Engraftment of Human Stem Cell-Derived Otic Progenitors in the Damaged Cochlea

Abstract

Most cases of sensorineural deafness are caused by degeneration of hair cells. Although stem/progenitor cell therapy is becoming a promising treatment strategy in a variety of organ systems, cell engraftment in the adult mammalian cochlea has not yet been demonstrated. In this study, we generated human otic progenitor cells (hOPCs) from induced pluripotent stem cells (iPSCs) in vitro and identified these cells by the expression of known otic markers. We showed successful cell transplantation of iPSC-derived-hOPCs in an in vivo adult guinea pig model of ototoxicity. The delivered hOPCs migrated throughout the cochlea, engrafted in non-sensory regions, and survived up to 4 weeks post-transplantation. Some of the engrafted hOPCs responded to environmental cues within the cochlear sensory epithelium and displayed molecular features of early sensory differentiation. We confirmed these results with hair cell progenitors derived from Atoh1-GFP mice as donor cells. These mouse otic progenitors transplanted using the same in vivo delivery system migrated into damaged cochlear sensory epithelium and adopted a partial sensory cell fate. This is the first report of the survival and differentiation of hOPCs in ototoxic-injured mature cochlear epithelium, and it should stimulate further research into cell-based therapies for treatment of deafness.

Keywords: animal model of ototoxicity; cell injection assay; engraftment; human induced pluripotent cells; in vitro otic model; migration; otic progenitor cells; sensory cell differentiation; transplantation.

Graphical abstract

Figure 1

A Model of Ototoxic Trauma-Induced Sensori-Neural Hearing Loss (A) Timeline of ototoxic drug lesion and electrophysiological measurements. (B) Auditory thresholds were measured by ABR. Pooled threshold values are represented by a dot (n ≥ 4 per group) and the SD indicated by a bar. After amikacin exposure, the ABR measurements showed a threshold shift of at least 20 dB at all the frequencies tested. Red arrows represent animals with an auditory threshold greater than 90 dB for frequencies from 16 to 32 kHz. Wilcoxon rank-sum test *p < 0.05, **p < 0.01, ***p < 0.001. (C) Confocal images of immunostaining for MYO7A (shown in red) and SOX2 (shown in green) on whole-mount surface preparations from second and basal cochlear turns (i.e., regions coding frequencies of 2, 4, and 8 kHz) of control-untreated and amikacin-treated animals. Amikacin exposure resulted in a severe loss of OHCs within the basal cochlear turn. Most of OHCs were missing, but a few MYO7A- and SOX2-immunopositive cells remained within the region of damaged cochlear sensory epithelium (arrows). Most of IHCs remain after amikacin treatment. IHCs, inner hair cells; OHCs, outer hair cells; TC, tunnel of Corti. Scale bars, 50 μm in all panels.

Figure 2

Surgical Approach for Cell Injection into the Scala Tympani (A) Timeline of human iPSC-derived otic progenitors in vitro and in vivo transplantation in adult guinea pig model of ototoxicity. (B–D) The anatomical landmarks used to ensure that the site of cochleostomy was consistent between animals are shown. A cochleostomy was performed at the base of the cochlea; dashed box in (B) and (C) and red arrow in (D) driving the cells into the scala tympani. (E) Schematic representation of sequential steps to inject hOPCs into the scala tympani by cochleostomy. (F) ABR measurements were performed on amikacin-untreated animals before cochleostomy (D19), 4 days after cochleostomy (D23), and 14 days after cochleostomy (D33). Auditory threshold values of pooled data (n ≥ 3 per group) are represented by the means (dot) ± SD (error bar). Wilcoxon rank-sum test, no statistically significant differences were found between groups before and after cochleostomy. RW, round window; ST, scala tympani. Scale bars, 500 μm.

