PA gel preparation

IKVAV PA gels (palmitoyl-VVAAEEEEGIKVAV-COOH) were synthesized as outlined by McClendon et al [12]. A PA gel with a scrambled IKVAV sequence (VVIAK: valine, valine, isoleucine, alanine, and valine) was also created as a control. PAs were synthesized using standard fluorenylmethoxycarbonyl (FMOC) solid-phase peptide synthesis and purified by preparative-scale reverse-phase HPLC with water and acetonitrile containing 0.1% NH₄OH. Solutions of PAs, composed of 0.5% E2 PA (palmitoyl-VVAAEE-NH₂) and either 0.125% IKVAV-PA or VVIAK-PA in 150 mM NaCl and 3 mM KCl (pH 7.4), were thermally annealed at 80°C for 30 minutes and slowly cooled to room temperature overnight to induce formation of aligned bundles of nanofibers suitable for shear-induced alignment [30]. Cells were mixed (as described below) with the PA solutions after the annealing and cooling steps. A PA covalently conjugated to the fluorophore TAMRA (Ex/Em 565/580 nm) was added at 0.001 wt % in PA gels for fluorescence imaging for the injection into human cadaveric temporal bones. 3-D visualization of the IKVAV molecule was constructed using the open-source MolView v2.4 software (molview.org).

Human ESC cultures

Frozen undifferentiated H1, H7, and H9 hESCs (passages 25–35) (WiCell Research Institute, Madison, Wisconsin, USA) were cultured in hESC culture medium as previously described (also see S1 Supporting Information) [8,31]. Maintenance was performed through daily media changes and passaging using Gentle Cell Dissociation Reagent (STEMCELL Technologies, Cambridge, Massachusetts, USA). Pluripotency was maintained via removal of differentiated colonies by standard picking and manual passage techniques, and verified prior to ONP differentiation by magnetic activated cell sorting for TRA-1-60⁺ cells (Miltenyi Biotec, Bergisch Gladbach, Germany). In human cadaveric temporal bone experiments, the resultant cell pellet was washed with phosphate-buffered saline (PBS) prior to staining for TRA-1-81 (undifferentiated hESC marker) [32].

Generation of hESC-derived ONPs

Differentiation of hESCs was performed following the protocol recently published by Matsuoka et al. [8]. Undifferentiated hESCs were passaged from the feeder layer and cultured in a 100-mm dish coated with Matrigel^™ (BD Pharmingen, San Jose, California, USA) using Essential 8^™ Medium (Life Technologies, Grand Island, New York, USA) or mTeSR^™1 (STEMCELL Technologies, Cambridge, Massachusetts, USA). In some cases, Geltrex^™ (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was substituted for Matrigel^™. The authors noted little difference between the two coatings in terms of ONP generation. Human ESCs were allowed to proliferate for 2 days prior to adding a chemically defined medium containing N2 and B27 supplements (N2B27-CDM). Ligands and growth factors were added in stepwise fashion to promote hESC differentiation to ONPs as shown in Fig 1, adapted from Matsuoka et al. and previously described [8]. Early-stage ONPs are defined as preplacodal ectoderm (PPE)-like cells treated with 100 ng mL^-1 human Wnt3a, 10 ng mL^-1 human fibroblast growth factor 2 (FGF2), and 50 ng mL^-1 insulin-like growth factor (1GF-1) (W/F/I) for 5 days. Mid-stage ONPs are defined as PPE-like cells treated with W/F/I for two additional days, followed by treatment with 500 ng mL^-1 Sonic hedgehog (SHH), 0.5 μM all-trans retinoic acid (ATRA), 10 ng mL^-1 FGF2, 20 ng mL^-1 EGF, and 50 ng mL^-1 IGF-1 (S/R/E/F/I) for 3 days. Late-stage ONPs are defined as mid-stage ONPs treated with S/R/E/F/I for 4 additional days (Fig 1). In a monolayer (2-D matrix) culture environment, mid-stage ONPs were neuronally differentiated into late-stage ONPs on Matrigel™. Alternatively, IKVAV-PA gels and VVIAK-PA gels were used to assess neuronal differentiation into late-stage ONPs within 3-D matrices. Mid-stage ONPs were seeded onto a 24-well plate at densities of 10,000–25,000 cells cm^-2 (poly-ornithine/laminin) or, for the PA gels, cell suspensions were mixed with PA solution (after the heating and cooling steps) at a 1:2 ratio (75 μL total volume per well, 5,000 cells μL^-1). Cells were allowed to grow for 7 additional days.

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Fig 1. Schematic summary of the protocol and timeline for deriving the SGN lineage from undifferentiated hESCs.

