1. Volume 18, Issue 1, Pages 68-81 (January 2017)
Cochlear Cell Modeling Using Disease-Specific iPSCs Unveils a Degenerative Phenotype and Suggests Treatments for Congenital Progressive Hearing Loss 
Makoto Hosoya, Masato Fujioka, Takefumi Sone, Satoshi Okamoto, Wado Akamatsu, Hideki Ukai, Hiroki R. Ueda, Kaoru Ogawa, Tatsuo Matsunaga, Hideyuki Okano 
Cell Reports 
Volume 18, Issue 1, Pages (January 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 68-81DOI: (10.1016/j.celrep.2016.12.020)
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3. Figure 1 Clinical Findings of Enrolled Patients
We established iPSCs from three enrolled patients harboring biallelic point mutations of the SLC26A4 gene.
(A–D) T410M lines were established from patient 1, a 4-year-old girl. She had bilateral severe sensorineural hearing loss (A). Direct sequencing analysis revealed a homozygous missense mutation, c.1229 C > T (p. Thr410Met) within the SLC26A4 gene (D).
(E–H) H723R lines were established from patient 2, a 7-year-old girl (E). She had bilateral mild sensorineural hearing loss. Direct sequencing analysis revealed a homozygous missense mutation, c.2168 A > G (p. His723Arg) within the SLC26A4 gene (H).
(I–L) M147V lines were established from patient 3, a 34-year-old woman (I). She had bilateral severe sensorineural hearing loss at moderate to high frequencies. Direct sequencing analysis revealed compound heterozygous missense mutations, c.439 A > G (p. Met147Val)/c.2168 A > G (p. His723Arg) within the SLC26A4 gene (L). CT scan results revealed mild malformations of the cochlea (arrow head in B, F, and J) with prominent EVA (arrow in C, G, and K) in all patients.
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4. Figure 2 Induction of hESCs or hiPSCs to Otic Progenitor Cells
(A) Schematic overview of the induction methods for OPCs.
(B–G) Induced OPCs derived from hiPSCs expressed OPC markers PAX8 (B and C), FOXG1 (B and F), PAX2 (C), SOX2 (B and C), OTX1 (D–G), GATA3 (E), and TBX1 (G). Scale bars represent 100 μm. The nuclei were counterstained with Hoechst (blue).
(H) The expression of PAX2, PAX8, GATA3, FOXG1, HMX3, DLX5, FBX2, EYA1, SIX1, OTX1, OTX2, and SOX2 was examined by RT-PCR.
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5. Figure 3 Induction of Cochlear Outer Sulcus Cells
(A) Schematic overview of the OSC induction method.
(B–E) Induced OSCs expressed cochlear OSC markers, including pendrin (B and C), CX30 (C), ATP6B1 (D), KIAA1199 (D), CRYM (D), and AQP4 (E). Scale bars for (B) represent 200 μm, for (C) represent 50 μm, and for (D) and (E) represent 100 μm. The nuclei were counterstained with Hoechst (blue).
(F and G) Induced OSCs expressed various mature cochlear OSC markers, including SLC26A4, KIAA1199, CRYM, DFNA5, WFS1, GRHL2, and AQP4, which were measured by RT-PCR (F), as well as pendrin, ATP6B1, CX26, CX31, and AQP4, which were measured by western blotting (G). Note that the expression level of SOX2, which is expressed in ES cells and OPCs, was dramatically reduced in differentiated OSCs (F). Western blotting revealed that pendrin protein was heavily glycosylated and could dimerize (G).
(H) The Cl–-dependent intracellular HCO3– distribution in the induced OSCs was examined using the fluorescent dye BCECF-AM. The addition of a specific Cl− blocker, niflumic acid, abolished the fluorescent signal, which indicates that this effect depends on anion exchange activity. ∗∗p < 0.01 ∗p < Bar and error bars represent the mean ± SEM (n = 16 per each).
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6. Figure 4
Patient-Derived Cells Showed Anion Exchange Activities and Mutated Pendrin Did Not Localize to the ER
(A) Cl–/HCO3– exchange activity on the whole-cell level did not significantly differ between control (WD39, 201B7, and H1) and patient (T410M, H723R, and M147V) cells.
(B) Induced OSCs expressed various Cl–/HCO3– exchangers. These results suggest that other SLC family members could compensate for SLC26A4 dysfunction in these cells.
(C–F) Mutated pendrin localized to the cytoplasm of induced OSCs derived from patients. Note that the protein was not retained in the ER. WD39: WD39 control iPS line (C), T410M (D), H723R (E), M147V (F): disease lines. Scale bars for (A)–(F) represent 50 μm. The nuclei were counterstained with Hoechst (blue).
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7. Figure 5
Patient Cells that Showed Intracellular Pendrin Aggregates Were More Susceptible to Stress-Induced Apoptosis
(A) Patient-derived cells showed intracellular pendrin aggregates (arrow).
(B) Significantly more patient-derived cells showed intracellular pendrin aggregates (n = 4).
(C) Patient-derived OSCs were more susceptible to proteasome inhibition by epoxomicin.
(D) The number of apoptotic cells increased in patient-derived cells when cells were stressed by exposure to epoxomicin.
∗∗p < 0.01, ∗p < Bars and error bars represent the mean ± SEM. Scale bar of a represent 100 μm. The nuclei were counterstained with Hoechst (blue).
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8. Figure 6 Site-Specific Gene Correction
(A) Schematic overview of the gene repair strategy for the human SLC26A4 gene. We designed SLC26A4-specific TALEN-mediated break points to be repaired by homologous recombination. The desired recombination event inserts a PGK promoter-puromycin resistance-TK cassette flanked by loxP sites. Arrows 1–6 indicate primer binding sites for PCR genotyping.
(B) Site-specific gene correction did not affect the molecular weight of pendrin.
(C and D) Site-specific gene correction reduced the intracellular aggregation of pendrin (arrow). Scale bars represent 100 μm. The nuclei were counterstained with Hoechst (blue).
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9. Figure 7
Rapamycin and Metformin Ameliorated PDS Patient-Specific Phenotypes
(A) Site-specific gene correction of the H723R #16 hiPSC line using TALENs. Four heterozygous corrected cell lines, and three homozygous corrected lines were obtained.
(B) Significant decreases in the numbers of the cells that showed intracellular pendrin aggregates were observed in heterozygous (yellow) and homozygous (green) corrected cell lines compared with the H723R #16 line (red). Note that the cell counts for the heterozygous, homozygous, and healthy control lines did not significantly differ (blue).
(C) Susceptibility to the cellular stress induced by proteasome inhibition was also ameliorated by site-specific gene correction. All genetically corrected lines showed increases in cell viability compared with the parental cell line. Notably, significant differences were observed between heterozygous lines and homozygous or control lines. These results demonstrate that the mutation is responsible for the degenerative phenotypes observed in PDS patient-derived OSCs. Furthermore, heterozygous mutations of the SLC26A4 gene may mildly sensitize cells to stress.
(D–F) Rapamycin ameliorated cell viability (D) and decreased apoptosis (E) in cells subjected to stress conditions for all nine patient-derived cell lines (F).
(G and H) Metformin treatment also resulted in a dose-dependent decrease in cellular stress susceptibility (Epox, epoxomicin; Metf, metformin).
(I) Schematic diagram of the proposed pathophysiology of PDS/DFNB4. Increased susceptibility with the intracellular aggregation of mutated pendrin results in hearing loss due to cochlear cell damage or death. Genetic or environmental backgrounds and EVA affects this process. ∗∗p < 0.01, ∗p < Error bars, mean ± SEM.
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