have reported that these protein or gene markers are expressed in cochlear supporting cells or fibrocytes. Based on these characteristics, iCX26GJCs are considered to be mammalian cochlear supporting cells or fibrocytes.

[0038] (2) iCX26GJC derived from patient iPSCs reproduced the pathology of GJB2-related hearing loss. In the mammalian cochlea, intercellular ion movement via connexin gap junctions maintains cochlear homeostasis. Previous studies using transfected COS-7 cells suggest that aberrant subcellular localization of CX26 protein induced by 235delC leads to loss of function and severe hearing impairment.

Claims

1. A method for producing inner ear cells having connexin 26 gap junctions, comprising culturing pluripotent stem cells in a medium supplemented with 1 to 20 μg / mL of insulin, and culturing the resulting embryoid bodies in the presence of cochlear-derived feeder cells.

2. 2. The method for producing inner ear cells according to claim 1, wherein the cochlea-derived feeder cells are cells formed by trypsinizing cochlear cells and culturing them to form colonies.

3. 3. The method for producing inner ear cells according to claim 1 or 2, wherein the pluripotent stem cells are iPS cells or ES cells.

4. The method for producing inner ear cells according to any one of claims 1 to 3, wherein the pluripotent stem cells are human iPS cells or human ES cells.

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