DIV: day in vitro; NNE: nonneuronal ectoderm; PPE: preplacodal ectoderm; ONP: otic neural progenitor; BMP4: bone morphogenetic protein 4; SHH: Sonic hedgehog; ATRA: all-trans retinoic acid; EGF: epidermal growth factor; BDNF: brain-derived neurotrophic factor; NT-3: neurotrophin 3; IGF-1: insulin-like growth factor 1; FGF2: fibroblast growth factor 2; E8: Essential 8^™ medium; N2B27-CDM: chemically defined medium containing N2 and B27 supplements; MACS: magnetic-activated cell sorting; FACS: fluorescence-activated cell sorting; p75: low-affinity neurotrophin receptor (p75^NTR); IKVAV-PA: IKVAV-containing peptide amphiphile; VVIAK-PA: VVIAK-containing peptide amphiphile. Protocol adapted and modified from Matsuoka et al. [8].

https://doi.org/10.1371/journal.pone.0190150.g001

Immunocytochemistry, image acquisition, and quantification of cells

Immunocytochemistry was performed using previously described standard techniques [11,18]. Briefly, cells were fixed with 4% paraformaldehyde solution, permeabilized with a nonionic surfactant, and blocked using bovine serum albumin or fetal bovine serum for gels. Cells were then incubated sequentially in primary and fluorescent secondary antibody solutions to allow visualization of targeted proteins by fluorescence microscopy (full details, antibody sources, and concentrations included in S1 Supporting information). Secondary antibody-only controls were performed by omitting the primary antibody during incubation (S1 Fig). For positive controls for immunofluorescence staining of ONPs refer to previous work [8]. Fluorescence imaging was performed on a Zeiss UV-LSM 510 META or a Nikon A1/C2 confocal microscope. Sequential scanning of channels was performed to prevent false-positive co-localization. ImageJ 1.51e 5 [33] was used to quantify images.

Analysis of neurite-bearing cells and growth

To confirm the neuronal origin of the processes extending from mid-stage ONPs treated with S/R/E/F/I and from late-stage ONPs with neuronal differentiation, immunostaining with an antibody against the neuron-specific marker β-III tubulin was used to visualize neurite-bearing cells and quantify their number and length. Images of randomly selected 20 areas of cells on Matrigel™ (control), within VVIAK-PA gels, and within IKVAV-PA gels were obtained at 40× magnification, digitized, and analyzed using ImageJ 1.51e 5 [33]. Neurite-bearing cells were quantified by counting the number of neurons in a field with neurite lengths at least twice the diameter of the cell body. The ratio of neurite-bearing cells to total number of cells was computed for each condition. Neurite length is determined by manually tracing the length of the longest neurite on randomly selected 20 late-stage ONPs using the NIH Image J software 1.51e 5 [33].

Live/dead cell viability assay and EdU cell proliferation assay

In vitro cell viability was assessed using a LIVE/DEAD Viability/Cytotoxicity kit (Life Technologies, Carlsbad, California, USA) with standard techniques. Briefly, cells were stained with both ethidium bromide and calcein as markers for abnormal cell membrane permeability (indicating cell death) and intracellular esterase activity (indicating healthy cell function), respectively. Click-iT^® EdU DNA incorporation assays (Invitrogen, Carlsbad, California, USA) were performed to confirm in vitro cell proliferation using standard techniques. For these assays, cells were fixed and permeabilized as for immunocytochemistry, stained with EdU reagent, and analyzed by fluorescence microscopy.

Generation of EGFP⁺ hESCs using a CRISPR-Cas9 system

EGFP was knocked into the AAVS1 safe harbor locus of H7 strain hESCs using a CRISPR-Cas9 based strategy as previously described with modifications [34,35]. Briefly, hESC line H7 was cultured in mTeSR^TM1 medium (STEMCELL Technologies, Cambridge, MA, USA) on multi-well culture plates coated with Matrigel^TM (BD Pharmigen, San Jose, California, USA) per manufacturer-provided protocols. Cells were fed daily with fresh media. Spontaneously differentiated cells were removed by regular inspection and manual dissection. Cells were passaged as clumps every 5–6 days with Accutase (EMD Millipore, Kankakee, Illinois, USA) at a split ratio of approximately one to twelve. Cells were pre-incubated with 10 μM ROCK Inhibitor Y-27632 (Stemgent, Cambridge, Massachusetts, USA) for 4 hours and dissociated to single cells with Accutase (EMD Millipore, Kankakee, Illinois, USA). The cell suspension was washed and resuspended in DPBS (Life Technologies, Carlsbad, California, USA) containing 1 μg hCas9 vector (Addgene #41815), 1 μg gRNA_AAVS1-T2 vector (Addgene #41818), and 2 μg AAV-CAGGS-EGFP vector (Addgene #22212). The suspension was transferred to an Amaxa electrode cuvette and nucleofected with a Nucleofector II/2b Device (Lonza, Valparaiso, Indiana, USA) using program B-016. 0.5 mL of pre-warmed mTeSR^TM1 media was added to the cells, and the mixture was transferred to conical vial containing 2 mL of additional media. The cells were centrifuged and resuspended in mTeSR^TM1 media supplemented with 10 μM ROCK Inhibitor Y-27632 and plated at a density of 20,000 cell cm^-2 on Matrigel^TM-coated plates. Cells were fed daily with fresh mTeSR^TM1 medium. Cells were selected by supplementing media with 0.5 μg mL^-1 of puromycin 72 hours after transfection. Individual puromycin-resistant hESC colonies were manually picked and expanded. Genomic DNA was isolated using the DNEasy Blood and Tissue Kit (Qiagen, Hilden, Germany), and on-target insertion of the EGFP cassette was confirmed by PCR using primers with complementarity within the AAVS1 homology arms: Forward primer: 5’-CCCCTTACCTCTCTAGTCTGTGC-3’; Reverse primer: 5’-CTCAGGTTCTGGGAGAGGGTAG-3’. The PPP1R12C locus was PCR-amplified with pfx polymerase (Life Technologies, Carlsbad, California, USA) and indicated primers (IDT, Coralville, IA, USA), and the product was analyzed by agarose-gel electrophoresis. Live cells were visualized by brightfield and epifluorescent microscopy (Nikon, Tokyo, Japan).

