Cell Therapy © The American Society of Gene & Cell Therapy
original article
Conditioning the Cochlea to Facilitate Survival and Integration of Exogenous Cells into the Auditory Epithelium Yong-Ho Park1,2,3, Kevin F. Wilson4, Yoshihisa Ueda5, Hiu Tung Wong1, Lisa A. Beyer1, Donald L. Swiderski1, David F. Dolan1and Yehoash Raphael1 1 Department of Otolaryngology-Head and Neck Surgery, KHRI, The University of Michigan, Ann Arbor, MI, USA; 2Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Chungnam National University, 33 Munwha Ro, Daesa Dong, Jung Gu, Daejeon, Korea; 3Brain Research Institute, College of Medicine, Chungnam National University, 33 Munwha Ro, Daesa Dong, Jung Gu, Daejeon, Korea; 4Division of Otolaryngology, University of Utah, 50 North Medical Dr.3C120 SOM, Salt Lake City, UT, USA; 5Department of Otolaryngology-Head and Neck Surgery, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka, Japan.
The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media. Received 8 July 2013; accepted 23 December 2013; advance online publication 4 February 2014. doi:10.1038/mt.2013.292
INTRODUCTION
In the auditory epithelium (AE) of the mammalian cochlea, hair cells (HCs) and supporting cells are formed during embryogenesis and once they mature, they cannot be replaced if lost. Because of the lack of regeneration of these cells, lesions in the AE lead to permanent hearing loss. One possible therapeutic approach for restoring hearing may be implantation of exogenous cells such as stem cells (SC) into the cochlea. This therapy would consist of two major stages: (i) producing the cells and (ii) inserting them into the target tissue, the AE. Progress in the first stage has provided
several methods for producing new HCs from different types of SCs.1–4 Here, we start to address the second stage, inserting and integrating exogenous cells into the native AE tissue. Inserting and integrating SCs into the AE is an essential practical step for therapeutic use of these cells for hearing restoration. Integration of exogenous cells in an epithelial layer is a complex and challenging task. Like other epithelia, the AE is a highly organized layer of confluent cells that are connected to each other by apical junctional complexes. The supporting cells of the AE rest on the basilar membrane, a thick layer of connective tissue immediately beneath the basement membrane. Cells injected into the fluid (perilymph) in the space under the basilar membrane, the scala tympani (ST), can survive but do not cross the basilar membrane into the AE. It is therefore necessary to attempt integrating cells by injecting them into the lumen of the cochlear duct (endolymph of the scala media (SM)). However, this fluid compartment is not hospitable to cells, because the luminal fluid (endolymph) contains high levels of potassium.5 Consequently, cells injected into SM endolymph do not survive.6 It is therefore a major challenge to help exogenous cells survive in the SM until they integrate into the AE. After establishing conditions that allow cells to survive, the next challenge is to induce their integration into the AE. This is difficult because the apical junctions between the AE cells are extremely robust, consisting of intermittent adherens/tight junctions,7,8 which make the organ of Corti an especially difficult barrier to breach. These elaborate junctions persist in the deafened AE depleted of HCs.9 The combination of this strong epithelial barrier and the overlying toxic fluids presents special challenges for inserting cells into this tissue. Not only must the highly resistant intercellular junctions be broken to permit intercalation of exogenous cells but doing so may expose neighboring tissues to the toxic endolymph. To facilitate survival of transplanted cells until they are integrated, and their integration into the AE without exposing surviving native cells to hazards, it is necessary to perform several simultaneous manipulations of multiple cochlear components.
