Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation - PubMed (original) (raw)

. 2016 Aug;23(8):1347-57.

doi: 10.1038/cdd.2016.16. Epub 2016 Feb 26.

Z-H Tang 1, J Zheng 2, H-S Shi 3, J Ding 1, X-D Qian 4, C Zhang 1, J-L Chen 1, C-C Wang 1, L Li 1, J-Z Chen 5, S-K Yin 3, J-Z Shao 1, T-S Huang 6, P Chen 7, M-X Guan 2, J-F Wang 1

Affiliations

Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation

J-R Chen et al. Cell Death Differ. 2016 Aug.

Abstract

Deafness or hearing loss is a major issue in human health. Inner ear hair cells are the main sensory receptors responsible for hearing. Defects in hair cells are one of the major causes of deafness. A combination of induced pluripotent stem cell (iPSC) technology with genome-editing technology may provide an attractive cell-based strategy to regenerate hair cells and treat hereditary deafness in humans. Here, we report the generation of iPSCs from members of a Chinese family carrying MYO15A c.4642G>A and c.8374G>A mutations and the induction of hair cell-like cells from those iPSCs. The compound heterozygous MYO15A mutations resulted in abnormal morphology and dysfunction of the derived hair cell-like cells. We used a CRISPR/Cas9 approach to genetically correct the MYO15A mutation in the iPSCs and rescued the morphology and function of the derived hair cell-like cells. Our data demonstrate the feasibility of generating inner ear hair cells from human iPSCs and the functional rescue of gene mutation-based deafness by using genetic correction.

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Figures

Figure 1

Figure 1

Identification of a Chinese family carrying MYO15A mutations. (a) The genogram outlining the inheritance of two MYO15A mutations, c.4642G>A (p.A1548T) and c.8374G>A (p.V2792M) was identified by gene screening of a panel of 69 genes. Family members marked by a * were donors for the next-generation sequencing of deafness genes, Sanger validation, and hearing tests. The 2-year-old male child (III:1) carries two MYO15A mutations. The inheritance of the MYO15A mutations in the family indicated that the child carries compound heterozygous MYO15A mutations. (b) Sanger sequencing confirmed the MYO15A mutations identified in the family members. Double peaks represent a heterozygous mutation. (c) Audiograms of the family carrying MYO15A mutations. Audiograms of the 2-year-old male carrying the compound heterozygous MYO15A mutations (III:1), the parents each carrying a single MYO15A mutation (II:1 and II:2), and the paternal grandparents with the grandfather carrying a single MYO15A mutation (I:1 and I:2). Note that the parents and grandmother have normal hearing. The grandfather has normal hearing below 3 KHz and normal conversational hearing with peak hearing loss at frequencies between 4–6 KHz. An X represents the audition of the left ear and a circle represents the audition of right ear

Figure 2

Figure 2

Generation and characterization of iPSCs from human dermal fibroblasts. (a) Phase contrast microscopy of dermal fibroblasts and derived iPSCs and AP staining of the four putative iPSCs. (b) Immunostaining for pluripotent markers OCT4, SOX2, NANOG, SSEA4, TRA-1–60, and TRA-1-81 in the four putative iPSCs. Nuclei were stained with DAPI (blue). (c) Embryoid bodies formed after 8 days of suspension culture. (d) Teratomas formed from the NOD-SCID mice and stained with hematoxylin and eosin. Tissue structure characteristics of the three germ layers were observed (gut epithelium for endoderm, cartilage for mesoderm, as well as neural rosettes and retinal pigment epithelium for ectoderm). Scale: 50 _μ_m in NANOG images; 100 _μ_m in the other images

Figure 3

Figure 3

Induction of human iPSCs toward otic progenitor differentiation. (a–c) Human iPSCs were induced toward otic progenitors and stained for the early otic markers, PAX8+NES (a), PAX8+PAX2 (b), and PAX8+SOX2 (c). Nuclei were stained with DAPI (blue). Scale: 30 _μ_m. (d) RT-PCR analysis of the expression of early otic marker genes. Expression of otic progenitor marker genes, PAX2, PAX8, GATA3, EYA1, SIX1, and DLX5, was detected in all four iPSCs induced toward the otic lineage. (e) The relative gene expression levels were quantified and plotted. The housekeeping gene, GAPDH, was used as an internal reference. Error bars represent the S.D. (_n_=3)