Percent EGFP retention after late-stage ONP differentiation was assessed by counting fluorescent and total cells in the same field captured by live cell epifluorescent microscopy (Nikon, Tokyo, Japan). Cell counting was performed manually using the NIH ImageJ 1.51e 5 [33]. Retention of fluorescence at this time point is presumed to follow a binomial distribution for error estimation: (σ: standard deviation, n: number of cells, p: probability that a cell has lost fluorescence after differentiation.)

RT-qPCR

RNA was extracted from undifferentiated hESCs (H7), hESC (H7)-derived late-stage ONPs, and EGFP⁺ hESC (H7) derived late-stage ONPs using the mirVANA™ miRNA isolation Kit (Life Techonologies, CA, USA). Twelve-point-five ng of RNA was reverse-transcribed with random primers using the iScript™ Reverse Trancription Superkit for RT-qPCR (Bio-Rad Laboratories, Hercules, California, USA) and the following cycling conditions: 25°C for 5 min, 46°C for 20 min, and 95°C for 1 min. The cDNA was amplified by qPCR using following primers (OCT3/4 forward: 5' ATG TGC AAG CTG AAG CCT TT 3'; OCT3/4 reverse: 5' GCT GCG ATC TTG TCT ATG CTC 3'; NANOG forward: 5' AAC TGG CCG AAG AAT AGC AA 3'; NANOG reverse: 5' CAT CCC TGG TGG TAG GAA GA 3'; GAPDH reverse: 5' TGT TCG TCA TGG GTG TGA AC 3'; GAPDH forward: 5' CTA AGC AGT TGG TGG TGC AG 3') on a Bio-Rad CFX384 (Bio-Rad Laboratories, Hercules, CA) with the following cycling conditions: 45 cycles: (95°C for 10 sec, 64°C for 15 sec, 72°C for 20 sec). C_Τ values were measured in two technical replicates and all values represent three biological replicates.

GAPDH was initially to be used as a reference gene for normalization. However, gene expression varied across conditions (S2 Fig). To avoid the introduction of bias, normalization was performed using the NORMA-gene method [36]. A normalization factor is computed for each cell type replicate based on the variation among replicates within the cell type across all target genes measured. The normalization factor is computed using a least square method, minimizing variability in the dataset between replicates. This normalization factor is added to each raw C_Τ value in order to yield a ΔC_Τ value. The relative expression in undifferentiated and differentiated hESCs was then analyzed using the ΔΔC_Τ method, comparing each ΔC_Τ to the mean ΔC_Τ value for each gene target for undifferentiated hESC samples [37].

Human ESC-derived ONP transplantation into X-SCID rat cochleae

X-SCID F344-Il2rg^em7kyo rats were generated by Dr. Tomoji Mashimo (Graduate School of Medicine, Kyoto University Institute of Laboratory Animals). This strain has severe combined immunodeficiency caused by a 7-bp deletion in exon 2 of the interleukin 2 receptor gamma (Il2rg) gene[29]. The rats were transferred to Northwestern University and housed under specific-pathogen-free conditions. The Institutional Animal Care and Use Committee of Northwestern University Feinberg School of Medicine approved our experiment procedures (IACUC Protocol number: IS00000379), which also met U.S. National Institutes of Health guidelines for animal care and use.

Twelve X-SCID rats (6 each for control and experimental groups), 6–8 weeks of age, were used as stem-cell transplant recipients using sterile techniques. A CRISPR-Cas9 system was used to generate EGFP⁺ hESCs for transplantation into X-SCID rat cochlea. A cochleostomy was performed posterior to the round window niche and a 23-gauge beveled needle was used to fenestrate the scala tympani medial wall for modiolar access. For injection, EGFP⁺ late-stage ONPs were first dissociated with Accutase (EMD Millipore, Kankakee, Illinois, USA) and suspended in PA solution at approximately 5,000 cells μL^-1. EGFP⁺ late-stage ONPs were also mixed culture medium (DMEM) at 5,000 cells μL^-1 (control). About 4 μL (2 x 10⁴ cells) were injected into the modiolus using a 10-μL Hamilton syringe (World Precision Instruments, Sarasota, Florida, USA). The rate of the injection was 1 μm min^-1. At that rate, we avoided damage to inner-ear tissues due to the injection [6,7,26]. The PA soluion (IKVAV-PA gels and VVIAK-PA gels) was injected as a liquid because it is technically not feasible to inject any material that has been already in gelled into the entire scala tympani, especially with the intent of filling the entire structure. Previous studies have already demonstrated that the self-assembly properties of the IKVAV-PA gel allowed for the liquid form of PA gels to establish the gelled state after injection in vivo as long as the local environment (the subarachnoid space) contains divalent cations such as calcium ions (Ca²⁺) [11,16–19]. Perilymph in the scala tympani has a similar ionic composition to CSF in mammals [38]. We have also performed an additional rheological study of the IKVAV-, VVIAK-PA hydrogels after gelation. Most importantly, the frequency sweep showed G’> G” (G’: the storage modulus; G”: the loss modulus) in all cases at all frequencies indicating the material is in a gelled state (see S1 and S2 Supporting Informations and S3 Fig for details). Furthermore, IKVAV nanofiber orientation is determined by tensile forces created during its ejection from the needle. Thus, to promote nanofiber alignment toward the modiolus, the injection needle was slowly withdrawn from the modiolar injection site during injection. Rats were allowed to survive 4 weeks post-implantation to assess surviving EGFP⁺ cells using an anti-EGFP antibody. Details of a similar transplantation surgery in gerbils have been reported elsewhere [6,7,26]. More detail is also available in S1 Supporting information.