Correspondence: Yehoash Raphael, Department of Otolaryngology-Head and Neck Surgery, KHRI, The University of Michigan, Ann Arbor, MI, USA. E-mail: [email protected] Molecular Therapy vol. 22 no. 4, 873–880 apr. 2014
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One possibility to accomplish insertion and integration of exogenous cells into the AE is to change the conditions in the cochlea in a transient fashion, to reduce or eliminate the obstacles to survival and integration of implanted cells. Specifically, it would be necessary to “condition” the cochlea by eliminating the high potassium and the endocochlear potential (EP) and by opening the cell–cell junctions of the AE in a reversible way. Here, we present results of experiments designed to test whether such conditioning of the cochlea can facilitate survival of exogenous cells in SM and their integration into the AE. Because the apical junctions also play an essential role in survival of the spiral ganglion neurons (SGNs) by maintaining the luminal barrier, we also evaluated the effects of opening the AE junctions on survival of the SGN. To condition the cochlea, we flushed away the endolymph and replaced it with artificial perilymph, blocked ion pumps in the stria vascularis (SV) with furosemide and applied sodium caprate, known to disrupt tight junctions. We then injected tagged HeLa cells into the SM. HeLa cells were selected as a stable and robust type of cell to test the conditioning approach and establish proof for the principle that cells can survive in the SM of the conditioned cochlea. The data demonstrate that conditioning the cochlea can prepare it to support survival of transplanted HeLa cells for at least 7 days.
RESULTS Cell survival in artificial cochlear fluids
To evaluate HeLa cell survival in the artificial cochlear fluids, HeLa cells grown in cultures were exposed to artificial endolymph, perilymph, or a 50:50 mixture of both. Culture plates were examined at 30 minutes, 1 hour, 3 hours, or 6 hours after the culture medium was replaced by artificial cochlear fluids. HeLa cells exposed to artificial endolymph promptly detached from the culture plate, assumed a spherical shape, and rapidly disintegrated. Floating debris from dead cells could be detected but intact floating cells were not seen. Reduction in cell density was evident as early as 1 hour (Figure 1d versus Figure 1a,e,f) and most cells died within 6 hours of being placed in the artificial endolymph (Figure 1j). Control
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In contrast, cells in artificial perilymph or in the 50:50 mixture survived longer than those in the endolymph only (Figure 1k and l). Quantitative analysis of cell survival over time determined that the number of surviving cells in the artificial endolymph was significantly decreased compared with cells exposed to artificial perilymph or the mixture of endolymph and perilymph (P < 0.01) (Figure 2).
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Figure 1 HeLa cell (mCherry fluorescence labeled) survival in culture dishes at 30 minutes. (a–c), 1 hour (d–f), 3 hours (g–i), or 6 hours (j–l) after dishes were filled with artificial endolymph (a, d, g, j), artificial perilymph (b, e, h, k), or mixture of endolymph and perilymph (c, f, i, l). In the artificial endolymph, HeLa cells begin to die within 30 minutes after exposure and most cells are dead within 6 hours. In the artificial perilymph and in the mixture of endolymph and perilymph, most HeLa cells survived at least 6 hours. Scale bar = 100 µm.
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Figure 2 Quantitative analysis of HeLa cell survival in the artificial cochlear fluids. At 1, 3 and 6 hours after artificial cochlear fluid exposure, survival of HeLa cells in endolymph is significantly reduced compared with that in perilymph or in a mixture of endolymph and perilymph. No differences in HeLa cell survival were observed between perilymph and the mixture of endolymph and perilymph at any time point. Statistical comparison was by t test; Asterisk indicates P < 0.01.
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Effect of sodium caprate on auditory epithelial junctions The concentration of sodium caprate selected to disrupt apical epithelial junctions, 10 mmol/l, was determined by preliminary experiments not reported here. To investigate the effect of sodium caprate on apical AE junctions in vivo, we injected sodium caprate dissolved in artificial perilymph into the SM of neomycin-deafened animals (7 days after inducing the ototoxic neomycin lesion). At 1 or 4 hours after the surgery, animals were sacrificed and whole mounts of the AE were stained with antibody against ZO-1 (a tight junction component) and prepared for epifluorescence. In untreated control ears, the AE was flat, with no remaining HCs or differentiated supporting cells. Cell–cell junctional areas in the AE appeared as a well-defined single line, suggesting tight junctions between cells were intact (Figure 3a). In ears injected with sodium caprate, the AE was also flat, but in contrast to controls, 1 hour after introducing sodium caprate into the SM, the staining pattern of ZO-1 in the AE included gaps and spaces between
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cells, and the overall appearance of the junctions was somewhat disrupted and less well defined. This suggested that sodium caprate induced deterioration of the apical junctions (Figure 3b). In tissues obtained at 4 hours after sodium caprate treatment, junctions appeared normal again, suggesting that after initially opening, the cell–cell contacts reorganized and resealed (Figure 3c).