Figure 4

Figure 4

Induction of otic epithelial progenitors toward inner ear hair cell differentiation. (a–c) Generation of inner ear hair cell-like cells from otic epithelial progenitors as revealed by immunostaining with antibodies for hair cell markers BRN3C+ATOH1 (a), BRN3C+MYO7A (b), and BRN3C+ESPN (c). Nuclei were stained with DAPI (blue). Scale: 100 _μ_m in MYO7A images, 30 _μ_m in ATOH1 images and 3 _μ_m in ESPN images. (d) Western blot analysis of the expression of hair cell marker genes. (e) The relative expression levels of hair cell marker proteins were quantified and plotted. The housekeeping gene, GAPDH, was used as an internal reference. Error bars represent the S.D. (_n_=3). (f) RT-PCR analysis of the expression of hair cell marker genes. Expression of inner ear hair cell marker genes such as BRN3C, ATOH1, MYO7A, as well as genes encoding stereocilia proteins, ESPN and MYO15A, were detected. CUSC represents chicken embryonic utricle stromal cells. (g) The relative expression levels of hair cell marker genes were quantified and plotted. The housekeeping gene, GAPDH, was used as an internal reference. Error bars represent the S.D. (_n_=3)

Figure 5

Figure 5

Hair cell-like cells from iPSCs have hair cell-like electrophysiological properties. (a) Cells stained with FM1-43 (green). Nuclei were stained with DAPI (blue). Cells with FM1-43-positive staining were sorted by FACS for electrophysiological recordings. Scale: 100 _μ_m. (b) The percentage of FM1-43 positive cells in the total number of induced cells. Error bars represent the S.E.M. (_n_=8), * represents statistically significant difference (P<0.05). (c) RT-PCR analysis of the expression of genes specific for hair cell mechanotransduction, including TMC1/2, TMHS, and TMIE. (d) The relative expression levels of genes specific for hair cell mechanotransduction were quantified and plotted. The housekeeping gene, GAPDH, was used as an internal reference. Error bars represent the S.D. (_n_=3). (e–f) Hair cell-like cells displayed voltage-dependent K+ currents, outward _I_K (c) and inward _I_K1 (d). (g) Voltage-dependent inward _I_Ca was recorded from hair cell-like cells. Currents were elicited by 10 voltage steps from the holding potential (c: –104 mV; d: –64 mV; e: −84), the peak current voltages are also indicated. (h) Statistical analysis of average peak current density in the hair cell-like cells induced from M+/+iPSCs, M+/−iPSCs, M−/−iPSCs, and MC/−iPSCs. Average peak current density is equal to the peak current divided by the capacitance. Details are listed in Supplementary Table S1. A two-tailed test was used for pair-wise comparison. ** represents P<0.01 compared with others. Error bars represent the S.E.M. (_n_=8)

Figure 6

Figure 6

Functional rescue of hair cell-like cells from MYO15A compound heterozygous mutant iPSCs by CRISPR/Cas9 genetic correction. (a) Immunostaining of F-actin in hair cell-like cells. F-actin stress fibers were visualized in hair cell-like cells derived from M+/+iPSCs, M+/−iPSCs and MC/−iPSCs by staining with F-actin dye phalloidin (green). White box: syncytia were observed in hair cell-like cells derived from M−/−iPSCs. Nuclei were stained with DAPI (blue). Arrow: stereocilia-like structure. Scale: 30 _μ_m in the left two columns, 6 _μ_m in the third column. (b) S.E.M. of hair cell-like cells derived from iPSCs. A stereocilia-like structure was observed. (c) The stereocilia length was measured using ImageJ software. The average stereocilia length of hair cell-like cells from M+/+iPSCs, M+/−iPSCs, and MC/−iPSCs was 3.52±0.29 _μ_m, 3.41±0.24 _μ_m, and 3.05±0.23 _μ_m, respectively. The stereocilia length of M−/−iPSCs was significantly shorter at 0.94±0.22 _μ_m. A two-tailed test was used for pair-wise comparison. *** represents a high statistically significant difference (P<0.001). Error bars represent the S.E.M. (_n_=8)

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