Compared to the gerbil surgery, several minor differences were accounted for [39]. The facial nerve in the rat middle ear is in a more superficial, anterior-rostral position than in the gerbil, and is thereby less protected. Care was taken during surgery to avoid injury causing paralysis to the facial nerve, which can adversely affect the animal’s general health. To safely perform the small cochleostomy, a three-flanged hand drill manufactured from quality stainless steel wire was used. Because localized damage to the basilar membrane in the lower basal turn can occur during cochleostomy near the round window, it is important to make the cochleostomy as posterior as possible to reduce the risk of intracochlear trauma.

To quantify EGFP⁺ ONP survival after transplantation, profile counts were generated from an image taken with the confocal and epifluorescent microscopes [40]. Due to the lack of a clear demarcation line segregating EGFP⁺ ONPs in the rat cochlea and the heterogeneous distribution of the EGFP⁺ ONPs, the profile count method was chosen over stereology-based methods. Six cochleae were injected with EGFP⁺ ONPs with IKVAV-PA gels and six cochleae were injected with control cells suspended in DMEM. EGFP⁺ cells and DAPI-stained cells were counted using the ITCN (image-based tool for counting nuclei) plugin for ImageJ [41]. The total profile number was calculated by counting profiles of the EGFP⁺ ONPs on the five most central modiolar sections. The profile number obtained from each of the five sections was multiplied by 4, as every fourth section was kept for analysis. The total profile number of the EGFP⁺ cells was determined in each of the five sections for each XSCID rat in the four anatomic subdivisions of the cochlea: the scala tympani (ST), scala media (SM), scala vestibule (SV), and modiolus (MO). One-factor factorial analysis of variance was used for statistical evaluation of the profile counts, and the significance of the difference in the profile count of transplanted EGFP⁺ ONPs across the compartments of the cochleae was tested using Tukey-Kramer’s test.

Ex vivo characterization of IKVAV-PA gels after injection into human cadaveric temporal bone

Two formalin-fixed cadaveric temporal bones underwent radical mastoidectomy for improved visualization of the inner ear in December 2013. Specimens were donated anonymously to Northwestern University Feinberg School of Medicine for scientific and educational use. The study fulfilled all the requirements of the Declaration of Helsinki regarding the ethical use of human cadaveric material [42]. Using a high-speed drill with 0.5–1.0 mm diamond burrs, a cochleostomy was created immediately anterior and inferior to the round window niche. A 33-gauge Hamilton needle (Bonaduz, GR, Switzerland) was used to breach the medial wall of the scala tympani adjacent to the modiolus. Next, using a middle cranial fossa approach, the bony covering of the IAC was drilled down to intact dura to access the nerve bundles and provide intracranial visualization. For 12 hr, the two whole-bone specimens (“A” and “B”) were soaked in a 25 mM CaCl₂ solution with 0.9% normal saline that approximated cerebrospinal fluid and promoted gel solidification after transmodiolar injection. The modiolar access was cannulated using a 25-gauge spinal needle attached to a 1 mL Hamilton syringe. For specimen A, 250 μL (i.e., approximate IAC volume [43]) of TAMRA-tagged IKVAV gel (0.25% E2 PA, 0.0625% IKVAV PA, 0.001% TAMRA PA) with hESC density of 5,000 cells mL^-1 and dilution ratio of 1:1 was injected into the modiolus. For specimen B, 300 μL of untagged IKVAV gel with TRA-1-81-tagged hESCs in N2B27-CDM was injected. Injections were performed manually over 2 minutes, with care taken to provide a constant fluid flow rate. A subsequent period of ~1 hr provided time for gel solidification. Overlying dura was removed to examine the VCN for red-wavelength fluorescence (excitation of TAMRA at 555 nm, Kodak Image Station In-Vivo F, DataMax v2.20) so as to assess passage of both gel and cellular components. Endoscopic images aligned with the injection pathway were obtained using a rigid 0° 16-mm Gyrus ACMI micro-endoscope (Gyrus ACMI Surgical Endoscopy Division, Southborough, Massachusetts, USA). This endoscope was also used to assist in the injection of ONPs into the modiolus in the cadaveric human bones, as it provided a favorable angle to the modiolus not attainable with a conventional operating microscope. Temporal bone imaging prior to PA gel injection was obtained for comparison to experimental procedures.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

TEM and SEM were performed using standard techniques. For TEM, tissues were sectioned and stored in paraformaldehyde with CaCl₂, then dehydrated in an ethanol series. Specimens were post-fixed with osmium tetroxide and stained with uranyl acetate, then embedded into an epoxy resin and sectioned on a microtome. Sections were again stained with uranyl acetate and lead citrate, further sectioned to a thickness of 70 nm, and imaged using a FEI Tecnai Spirit G2 electron microscope. SEM samples were prepared by dehydration through an ethanol series and critical point drying process followed by osmium coating using an osmium plasma coater. SEM images were taken on a Hitachi S-4800 field emission scanning electron microscope.