Survival of transplanted cells in the SM To evaluate transplanted HeLa cell survival in vivo, mCherry fluorescence labeled HeLa cells were transplanted into the SM in untreated control and conditioned (intravenous furosemide injection and endolymph flushed with sodium caprate in artificial perilymph) animals. At 4 hours, 1 day, or 7 days after transplantation, animals were sacrificed, and cochlear tissues were examined under a fluorescence stereomicroscope. Upon dissection of the tissues, no signs of inflammatory response were noted in the cochlea. In untreated control ears, cochleae were devoid of red fluorescence signal (Figure 4a), suggesting that the implanted cells did not survive. However, in conditioned cochleae, bright red fluorescence signals were observed inside the cochlea as much as 7 days after HeLa cell transplantation (Figure 4b–d). To examine the areas with red fluorescence more closely, cochleae were dissected and stained to label tight junctions (ZO-1 antibody, green) and nuclei (Hoechst 33342, blue) in whole mounts. Under epifluorescence combined with phase contrast microscopy, transplanted HeLa cells were observed around the
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Figure 3 Whole-mounts of the cochlear sensory epithelium in neomycin-deafened control ear (untreated, a) and deafened ears obtained 1 hour (b), or 4 hours (c) after sodium caprate treatment, stained for ZO-1 (green) and photographed with epifluorescence. (a) In neomycin-deafened control ear without sodium caprate treatment, there are no gaps between cells in the auditory epithelium. (b) One hour after sodium caprate treatment, cell junctions have irregular contours and gaps (arrow-heads) suggesting disruption of apical junction complexes. (c) Four hours after sodium caprate treatment, a few gaps remain between cells, but most of the tissue appears to have reorganized to resemble the control ear without sodium caprate treatment. Scale bar = 30 µm.
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Figure 4 Fluorescence stereomicroscopy images of partially dissected cochleae that were implanted with HeLa cells (red) 4 hours after injection into unconditioned cochlea (a) or several time points after injection into cochleae conditioned with infusion of sodium caprate in artificial perilymph and systemic furosemide (b, 4 hours; c, 1 day; and d, 7 days). (a) Absence of red fluorescence at 4 hours after injection of cells into unconditioned cochlea suggests rapid demise of implanted cells. (b–d) Bright red fluorescence detected in conditioned ears indicates survival of implanted cells up to 7 days after transplantation. Scale bar = 500 µm.
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AE and in other areas adjacent to the epithelium as late as 7 days after injection into the SM (Figure 5a–c). Transplanted HeLa cells displayed intact nuclei (Figure 5b,d,f), suggesting that they were living cells. To determine if surviving HeLa cells were integrated into the AE, we looked for presence of labeled ZO-1 in junctions between the implanted cells and native cells in the AE. We found that some of the red cells displayed ZO-1 and others did not, suggesting that at least some of the transplanted cells formed junctional contacts with native cells (Figure 5b,d,f). To further assess the extent of integration of the implanted cells in the AE, we used confocal microscopy and compared the focal plane of the red cells to that of the native cochlear cells. We determined that some of the implanted cells appeared to adhere to the AE and reside above it (Figure 6a), whereas others appeared at the same focal plane as the native AE cells, suggesting that the exogenous cells have integrated into the tissue (Figure 6b,c). In some cases, we observed red cells positioned between green (ZO-1) labeled cells, based on 3D reconstruction of optical sections (Figure 6d1,d2), providing further evidence that exogenous cells have integrated into the AE.
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Effect of sodium caprate on spiral ganglion neuron survival Opening junctions in the AE may pose a risk to neuronal survival because potassium from SM can potentially reach the spiral ganglion and induce nerve degeneration. To investigate the effect of opening AE junctions on SGN survival in vivo, we investigated the worst case scenario, where junctions are opened but SV pumps were not poisoned by furosemide. To that end, we injected sodium caprate dissolved in artificial perilymph into the SM of left cochleae of normal control animals. We elected to use normal (nondeafened) animals because their population of SGNs is complete and comparable with many human patients who will be future candidates for SC therapy. Seven days after surgery, animals were sacrificed and plastic cross-sections of the cochlea were prepared. We compared SGN number between normal ears and sodium caprate–treated ears. Rosenthal’s canal appeared to contain a confluent population of neurons in first and second turns of untreated control (right) cochleae (Figure 7a,c) and also sodium caprate–treated ears (Figure 7b,d). Quantitative analysis revealed that SGN densities were decreased only in the upper basal turn of sodium caprate– treated ears, but not in other areas (Figure 7e). Thus, the overall pattern of the SGN density data indicates that transient opening of junctions in the AE did not induce a drastic SGN degeneration throughout the entire cochlea.
