Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea

Abstract

Stem-cell-based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe-to-profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non-invasive in vivo visualization of cells and long-term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co-expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase-expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427-440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Keywords: bioluminescence imaging; cochlea; firefly luciferase; guinea pig; hair-follicle-bulge-derived stem cells.

Figure 1

Schematic drawing explaining the basic principles of dual‐reporter gene expression in genetically engineered cells. The lentiviral gene construct is designed to stably co‐express copepod green fluorescent protein (copGFP; emitting at ∼502 nm) and codon‐optimized firefly luciferase Luc2 (emitting at ∼560 nm). The copGFP‐Luc2 construct is composed of the EF1 promotor and genes coding for copGFP and Luc2. Both genes are coupled via a T2A‐like sequence, which mediates co‐translational cleavage (ribosome skipping) and, hence, results in bicistronic expression. The inserts are flanked by long terminal repeats (LTR). TF: transcription factors; RNA Pol II: RNA polymerase II.

Figure 2

Illustration of the three different injection routes. (A) Injection into the internal auditory meatus. (B) Injection into the scala tympani via the round window membrane. (C) Injection into Rosenthal's canal via the round window membrane. IAM: internal auditory meatus; ST: scala tympani; SV: scala vestibuli; RC: Rosenthal's canal.

Figure 3

Detection of bioluminescent signals emanating from the cochlea in situ and verification of injection. (A, B) Pseudocolor light emission images of an auditory bulla after injection of HEK293‐copGFP‐CBG99 cells (5 × 10⁴ cells in 10 μL PBS containing D‐luciferin at a final concentration of 15 mg/mL) directly into the internal auditory meatus. Bioluminescence is expressed as radiance (photons/second/cm²/sr). Optical imaging of the auditory bulla after its removal from the skull demonstrates that the bioluminescent signal can pass through the tympanic membrane (arrow) and crevices (*) in the bony wall of the auditory bulla (A). After removing part of the bony wall of the auditory bulla (B), the cochlea can be discerned and a bright bioluminescent signal is observed at the basal cochlear turn, localized near the round window. (C) Photomicrograph of a cleared cochlea after injection of a methylene blue solution directly into the internal auditory meatus. Myelin sheaths of the neurons in the basal and middle turns are stained blue, allowing visualization of Rosenthal's canal (scale bar: 1 mm).

Figure 4

Comparison of the different application routes. (A, B) Pseudocolor light emission images of guinea pig cadaver heads after injection of HEK293‐copGFP‐CBG99 cells (5 × 10⁴ cells in 10 μL PBS containing D‐luciferin at a final concentration of 15 mg/mL) into the internal auditory meatus. (A) Ventrolateral view of the cadaver head from the left side. Bioluminescent signal is located at the cavum conchae of the auricle. (B) Ventral view of the same head seen from the rear. A distinct bioluminescent signal is seen emanating from the injection site, i.e., the internal auditory meatus. (C, D) Injection of HEK293‐copGFP‐CBG99 cells (5 × 10⁴ cells in 5 μL PBS containing D‐luciferin) directly into the scala tympani via the round window membrane. (C) Lateral view from the right side showing bioluminescent signal located at the cavum conchae of the auricle. (D) Injection of transduced cells into the left masseter muscle results in a considerably lower signal, due to light absorption by tissue chromophores. (E, F) Injection of HEK293‐copGFP‐CBG99 cells (5 × 10⁴ cells in 5 μL PBS containing D‐luciferin) directly into the modiolus of the basal cochlear turn. After an initial injection in the right ear (E) followed by a second injection in the left ear (F), in both ears a bright bioluminescent signal was invariably located near the cavum conchae of the auricle, indicating that the light emanating from the cochlea is passing through the tympanic membrane and the external auditory meatus. Bioluminescence is expressed as radiance (photons/second/cm²/sr).

Figure 5

Detection of reporter molecule expression in HFBSCs expressing copGFP‐Luc2. Light micrographs showing reporter molecule expression in cultures of transduced HFBSCs containing the copGFP‐Luc2 construct. (A–C) Fluorescence microscopy: Fluorescence (A) and bright‐field (B) images of a representative area in the culture were merged and the resulting overlay (C) shows that nearly all cells express the fluorescent reporter molecule copGFP (scale bar: 100 μm). (D–F) Bioluminescence microscopy: From the overlay (F) of the bioluminescence (D) and bright‐field (E) images it is clear that most (i.e., viable) cells express the bioluminescent reporter molecule Luc2 (scale bar: 200 μm).

Figure 6

Time course of the bioluminescent signal observed in cultures of transduced HFBSCs. The signal of transduced HFBSCs containing the copGFP‐Luc2 construct gradually increases after addition of D‐luciferin (final concentration: 0.5 mM) and reaches its plateau phase after approximately 15 min (red line), irrespective of the amount of plated cells (dotted line: 5 × 10⁴ cells; solid line: 2.5 × 10⁴ cells) studied. Values for each time point and cell amount represent the averaged data sets from six wells. Bioluminescence is expressed as photon flux (ph/s: photons/second).

Figure 7

Cell dilution series. (A–D) To determine the amount of transduced HFBSCs (containing the copGFP‐Luc2 construct) needed to reach signal threshold for bioluminescence imaging a cell dilution series was performed. Bioluminescence is expressed as radiance (photons/second/cm²/sr). Cells were resuspended in PBS at a concentration of 1 × 10⁶ cells/100 μL followed by addition of D‐luciferin (final concentration: 15 mg/mL). Different amounts of cells were injected into the modiolus of the basal cochlear turn in the right ear. A bright bioluminescent signal was seen after injection of 5 × 10⁴ cells (A). Considerably lower signals were detected after injection of 2.5 × 10⁴ cells (B) and 1.25 × 10⁴ cells (C). Injection of 0.5 × 10⁴ cells (D) did not result in a detectable bioluminescent signal. (E) Quantitative measurement of the bioluminescent signal shows that the threshold for bioluminescence imaging lies between 0.5 × 10⁴ cells and 1.25 × 10⁴ cells. Bioluminescence is expressed as photon flux (ph/s: photons/second).

Figure 8

Perls' Prussian blue staining of transduced HFBSCs containing ferumoxytol. Histochemical visualization of non‐heme iron in transduced HFBSCs containing iron oxide nanoparticles (ferumoxytol) using the method with DAB intensification. Dark brown precipitates represent the presence of intracellular iron accumulations. (A) Iron accumulations in transduced iron‐containing HFBSCs (arrows) after injection into the scala tympani (ST) of the cochlea. The spiral ganglion (SG) does not contain iron accumulations. (B) Same section, but with overlay of grayscale bright field image with the green‐fluorescent signal, demonstrating that iron‐containing HFBSCs also express copGFP (arrows). However, spiral ganglion cells, myelin sheath, bone matrix, and erythrocytes (*) demonstrate autofluorescence at the same wavelengths. (C) Iron accumulations in transduced HFBSCs in culture dishes after uploading with ferumoxytol nanoparticles. (D) Transduced HFBSCs (control) that have not been uploaded with ferumoxytol nanoparticles do not demonstrate any intracellular deposits after treatment with the Perls' Prussian blue method and DAB intensification (scale bar: 100 μm).