Statistical analysis

Statistical analysis was performed with one-way ANOVA (with Tukey-Kramer’s post hoc test to identify significant differences between means while controlling the family-wise error rate) or with a two-tailed, unpaired Student's t-test. Equal population variance was not assumed in t-tests in order to perform more stringent statistical analysis, but was assumed during ANOVA. A free statistical software package, R (version 3.2.5), was used to perform these tests [44]. Mean values are typically expressed as mean +/- standard error. A significant p-value is indicative of a significant difference where the probability is less than (p < 0.05*), 0.01 (p < 0.01**), and 0.001 (p < 0.001***).

Differentiation of ONPs in vitro

Immunocytochemistry.

The sequential differentiation of hESCs into mid-stage ONPs used the protocol of Matsuoka et al. [8] is summarized and modified in Fig 1 to include details regarding the IKVAV-PA scaffold and the controlled scaffold. ONPs were then cultured on a 2-D matrix (Matrigel™), embedded in 3-D scrambled control PA gels (VVIAK), or experimental PA gels (IKVAV). Derived mid-ONPs plated on Matrigel™ and treated with S/R/E/F/I for seven days (Fig 2A) expressed neuronal progenitor markers (NEUROD1 [45] and SOX2 [46]) and otic markers (PAX2 [47]), indicating that the cells belong to the ONP lineage (Fig 2B, 2C and 2D) [8]. In addition, cells were positive for GATA3 [48] and PAX8 [8,49] showing that they are also at the later stage of ONPs (Fig 2D and 2E). Furthermore, these cells did not express NANOG, SOX10, GFAP, PAX6, or E-cadherin, distinguishing them from hair cells, pluripotent cells, and progenitors fated for neural crest, astroglial, or central neuron lineages [50–53]. A more complete immunocytochemical characterization of late-stage ONPs using H1, H7, and H9 hESCs is provided in Matsuoka et al. [8].

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Fig 2. Immunocytochemical assessment of differentiation of mid-stage hESC-derived ONPs into late-stage ONPs.

(A): An experimental paradigm. Immunocytochemistry of late-stage ONPs derived from H1, H7, and H9 undifferentiated ESCs for SOX2 (B), PAX2 (C), PAX8/NEUROD1 (D), and GATA3/PAX8 (E). Human hESC cell lines used were H7 (C), H9 (B), and H1 (D, E). Scale bars: 50 μm (B and D) and 20 μm (C and E). m-ONP: mid-stage ONPs; l-ONP: late-stage ONPs; SGN: spiral ganglion neurons; S/R/E/F/I: SHH/ATRA/EGF/FGF2/IGF-1.

https://doi.org/10.1371/journal.pone.0190150.g002

Fig 3A(a) diagrams how IKVAV-PA gels were used to create 3-D matrices in vitro. VVIAK-PA gels, which form the same 3-D structure but do not activate Integrin signaling, was used a control [11]. Matrigel^™ was also used to compare differentiation to our previously established 2-D culture system [8]. Fig 3A(b) shows a phase contrast photomicrograph of a hESC-derived mid-stage ONP treated with S/R/E/F/I for seven days in IKVAV-PA gels. The neurite-bearing cell displays the typical bipolar morphology of the otic neuronal lineage. Immunocytochemical characterization of late-stage ONPs embedded into IKVAV-PA gels is shown in Fig 3B; these bipolar cells also have extended neurites (white arrows in Fig 3B(b), 3B(c) and 3B(d)). Quantification of β-III tubulin expression and of neurite extension demonstrate the advantages of the IKVAV-PA matrix. With Matrigel^™, only 42.4% ± 3.40 of these late-stage ONPs expressed the neuronal marker, β-III tubulin [54] (Fig 3C). Cells embedded in “scrambled” (VVIAK-PA) gels generated late-stage ONPs with strong nestin positivity (Fig 3C); however, only 39.6% ± 4.17 of cells expressed β -III tubulin (Fig 3C). In contrast, almost all cells in IKVAV-PA gels expressed strong positivity for both β -III tubulin (97.2% ± 1.83) (p< 0.01) and nestin (88.0% ± 3.74) (Fig 3C), suggesting that this matrix enhanced neuronal differentiation. The percentage of cells extending neurites (Fig 3D) was significantly higher in IKVAV-PA gels than in VVIAK-PA (p < 0.01) or Matrigel™ (p < 0.01) matrices (18.6% ± 5.05 for IKVAV-PA gels; 5.00% ± 0.71 for VVIAK-PA gels, and 1.80% ± 0.97 for Matrigel™). The average length of neurites (Fig 3E) was also about twice as long in IKVAV-PA gels than in the two controls (19.6 μm ± 2.36 for IKVAV-PA; 9.60 μm ± 1.72 for VVIAK-PA (p < 0.05); and 11.4 μm ± 2.16 for Matrigel™ (p < 0.05)). The combined enhancement in β -III tubulin expression, the number of neurite bearing cells, and also in neurite length indicate that the IKVAV-PA gels promote neuronal differentiation of hESC-derived ONPs.