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Figure 5 Whole-mounts of the AE obtained at different time points after scala media cell transplantation into conditioned cochleae, imaged with epifluorescence of mCherry labeled HeLa cells combined with a phase contrast image (a, c, e) or epifluorescence labeling ZO-1 (green) and nuclei (blue) (b, d, f). The medial aspect of the whole-mounts (modiolar side) faces the bottom in each image. Tissues were obtained 4 hours (a, b), 1 day (c, d) or 7 days (e, f) after HeLa cell injection. (a, c, e) Numerous transplanted HeLa cells were observed in close association with the AE. (b, d, f) The red HeLa cells display intact nuclei and some of them displayed ZO-1 (arrowhead), suggesting that living HeLa cells have a junctional contact with native AE cells. Scale bar = 30 µm. AE, auditory epithelium.
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Figure 6 Stacks of confocal optical sections of whole-mounts of the AE 4 hours after HeLa cell (red) transplantation into scala media of conditioned cochlea stained for ZO-1 (green). Images (sequential focal planes of the same stack) were obtained from top (luminal) to bottom of the AE (a, b, c) or, in another stack (d), cross-sectional images were reconstructed (d1 and d2). (a) The red HeLa cells appear to be attached to the luminal aspect of the AE (the endolymphatic side). (b, c) Some of the red cells appear at the same focal plane as AE cells, suggesting red cells have integrated into the AE. (d) In another area, red cells are seen between ZO-1 positive cells (green label) in reconstructed optical sections (d1, d2, arrow-head), suggesting red cells have integrated into the AE. Scale bar = 30 µm. AE, auditory epithelium.
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in the middle ear which partly covered the otic capsule. This fluid was noted in deafened animals regardless of the conditioning and inoculation protocols, indicating that it resulted from the injection of neomycin. The fluids of the cochlea appeared clear. The recovery of EP following furosemide injection is an important parameter for better understanding the conditions to which inoculated cells can adapt and for determining the functionality of the SV after the cochlear conditioning protocol. To that end, we measured the EP in two animals that received furosemide. In one animal EP was recorded 1 week after furosemide injection and in another 10 days after the injection. EP was 50 and 79 mV, respectively, demonstrating that it can recover following the diuretic treatment.
DISCUSSION
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Figure 7 Comparison of SGN density in normal and conditioned cochleae. Light micrographs of cross-sections through Rosenthal’s canal in the first (a,b) and second turn (c and d) of a normal ear (a and c), or 7 days after sodium caprate in artificial perilymph injection into scala media (b and d), and density of SGNs (e). (a–e) Rosenthal’s canal in sodium caprate in artificial perilymph-injected ears exhibits a confluent population of neurons similar to normal ears. Scale bar = 50 µm. (e) Quantitative analysis indicates SGN density is not significantly reduced following treatment with sodium caprate dissolved in artificial perilymph (black bars) compared with normal ears (gray bars) except in the upper basal region. Statistical comparison was by t test; asterisk indicates P < 0.05. (L = Lower and U = Upper). SGN, spiral ganglion neurons.