Figure 3

Schematic Illustration of the Generation of Human Otic Progenitors from iPSCs Using a Monolayer Culture Protocol (A) Schematic representation of otic progenitor cells generation from hiPSCs by exposure to FGF3 and FGF10 (50 ng/mL each) in DFNB medium for 13 days in vitro. In some FGF-treated cultures, hOPCs at day 13 in vitro were differentiated into late otic sensory cells by exposure to GSI (5 μM) until day 20. (B) QPCR analysis of a panel of known otic progenitor (pax2, gata3, dlx5, bmp4, pax8) gene markers in FGF-treated cultures at day 13 as compared to their relative expression levels in undifferentiated hiPSCs at day 0. Relative gene expression levels were normalized against gapdh gene and compared to hiPSCs at day 0. Group means and SD are shown. An unpaired Wilcoxon matched-pairs signed-rank test was used for single comparisons; statistical differences are indicated by *p < 0.05, **p < 0.01; ns, no statistically significant difference (n = 3). (C–H) Representative immunostainings for EYA1 (D) PAX2 (F), and SOX2 (G) in day 13 FGF-treated cultures. A population of PAX2 (shown in white), SOX2 (shown in red), and EYA1 (shown in red) immunopositive cells are observed in these differentiated cultures. DAPI staining is shown in blue. (I–K) A sample of differentiated cells at day 13 was labeled with the Vybrant-Dil (J) tracer and immunostained with PAX2 antibody (I). Double labeling (K) indicated that virtually all Dil-labeled cells (shown in red) are immunopositive for PAX2 (shown in green). DAPI staining is shown in blue. FGF, fibroblast growth factor; DFNB, DMEM/F12 with N2/B27; GSI, gamma secretase inhibitor. Scale bars, 20 μm in all panels.

Figure 4

Human Otic Progenitors Trigger an Immune Reaction after Transplantation (A) Timeline of experimental design for hOPC transplantation into control and implanted drug-exposed cochleae. (B–D) IBA1-immunopositive cells (shown in green) are revealed by immunohistochemistry on transverse sections from cochleae of control untreated (B), no-implanted, 4 days post-implanted (C), and 14 days post-implanted drug-treated animals (D). The Vybrant-Dil-labeled cells are shown in red. Some of IBA1-immunopositive cells are observed at the basal part of the damaged organs of Corti (arrows). A slight IBA1 immunoreactivity was detected in some hair cells mostly those of amikacin-treated day 4 post-grafted cochlea (C, asterisks). Arrowheads show Dil-positive/IBA1 negative cells in (D). (E–G) Representative images from the modiolus areas of amikacin-untreated ungrafted animals (E) and amikacin-treated day 33 post-grafted animals (G) showing expression of IBA1 in many cells. (F) In the modiolus of amikacin-treated day 4 post-grafted animals, few Dil-labeled cells (shown in red, arrows) were surrounded by IBA1 immunostaining (shown in green, arrows). Nuclei were revealed by DAPI staining (shown in blue). Scale bars, 20 μm in all panels. SM, scala media; ST, scala tympani; TC, tunnel of Corti.

Figure 5

Stereological Imaging of Fluorescent Dil-Labeled Otic Progenitors in Ototoxic Damaged Cochlea (A–D) The distribution of Dil-labeled cells was analyzed on grafted cleared mature guinea pig cochlea imaged through its entire volume. (A) Representative 3D reconstruction obtained from cochlea dissected at 14 days post-implantation after tissue clearing. Dil-labeled cells (shown in red) were observed at different cochlear turns. (B) Magnification of dotted area in (A) allowed to visualize Dil-labeled cells (in red) within the cochlear epithelium. (C) Virtual transversal slice obtained from (A), where it is possible to observe some Dil-labeled cells at level of organ of Corti region. (D) Magnification of dotted area in (C) showing the organ of Corti region. (E) Distribution of Dil-labeled cells was analyzed on cochlear whole-mount surface preparations. The X (in red) and Y (in green) axes are represented in the image. Yellow dotted line indicates the area of the stria vascularis. (F) Virtual 3D reconstruction of the cochlear sensory epithelium was obtained by transposition of Z-stack images acquired from whole-mount preparations with confocal microscopy. The X (red line), Y (green line), and Z (blue line) axes are represented in the image. (G) MYO7A-immunopositive cells (shown in green) were revealed at the level of IHCs row by immunohistochemistry. Some Dil-positive cells were also observed in the OHC region. Nuclei were revealed by DAPI staining (shown in blue). Scale bars, 100 μm (B and C), 200 μm (E), and 20 μm (G). TC, tunnel of Corti; IHCs, inner hair cells; OHCs, outer hair cells.

