Fig 1.
Neuronal differentiation protocol and phenotype analysis.
(A) Illustration of the neuronal differentiation protocol. (B–D) Representative images of H7 hESC derived precursors after formation of EBs (B), neuronal rosettes (C) and NPCs (D). (E–G) qPCR analysis of gene expression during EB (green, n = 3), neuronal rosette (blue, n = 4) and NPC (grey, n = 3) stages compared to undifferentiated hESC (red, n = 3). Error bars indicate standard error of the mean. (H–J) Representative images of protein expression 4 weeks after terminal differentiation. Nuclei are counterstained with Hoechst. (H) Differentiated cells express the neuronal marker TUJ1 and the vesicular glutamate transporter VGLUT1. (I) MAP-2 expression indicates maturation of derived neurons. (J) Neurons stained positive for the synaptic protein synaptophysin (SYP, counterstained with TUJ1). Scale bars represent 200 μm in B, 50 μm C and J, and 100 μm in D, H, and I.
Fig 2.
Neurite alignment on nanofiber mats in vitro.
(A–C) Representative images of TUJ1 stained terminally differentiated H7 hESC derived neurons (green) on PS coverslips (A) and unaligned (B) and aligned (C) nanofiber mats. Insets in B’ and C’ show light microscopic images of unaligned and aligned nanofiber mats. (D–F) Plot of image intensity as a function of angle with corresponding FWHM values after Fourier transformation of fluorescence images of A, B and C illustrating the extent of alignment. A low FWHM indicates high alignment. (G) Quantification of FWHM values of coverslips (n = 8) and unaligned (n = 7) and aligned (n = 6) fibers terminally differentiated for 2–6 weeks. For random samples, only one out of 7 samples allowed FWHM calculation. (H) Immunohistochemistry for TUJ1 (green) and VGLUT1 (red) shows that neurons maintain a glutamatergic fate on fiber mats. Scale bars represent 100 μm in A, B, C and H and 50 μm in B’ and C’.
Fig 3.
Nanofibrous scaffold assembly, NPC seeding and pre-implantation adhesion.
(A and B) High resolution images of single (A, SEM image) and assorted (B, light microscopic image) PLLA:PCL nanofibers. (C and D) SEM images of assembled scaffolds consisting of nanofiber bundle and PCL sheath. (E) Illustration of procedures for seeding NPCs on nanofibers for implantation. A long nanofiber bundle with sheath was attached to a coverslip. (1) Concentrated cell suspension (10,000 cells/μl) was deposited on a nanofiber portion outside the sheath and the cells allowed to settle. (2) The sheath was shifted and positioned over the nanofiber area with attached cells. (3) The excess length of nanofibers was cut, releasing the scaffold for implantation. (F) Nuclear Hoechst stain of NPCs one hour after implantation. (G) TUJ1 stain (green, counterstained with Hoechst) shows fiber extension and alignment on Nanofiber bundles as early as 24h after seeding. (H) Quantification of Hoechst stain based cell counts on coverslips and bundles of parallel cultures. Error bars represent standard error of the mean. (I) Neurite extension aligned with nanofiber orientation 6 weeks after seeding (green: TUJ1, red: Nestin, blue: Hoechst). Scale bars represent 1 μm in A, 100 μm in B, D, F, G and I and 1 mm in C.
Fig 4.
Efficacy of ouabain deafening in guinea pigs pre-implantation.
A. Image collage of a mid-modiolar plastic section with positions of Rosenthal’s canal cross sections labelled from P1 to P8. (B) Representative images of spiral ganglia and the organ of Corti in untreated control (left) and ouabain deafened specimen (right, scale bar = 50 μm). SGN somata can be seen as dark round circles within the bony structure of the Rosenthal’s canal in control, but are depleted following ouabain treatment. Accompanying the loss of somata is a loss of fiber structures, which in control subjects project to organ of Corti and modiolus. (C) Quantification of SGN depletion along the cochlear axis from apex to base (control: n = 4, deafened: n = 5, * p < 0.001 for each region). (D) Click aABR waveforms elicited at increasing sound pressure levels (SPL) in a guinea pig pre- and post-deafening. (E) Quantification of aABR thresholds shows significant threshold elevation by ouabain treatment (* p = 0.01, n = 19), confirming efficient deafening at a physiological level. The pre-implantation deafening data include animals from sham, 1 and 2 month cell-free scaffold and 1 month NPC-scaffold groups. Error bars represent standard error of the mean.
Fig 5.
Surgical approach and histological assessment of placement and tissue response.
(A–C) Guinea pig temporal bone showing the access to and the scaffold placement in the IAM. (A) The scaffold was advanced into the IAM through a cochleostomy in the base of the cochlea. (B) Positioning of the electrode co-implanted for eABR recordings. (C) View on the positioned scaffold from the brain side. (D and E) Plastic cross-sections of the IAM with implanted cell-free scaffold 15 days post-implantation. (D) Cross section showing the IAM with its bony wall and the scaffold (arrowhead; sheath shaded in blue) embedded in host tissue. Host tissue was found to infiltrate the full length of the scaffold. (E) Higher magnification of the scaffold’s interior with host tissue embedding the nanofibers (shaded in green), which adopt a chain-like arrangement. (F) IAM immunohistochemistry for the hematopoietic lineage marker CD45 shows few immune cells within the IAM 4 days after implantation with an NPC-seeded scaffold. (G) Additional samples 1 month post-implantation were examined for CD45 labeling, including sham, cell-free scaffold, and NPC scaffold animal groups, as well as untreated controls. Few immune-positive cells were identified in these sections indicating that the surgery, scaffolds, and NPCs did not trigger a significant immune response. (H) Immunohistochemistry for TUJ1 (neurons), GFAP (glia) and vimentin (Schwann cells/fibroblasts) in control, sham and cell-free scaffold (1-month post implant) shows involvement of vimentin and GFAP positive cells in tissue repair. Scale bars represent 1 mm in A—C, 100 μm in D—H.
Fig 6.
Post-implant physiology and histological assessment of H9-GFP derived NPC survival.
(A) Representative examples of in vivo threshold eABR waveforms of cell-free and NPC scaffolds at post implantation days indicated. In both groups, waveforms are flattened after deafening and implantation. Profiles of distinct waves partially recover over the one month observation period. All responses were evoked with a 300 μA stimulus, except for the day-9 time point of the cell-free scaffold subject (indicated by *, 300 μA was below threshold and thus a 370 μA response is shown for illustration). (B) Course of post implantation eABR thresholds for individual animals implanted with cell-free scaffolds (n = 4) and NPC scaffolds (n = 9) is shown alongside the mean thresholds for NPC-only injections (n = 3). Gray triangle symbols indicate two animals implanted with dye-labeled NPC-scaffolds; the elevated thresholds in these animals may indicate a negative impact from the FluoroRuby dye on NPC health. Data points are slightly shifted in time for clarity. (C) hrGFP immunohistochemistry for the detection of H9-GFP derived NPCs in sections of control and implanted specimen collected 4 days post implantation. The excerpt shows a magnification of the interior of the scaffold with hrGFP expression in two Hoechst-positive cells (blue channel enhanced for illustration). (D) There was no hrGFP signal detected in 1 month implantations. TUJ1 stain shows interspersed fibers both in cell-free and NPC-seeded conditions. Scale bars represent 100 μm in C and D and 50 μm in the excerpt of C.