General response to conditioning and cell inoculation procedures There were no mortalities in animals used in this set of experiments. Morbidity was limited to signs that are typical after neomycin ototoxicity, including unsteadiness and occasional head tilt. These resolved within a few days and did not worsen after the inoculation of the exogenous cells. Upon dissection of the temporal bone, some of the animals exhibited a whitish fluid Molecular Therapy vol. 22 no. 4 apr. 2014
The goal of this project was to devise means of enhancing survival and integration of cells injected into the hostile fluid environment of the SM. Our in vitro experiments with synthetic endolymph and perilymph confirm that endolymph is toxic and that dilution with an equal amount of perilymph is sufficient to greatly enhance cell survival for at least 6 hours. Based on this result, we developed a transplant protocol that included replacement of endolymph with artificial perilymph and furosemide injection to temporarily inhibit SV pumps. In addition, we sought to enhance integration of the transplanted cells into the deafened AE by using sodium caprate to temporarily weaken cell junctions in the host tissue. Results of our in vivo experiments in guinea pigs demonstrate that the combination of treatments did increase survival of HeLa cells in the cochlea relative to untreated control ears. Moreover, the surviving cells were frequently observed to be embedded within the native cell layer, not merely attached to its surface. We also show that injury to SGN is restricted to the basal turn, that EP can recover to near normal levels and that no other pathology could be observed after administration of sodium caprate in normal ears. The survival of exogenous cells injected into the SM after conditioning the cochlea is an all-or-none outcome, since nonconditioned cochleae contain no surviving exogenous cells. It therefore appears that the three-component conditioning accounts for the survival of the implanted cells. Nevertheless, the exact contribution and impact of each of the three conditioning components (fluid flush, furosemide, and sodium caprate) needs to be evaluated and considered independently. In addition, indirect consequences of the conditioning procedures need to be considered. For instance, it is possible that the procedures to flush the endolymph and inject the cells resulted in injury to Reissner’s membrane, creating a less toxic fluid solution due to mixing of endolymph and perilymph. It is also possible that one of the two procedures for reducing potassium levels, by itself, would have sufficed to promote cell survival over the first critical hours after injection. While these details will need to be resolved in future studies, the results presented here serve to demonstrate the general concept that manipulation of conditions in SM can enhance survival of transplanted cells. Considering the transient effects of the conditioning, it is unclear why cells could survive in the SM for 7 days. The transient nature of furosemide effects have been shown to last for hours10,11 877
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but certainly not for 7 days, which is the longest survival time we examined. In addition, we have shown here that the EP in normal animals that receive the conditioning treatment can recover. Thus, once the effects of furosemide subside and the SV regains its function, potassium levels in the SM begin to recover and return to normal within hours11–13 as does the EP.13–15 Our finding that implanted cells survive despite the return of a potassium rich fluid around them suggests that gradual return to normal conditions in SM is compatible with survival of transplanted cells. It is unclear whether this survival is possible because it is gradual, allowing the cells to slowly adapt to the extracellular potassium, or because cells integrate into the AE, leaving only their apical membrane exposed to endolymph, similar to the native cochlear cells. Additional work in cultures may be able to shed light on the ability of cells to survive in SM-like conditions that are introduced slowly, over hours. Such studies should also characterize the ability of cells to gradually change channels on their plasma membrane and become less susceptible to the high potassium. Alternatives to SM as target for cell transplantation should be considered. For instance, ST is an easier target to reach surgically, especially in the human ear. Previous attempts to inject exogenous cells into the perilymph showed some success and an increased survival and differentiation of transplanted cells in damaged inner ears, compared with uninjured controls.16,17 However, cells did not find their way into SM. It appears that the basilar membrane forms a barrier that, while permeable to small molecules, prevents migration of cells from ST to the AE. Procedures for a transient degradation of the basilar membrane may facilitate targeting cells into the AE after a perilymph inoculation. Once the procedure of cell transplantation is extended to successfully include SCs, it will be necessary to consider practical applicability to the human ear. This may present a challenge because access to the SM in human ears is complex, and the ability to replace the entire endolymph with low potassium fluid like perilymph is not readily available. A potential way to overcome this difficulty may be to induce a longer block of the SV that will last several hours before cells are injected allowing potassium to be naturally depleted prior to surgery. While our data present a concept for enhancing cell survival and integration, further details and optimization steps are needed. We selected sodium caprate for transient disruption of the junctional complexes based on the ability of this compound to increase intestinal absorption of drugs and therapeutic oligonucleotides in various mammals via apical junction disruption.18,19 Previous studies have shown that sodium caprate increases paracellular permeability by triggering a phospholipase C-dependent release of intracellular calcium, which results in withdrawal of actin from peri-apical adherens junction rings, reduction of occludin and ZO-1 density in tight junctions, and increased space between cells at the tight junctions.20,21 The effect of sodium caprate on cell–cell junction permeability is also dose-dependent and time-reversible. We showed with ZO-1 staining that after sodium caprate treatment, the junctions between cells in the AE appeared somewhat disrupted and reorganized 4 hours after. The indication for junction disruption was based on epifluorescence observation, where cells appeared to have an irregular pattern of junctional lines when stained for ZO-1, in contrast to well-defined lines in 878