Figure 6

Number of Surviving Transplanted Cells Increased in Animals Pre-treated with Cyclosporin (A) Timeline of experimental setup for hOPC transplantation in cyclosporin-treated versus cyclosporin-untreated grafted animals. (B) Representative images of the whole-mount surface preparation of cochlear epithelia at 14 days post-implantation. Dil-labeled cells (red) correspond to grafted hOPCs and nuclei were stained with DAPI (blue). (C) The number of Dil-labeled cells were quantified in cochlear sensory epithelium area at different cochlear turns. The CSE was represented in the space between dotted line in (B). Cyclosporin-treated group is represented in green color, and cyclosporin-untreated group is represented in orange color. Each floating bar represents group means (line) and min to max (box) values (n = 3). ANOVA test was used for multiple comparisons; different letters indicate significant differences between groups (**p < 0.005). (D) Virtual transversal images of cochlear sensory epithelium were obtained by 3D reconstructions of Z-stack series. Schematic reference to basilar membrane (BM) and reticular lamina (RL) is shown with dotted lines. The distance of Dil-labeled cells from basilar membrane was determined by Zen software. (E) The percentages of cells found at different distances was determined by the ratio of number of cells quantified in a delimited distance/total number of cells quantified into the entire turn of the cochlea. Data shown correspond to the basal turn of the cochlea from cyclosporin-treated (shown in green) and cyclosporin-untreated (shown in orange) animals. CSE, cochlear sensory epithelium; OHC, outer hair cell; IHC, inner hair cell; BM, basilar membrane; RL, reticular lamina. Scale bar, 20 μm.

Figure 7

Human Otic Progenitors Expressed Late Otic Sensory Markers 14 Days Post-implantation In Vivo (A) Transplanted Dil-hOPCs in a transverse section of drug-treated cochlea. A projection of a confocal image stack showing MYO7A (shown in green) and SOX2 (shown in white) immunoreactivity, Vybrant-Dil (shown in red), and nuclear DAPI staining (shown in blue). We observed a complete loss of OHCs, but the IHC (arrow) remained in the ototoxin-damaged organ of Corti (OC, dotted line). (B–B″) Enlarged region of boxed area in (A) showing MYO7A/Dil-positive cells (arrows) at the basal part of the organ of Corti. (C) Area from whole-mount surface preparation of another ototoxin-damaged and grafted cochlea showing two Dil-labeled cells (red) immunopositive for MYO7A (green). (D–D″) Magnification of a dotted area in (C) with separated channel combinations: Dil-labeled cells (red), MYO7A-immunopositive cells (green), merge (yellow). (E) A confocal image of another ototoxin-damaged cochlea showing implanted Dil-labeled cells and nuclear SOX2 immunostaining (white). (F–F″) A magnification of boxed area in (E) revealed some SOX2/Dil cells at the basal area of the organ of Corti (arrows). Scale bars, 20 μm (A and E), 10 μm (C and F), and 5 μm (B′–B″, D′–D″, and F′–F″).

Figure 8

Survival, Distribution, and Cell Fate of Mouse Atoh1-GFP Progenitor Cells 4 Weeks Post-transplantation (A) Timeline of the experimental design used for Atoh1-GFP progenitor cell transplantation including cyclosporin treatment. (B) The HC markers MYO7A (shown in red) and POU4F3 (shown in white) were observed on cochlear whole-mount surface preparations as revealed by immunohistochemistry. Few Atoh1-GFP-injected cells (shown in green) migrated to the cochlear sensory epithelium and were found below the IHC region (MYO7A/POU4F3) immunopositive cells. (C) Engrafted Atoh1-GFP cells (green) moderately expressed the POU4F3 (shown in white, arrows). (D) 3D reconstitution of whole-mount preparation outlining the X (in red), Y (in green), and Z (in blue) axes. (E) Virtual transverse image confirming the spatial localization of Atoh1-GFP engrafted cells (arrows) in the area below the MYO7A immunopositive endogenous residual IHC. Scale bar, 10 μm (B and E) and 5 μm (C). IHC, inner hair cell; BM, basilar membrane.