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Fig 3. Neuronal differentiation of hESC-derived late-stage ONPs in IKVAV and VVIAK 3D matrices.

(Aa): An experimental paradigm on neuronal differentiation of hESC-derived mid-stage ONPs treated with S/R/E/F/I in IKVAV & VVIAK PA gels (3-D) and Matrigel™ (2-D) for seven days. DIV: days in vitro. (Ab): A photomicrograph of phase-contrasted image of a mid ONP treated with S/R/E/F/I in IKVAV-PA gels. A White arrow indicates a neurite. Scale = 20 μm. (B): Immunocytochemistry of nestin and β-III tubulin for hESC-derived mid-ONPs treated with S/R/E/F/I in IKVAV-PA gels. DAPI stain is shown in blue. A white arrow indicates neurites. Scale bar: 20 μm. (C): Quantification of nestin and β-III tubulin-immunopositive cells on derived mid-stage ONPs treated with S/R/E/F/I in IKVAV-PA gels, VVIAK-PA gels, and Matrigel™ for 7 days. β-III: β-III tubulin. (D): Quantification of neurite-bearing cells on derived mid-stage ONPs treated with S/R/E/F/I. in IKVAV-PA gels, VVIAK-PA gels, and Matrigel™. (E): Quantification of average neurite length on S/R/E/F/I-treated hESC-derived mid-stage ONPs. ** p < 0.01, * p < 0.05 by one-way ANOVA with Tukey-Kramer’s post-hoc test.

https://doi.org/10.1371/journal.pone.0190150.g003

In vivo hESC-derived late-stage ONP transplantation into X-SCID rats

In vitro characterization of EGFP⁺-labeled hESCs generated by the CRISPR-Cas9 system with EGFP⁺ constitutively expressed from the AAVS1 safe harbor locus are shown in Fig 4. Fig 4A shows a schematic of the AAVS1 safe harbor locus within the PPP1R12C gene and a targeting vector containing a puromycin-resistance cassette (puro^®), CAG enhancer, and EGFP within 5’ and 3’ homology arms (HA). EGFP targeting was validated by PCR (Fig 4B). The first intron of PPP1R12C was PCR amplified with forward and reverse primers complementary to 5' and 3' homology arms, respectively. Correctly edited cells (clone 1, 2 and 4 in Fig 4B) exhibited a shift in amplicon size to 4.7 kb attributable to insertion of the transgene elements (i.e., the exogenous EGFP into the AAVS1 locus) compared to 0.5 kb in targeted, but unedited cells (Clone 3 and 5). Note that Clone 6 was untargeted H7 cells used as a control.

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Fig 4. CRISPR-Cas9-modified EGFP⁺ hESCs that differentiated towards late-stage ONP lineage possess late-stage otic neuronal progenitor characteristics.

(A): Schematic of AAVS1 safe harbor locus within PPP1R12C gene and targeting vector containing puromycin-resistance cassette (puro^®), CAG enhancer, and EGFP within 5’ and 3’ homology arms (HA). Arrows indicate PCR primers. (B): EGFP targeting was validated by PCR. H7: H7 human ESCs were used as a control (untargeted clone). (C): CRISPR-Cas9 modified EGFP⁺ undifferentiated hESCs in culture. (Ca): phase contrast, (Cb): fluorescence (EGFP⁺) imaging. CRISPR-CAs9 modified EGFP⁺ late-stage ONPs: (Cc): phase contrast, (Cd): fluorescence imaging. Scale bar = 100 μm. (D): RT-PCR quantification of OCT3/4 and NANOG expression measurements for H7 hESC, H7 late-stage ONPs (lONPs), and CRISPR-Cas9 modified EGFP⁺ late-stage ONPs. n.s.: not statistically significant, *** p < 0.001, and ** p < 0.01 by one-way ANOVA with Tukey-Kramer’s post-hoc test. (E): Immunocytochemistry of CRISPR-Cas9 modified EGFP⁺ late-stage ONPs for NEUROD1 (Ea), nestin (Eb), SOX2, and PAX8 (Ec). Scale bar = 25 μm.

https://doi.org/10.1371/journal.pone.0190150.g004

We then characterized EGFP positivity in these cells (Fig 4C, 4D and 4E). Immunocytochemical analysis demonstrates that 55.0% ± 2.2 of EGFP⁺ hESCs retained positivity after differentiation to the late ONP stage (Fig 4C(d)). As expression of EGFP comes at a metabolic cost to the cell, it is anticipated that there will be selective pressure over time during differentiation, reducing the percentage of EGFP⁺ cells (Fig 4C(b) and 4C(d)). RT-qPCR demonstrated significantly decreased mRNA levels of the stem cell markers, NANOG [55] and OCT3/4 [55], suggesting appropriate lineage differentiation of the hESC-derived CRISPR-Cas9-modified EGFP⁺ late-stage ONPs.