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normal junctions. The actual morphology of the cell-to-cell contact area after sodium caprate application will need to be studied in more detail using transmission electron microscopy. The procedure we used to disrupt apical junctions in the AE might induce negative consequences for neuronal survival because leakage of potassium from the SM could induce degeneration of SGNs. We therefore tested the effects of transient junctional disruption with sodium caprate on SGN survival. We determined that the density of SGNs in the area near the injection was reduced compared with normal ears, but overall data suggest that this transient junctional opening in the AE may not induce SGN degeneration throughout the cochlea. The results we present demonstrate that transient conditioning of the deaf cochlea facilitates survival and integration of transplanted cells. This is the first step toward introducing cells for therapy in a deaf ear. HeLa cells may be considered for future therapies that provide secreted molecules like neurotrophins. However, to replace missing supporting cells or HCs, it will be necessary to further develop the technology to include actual SCs and to design a method to guide their differentiation after they are integrated in the tissue. In addition, it will be necessary to “home” the transplanted cells towards the appropriate location within the AE, along the length or width of the cochlea. For HC transplantation, it will also be important to enhance formation of functional synapses with SGNs. This task might be resolved by gene therapy with the neurotrophic factors such as neurotrophin 3 and brainderived neurotrophic factor.9 Our data on implantation and integration of exogenous cells in the AE may also be applicable for SC therapy in other epithelial tissues that require therapies based on cell replacement. While our data are first to show survival of implanted cells in the SM, the results have to be extended from use of HeLa cells, as done here, to actual SCs. To achieve this aim, it will be important to consider whether injected cells will be undifferentiated SCs that will be guided to differentiate to the needed cell types after their integration, or differentiated SCs. Each of these two approaches poses challenges. In addition, when designing the cells to be transplanted, one could also consider producing cells that are more tolerant of the high potassium in SM. When potassium-tolerating cells are available, perhaps fewer steps will be needed for conditioning the cochlea. In conclusion, our data show that without specific steps to facilitate survival and integration of implanted cells in the SM compartment of the cochlea, exogenous cells can be expected to degenerate rapidly after transplantation. However, with specially designed measures to condition the cochlea, the recipient tissue can be transiently modified to enhance survival and integration of exogenous cells after injection into SM. Extending the conditioning to also support integration of actual SCs may necessitate additional steps.
MATERIALS AND METHODS
Cells and culture in artificial cochlear fluids. HeLa cells were selected for these experiments because they are robust and stable, yet without conditioning they cannot survive in SM. The HeLa cells we used were stably transfected with mCherry for red fluorescence. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS, Mediatech Inc.,
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Manassas, VA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml puromycin in a humidified chamber with 5% CO2 at 37 °C. The cells (4 × 105) were seeded in 35 mm cell culture microplates and grown to confluence. To evaluate HeLa cell survival in the artificial cochlear fluids, culture media was removed and replaced with artificial endolymph (KCl 140 mmol/l and KHCO3 25 mmol/l), artificial perilymph (NaCl 145 mmol/l, KCl 2.7 mmol/l, MgSO4 2.0 mmol/l, CaCl2 1.2 mmol/l and HEPES C8H18N2O4S 5.0 mmol/l) or a 50:50 mixture of the two. At 30 min, 1, 3, or 6 hours after exposure to the artificial cochlear fluids, the culture microplates were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS and then covered with round cover glasses using CrystalMount. The fixed cells were examined using a Leitz Fluovert FS fluorescence microscope with a digital monochrome Spot camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Animals. All animal experiments were approved by the University of
Michigan, University Committee on the Use and Care of Animals and performed using accepted veterinary standards. We used young adult pigmented guinea pigs from our breeding colony at the University of Michigan. At the beginning of the experiment, all animals had normal Preyer’s reflex and weighed 300–350 g. A total of 48 guinea pigs were enrolled this study. Forty two animals were deafened bilaterally with neomycin (see below): left ears were used for the experimental group and right ears were used for control. The other six animals were not deafened and were used to evaluate effect of junctional disruption with sodium caprate on SGN survival (N = 4) or to measure EP following furosemide injection (N = 2).
Surgical procedure for deafening, conditioning, cell transplantation and EP measurement. Before surgery, the animals were anesthetized with ket-
amine HCl 40 mg/kg (Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) and xylazine 10 mg/kg (AnaSed, Shenandoash, IA) and 0.5 ml of 1% lidocaine HCl was injected subcutaneously in the postauricular area for local anesthesia. The animals were placed in a prone position on a thermoregulated heated pad. The temporal bone was exposed via a postauricular incision and opened to visualize the round window membrane. A small cochleostomy was made in the bone near the round window with a sharp pick. Using a microcannula connected to the tip of a 30 gauge needle and Hamilton syringe, 10 µl of 5% neomycin sulphate solution (Neo-Rx, Pharma-Tek, in sterile distilled water) were injected into the ST through the cochleostomy using an infusion pump for 2 minutes. After removal of the microcannula, the cochleostomy and bulla wall were sealed with tissue adhesive (Vetbond, 3M) and carboxylate cement (Durelon, ESPE). The skin incision was closed in two layers. At 7 days after the deafening surgery, animals were given a second surgery for cell transplantation. Under anesthesia (as above), the animals were placed in a supine position on a thermoregulated heated pad. Furosemide 60 mg/kg (Hospira Inc., Lake forest, IL) was injected intravenously and the temporal bone bulla was exposed via a ventral approach. After removal of the ventral part of the bulla, the medial aspect of the cochlea could be visualized from apex to base. The pigment in the SV was used as a landmark for accessing the SM. The otic capsule was
perforated and opened at two locations, (in the second and third turns) to access the SM. 2 µl of 10 mmol/l sodium caprate (Sodium decanoate, Sigma–Aldrich Co., St. Louis, MO) dissolved in artificial perilymph was injected into the SM through the second turn cochleostomy using an infusion pump for 2 minutes while excess fluid was permitted to escape through the third turn opening. Then, 30 minutes later, a bolus of dissociated HeLa cells with Cherry red fluorescence tags (21 × 106 cells/ml) was injected through the second turn cochleostomy. The cochleostomies and bulla wall were sealed in the same manner as above. In 20 animals, mCherry fluorescence labeled HeLa cells were transplanted into the SM after conditioning (intravenous furosemide injection and flushing endolymph with sodium caprate in artificial perilymph) and in eight animals, HeLa cells were transplanted into the SM without conditioning. Another 14 animals were deafened and conditioned but not given HeLa cells; these animals were used to assess the effect of conditioning on intercellular junctions. In addition, four animals were given sodium caprate but not cells or furosemide and were not flushed, and these were used to assess the effect of junction disruption on survival of SGN. The time-lines for these experiments are shown in Figure 8. EP was measured as described.22 Tissue preparation and immunohistochemistry. To assess the intercellular junctions of the AE, animals were sacrificed 1 or 4 hours after injection of furosemide intravenously and sodium caprate into SM. Following decapitation under deep anesthesia, temporal bones were removed and the fluid spaces of the inner ear perfused with 4% paraformaldehyde in PBS for 1 hour at room temperature. After removal of cochlear bony walls and lateral wall tissues, cochlear tissues were prepared for immunostaining. Tissues were permeabilized with 0.3% Triton X-100 (Sigma–Aldrich Co., St. Louise, MO) for 10 minutes and rinsed in PBS and were incubated in a solution of 5% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) in PBS for 1 hour to reduce nonspecific antibody binding. After rinsing in PBS, tissues were incubated with monoclonal anti-ZO-1 antibody (Zymed Laboratories, Inc., San Francisco, CA) at 1:100 dilution overnight at 4 °C. Specific binding of this antibody in the guinea pig cochlea has previously been determined.23 After rinsing in PBS for 10 minutes, tissues were incubated with AlexaFluor 488 goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR) at 1:200 dilution for 1 hour at room temperature. After rinsing in PBS, the specimens were further dissected to separate individual cochlear turns and mounted on glass slides using CrystalMount (Biomeda, Foster City, CA). The specimens were observed using a Leica DMRB epifluorescence microscope (Leica, Eaton, PA) with a digital monochrome Spot camera. To assess survival of implanted HeLa cells in the cochlea, animals were sacrificed 4 hours, 1 day, or 7 days after transplantation. After fixation and partial removal of the cochlear bony walls and lateral wall tissues, cochleae were examined using a Leica M205 FA automated fluorescence stereomicroscope (Leica, Eaton, PA). Dissection was then completed and the tissues were stained as above except that a DNA intercalating fluorescent probe (Hoechst 33342, Molecular Probes, Eugene, OR) diluted 10 µg/ml in PBS was added to stain nuclei. The whole mount specimens
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Figure 8 Schematic time-line of in vivo cell transplantation experiment. At 7 days after deafening, the cochlea was conditioned (intravenous furosemide injection and endolymph flushed with sodium caprate dissolved in artificial perilymph). Thirty minutes after conditioning, HeLa cells (mCherry fluorescence labeled) were transplanted into scala media. Animals were killed to evaluate presence and integration of transplanted HeLa cells at day 8 or 14.