The EGFP⁺ labeling did not alter the ability of ONPs to differentiate into late-stage cells in culture (Fig 4E). Representative immunocytochemical analyses of hESC-derived EGFP⁺ late-stage ONPs (H7) demonstrate that the cells have differentiated into neuronal progenitors (NEUROD1 [45], nestin [56], and SOX2 [46]) and also into the late-stage otic neuronal lineage (PAX8 [8,49]). Note that immunocytochemical photomicrographs for PAX8 in Fig 4E are pseudo-colored in green for readers’ convenience. The EGFP⁺ late-state ONPs did not express SOX10 [50], GFAP [52], PAX6 [51], or E-cadherin [53] indicating that the cells have differentiated specifically toward the otic neuronal lineage. These findings are consistent with the late-stage of ONP development described in Fig 2 and also with our previous report [8]. Thus, the CRISPR-Cas9-modified EGFP⁺ hESCs differentiated towards the late-stage ONP lineage, possess late-stage otic neuronal progenitor characteristics (PAX8⁺, SOX2⁺, NEUROD1⁺, and nestin⁺) at the protein level, and show a loss of pluripotency (down-regulation of NANOG and OCT3/4 at the mRNA level).

Fig 5A shows our experimental paradigm for transplantation of the EGFP⁺ late-stage ONPs into X-SCID rat cochlea. EGFP⁺ ONPs transplanted with IKVAV-PA gels were found within the cochlea 28 days after transplantation (Fig 5B). DMEM-only injection was performed as a control (Fig 5C). Immunohistochemistry from a sectioned cochlea (Fig 5B) shows expression of EGFP positivity specific to the intracochlear regions of the scala tympani (outlined regions in yellow in Fig 5B). High-power imaging resolved individual cells with (short white arrow) and without (long white arrow) EGFP⁺ staining (Fig 5D); such high-power images facilitated quantitative analyses, summarized in Fig 5E and 5F. Use of IKVAV-PA gels resulted in a significantly greater (p < 0.01) number of intracochlear EGFP⁺ profiles (Fig 5E). There also were significantly more EGFP⁺ profiles in the modiolus and basal turn after transplantation with IKVAV-PA gels and significantly less cell migration to higher cochlear turns such as the mid turn and apical turn (* p < 0.05, ** p < 0.01).

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Fig 5. IKVAV-PA gels enhance survival of transplanted EGFP⁺ in the X-SCID rat cochlea.

(A): An experimental paradigm. DPI: days post injection. (B): Anti-EGFP antibody labeling of EGFP⁺ cells in the basal turn of the XSCID cochlea. BT: basal turn. Scale bar = 200 μm. (C): Control experiment showing DMEM only injection into the cochlea. Scale bar = 200 μm. (D): Example high-power image of DAPI and EGFP profiles at cellular level used for subsequent quantification. Scale bar = 20 μm. (E): The percentage of EGFP⁺ profiles per section injected with IKVAV-PA gels in and with DMEM (n = 6 for each condition). (F): Comparison of EGFP⁺ profiles in each turn and the modiolus injected with IKVAV-PA gels in and with DMEM (n = 6 for each condition). Abbreviation: MO: modiolus; BT: basal turn; MT: mid turn; and AT: apical turn. * p < 0.05, ** p < 0.01, and *** p < 0.001.

https://doi.org/10.1371/journal.pone.0190150.g005

Ex vivo cadaveric human temporal bone study

We then tested the potential clinical feasibility of transmastoid injection of hESCs embedded in IKVAV-PA gels using cadaveric human cochleae. Fig 6 summarizes anatomical landmarks and the presence of IKVAV-PA gel in injected cadaveric human cochleae. A schematic of a coronal section of a human cochlea at the level of mid-modiolus is shown in Fig 6A. Note the trajectory of the needle accessing the modiolus. The same trajectory is illustrated in human cadaveric temporal bone in Fig 6B, which shows the basal turn of the left cochlea, revealed after tympanomastoidectomy in the cadaveric temporal bone. The cochleostomy (C) provided access to the scala tympani (ST) and scala vestibule (SV). For comparison, Fig 6C is an intraoperative image of the basal turn of another left human cochlea from a patient who underwent a mastoidectomy. The black arrows in Fig 6B and 6C indicate a favorable direction for cell injection into the modiolus through the cochleostomy site (blue dotted line).

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Fig 6. Ex vivo cadaveric human temporal bone study.

(A): Schematized midmodiolar cross-section of a mammalian cochlea, showing injection needle approaching the modiolus. (B-C): Photomicrograph of the basal turn of the left cochlea in a cadaveric temporal bone (B) and in humans (C). Black arrows indicate direction of injection with IKVAV-PA gels. Note the direction of the fine needle for injection of IKVAV gel containing hESCs. A blue-dotted line shows the cochleostomy site. A white dotted line: round window. (D and E): Endoscopic IKVAV-PA gel injection into human modiolus. View of cochleostomy site using a 16-mm 0° rigid endoscope. A black dashed line marks cochleostomy boundary. A white dashed line indicates presumed plane of the scala vestibuli. (F): Artist’s rendition of the superior view of the human skull base, with black arrow showing the direction of injection of IKVAV gels with hESCs and anatomical landmarks. Black square corresponds to sectioned area in subsequent figures. (G-L): IKVAV-PA gels with hESCs in the IAC in two sets of human cadaveric temporal bones. (G and J): Middle cranial fossa view of the human cadaveric temporal bone before the hESC injection. TAMRA-tagged IKVAV gels (H) and corresponding autofluorescence measurement observed in normal temporal bone tissue abutting the IAC (I). TRA-1-81 tagged hESCs with magnified inset (K) and corresponding autofluorescence measurement (L). Abbreviations: RW: round window; C: cochleostomy site; ST: scala tympani; SV: scala vestibule; M: modiolus; N: spinal needle; ACF: anterior cranial fossa; MCF: middle cranial fossa; PCF: posterior cranial fossa. Cranial nerves are denoted by Roman numerals.

https://doi.org/10.1371/journal.pone.0190150.g006

Based on the requirement for this specific 3-D approach in human temporal bone, we chose a 16-mm 0° rigid micro-endoscope, which provides a favorable angle to the modiolus for the injection of hESCs into the cochlea. It should be noted that a conventional operating microscope would be unable to provide this angle of approach. Using the micro-endoscope, we again performed a cochleostomy on the basal turn of a human cadaveric cochlea. Endoscopic photomicrographs show the injection site at the medial aspect of the scala tympani in Fig 6D (with no needle) and 6E (with the injection needle). Note that the needle trajectory was central through the medial wall of scala tympani to access the modiolus (Fig 6A, 6D and 6E), indicating the successful transcochlear approach to the modiolus and IAC without disruption of the other scalae. Fig 6F, which depicts an artist’s rendition of the human skull base indicating the region examined in subsequent images, provides the trajectory of the injection needle in relation to the human skull base. Note the black arrow for the trajectory of the injection.

Having identified the specific injection trajectory, we then performed injection of IKVAV-PA gels with/without hESCs. Undifferentiated hESCs were used for this proof-of-concept study of our surgical approach as they could be easily identified by detection of the cell surface marker, TRA-1-81. Fig 6G shows the middle cranial fossa of a human cadaveric temporal bone before injection. We sought confirmation that TAMRA-tagged IKVAV-PA was delivered into the IAC of the cadaveric temporal bone through the transmodiolar injection. One hour after injection, endoscopic examination revealed orange fluorescence corresponding to the fluorophore-tagged IKVAV-PA medial to the IAC fundus (Fig 6H). For comparison, autofluorescence of the bone in absence of TAMRA-tagged gel is evident in Fig 6I. Passage of IKVAV-PA gel from the modiolar injection to the inner confines of the IAC was observed within 1–2 minutes. We also visualized the location of the hESCs in the gel delivered to the IAC. Following similar injections in different human cadaveric temporal bones, fluorescent TRA-1-81⁺ hESCs in IKVAV-PA gel were also found overlying the medial aspect of the VII/VIII nerve complex (Fig 6J and 6K). Of note, a moderate amount of autofluorescence was observed in normal temporal bone tissue abutting the IAC using in vitro imaging, and this was used as a control condition to verify positive TRA-1-81 staining of hESCs prior to transmodiolar injection (Fig 6L). Taken together, both TAMRA-tagged IKVAV-PA gels and TRA-1-81 tagged hESCs were successfully delivered into the IAC on the middle cranial fossa side of the temporal bone using transmastoid injection.

Nanofiber characteristics in vitro and ex vivo after injection of IKVAV-PA Gels

IKVAV-PA gel forms nanofibers in vitro that align parallel to an applied shear force when laminar flow is maintained during processing (Fig 7A). However, much of this alignment is lost if laminar flow is not maintained (Fig 7B). We used transmission electron microscopy (TEM) to determine whether fiber alignment is maintained after injection of IKVAV-PA gels into cadaveric human IAC containing the auditory nerve. Examination of the nerve complex following IKVAV-PA injection revealed a parallel alignment of self-assembled nanofibers within the gel that formed upon injection (Fig 7C). These fibers also appeared to orient in the direction of the applied shear force (radially along the longitudinal axis of the nerve), thus mirroring the anatomic arrangement of the VII/VIII nerve complex directed toward the cochlear nucleus. No evidence of similar peptide scaffolds was noted in high-powered images of control nerve tissue (e.g., Fig 7D).

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Fig 7. Electron microscopy study of IKVAV-PA gels in vitro and ex vivo human cadaveric tissue.

(A): SEM image of aligned IKVAV nanofibers in vitro (note predominant orientation in the direction of the shear force, indicated by red arrow). Scale bar = 1 μm. (B): Non-aligned IKVAV nanofibers in vitro showing a randomly oriented configuration due to fluid turbulence. Scale bar = 1 μm. (C): TEM image of self-assembled IKVAV-PA gel overlying the facial-vestibulocochlear nerve complex (indicated by a black circle and black arrows) following endoscopic transmastoid injection into the modiolus. Note the parallel alignment of self-assembled peptides indicated by the red arrow (11,000× magnification). Scale bar = 1 μm. (D): Control nerve specimen with no evidence of peptide scaffold for comparison (1,900× magnification). Scale bar = 1 μm. Note putative epineurium of the facial-vestibulocochlear nerve complex indicated by the white dotted line (C & D). Abbreviations: VIIVIII: facial-vestibulocochlear nerve complex.

https://doi.org/10.1371/journal.pone.0190150.g007