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were observed using a Leica DMRB epifluorescence microscope with a digital monochrome Spot camera or a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany). Tissue preparation for plastic cross-sections. SGN survival after junctional disruption of AE was evaluated in four animals receiving only sodium caprate and sacrificed (as above) 1 week later. After fixation, the cochleae were decalcified in 3% EDTA in PBS with 0.25% glutaraldehyde (typically 3 weeks), dehydrated in ethanol and embedded in JB-4 resin (Electron Microscopy Sciences, Hatfield, PA). Blocks were sectioned for light microscopy at a thickness of 3 µm on a Leica Ultracut R microtome. Sections were stained with toluidine blue and photographed using a Leica DMRB upright photomicroscope. Image processing and statistical analysis. Adjustment of image contrast,
superimposition of images, and colorization of monochrome fluorescence images were performed using Adobe Photoshop (version 7.0). To evaluate HeLa cell survival in the artificial cochlear fluids, images of five random areas were selected for each culture microplate. The number of HeLa cells exhibiting bright red fluorescence was counted. We compared the differences of average number of surviving HeLa cells in each artificial cochlear fluid using the Student’s t test at each time point. To evaluate SGN survival after sodium caprate application, images of five cross-sections were randomly selected out of the total of ~40 sections obtained for each cochlea. SGNs that exhibited a clear nucleus and cytoplasm were counted by an observer blinded to experimental group. The area of Rosenthal’s canal was measured using tpsDig2 software (F. James Rohlf, Ecology & Evolution, SUNY at Stony Brook) and the number of SGN per 10,000 µm2 was calculated. We compared the difference of average SGN density between normal and sodium caprate-treated ears using Student’s t test for paired samples, with Bonferroni adjustment for multiple comparisons. Corrected P values
original article
Conditioning the Cochlea to Facilitate Survival and Integration of Exogenous Cells into the Auditory Epithelium Yong-Ho Park1,2,3, Kevin F. Wilson4, Yoshihisa Ueda5, Hiu Tung Wong1, Lisa A. Beyer1, Donald L. Swiderski1, David F. Dolan1and Yehoash Raphael1 1 Department of Otolaryngology-Head and Neck Surgery, KHRI, The University of Michigan, Ann Arbor, MI, USA; 2Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Chungnam National University, 33 Munwha Ro, Daesa Dong, Jung Gu, Daejeon, Korea; 3Brain Research Institute, College of Medicine, Chungnam National University, 33 Munwha Ro, Daesa Dong, Jung Gu, Daejeon, Korea; 4Division of Otolaryngology, University of Utah, 50 North Medical Dr.3C120 SOM, Salt Lake City, UT, USA; 5Department of Otolaryngology-Head and Neck Surgery, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka, Japan.
The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media. Received 8 July 2013; accepted 23 December 2013; advance online publication 4 February 2014. doi:10.1038/mt.2013.292
INTRODUCTION
In the auditory epithelium (AE) of the mammalian cochlea, hair cells (HCs) and supporting cells are formed during embryogenesis and once they mature, they cannot be replaced if lost. Because of the lack of regeneration of these cells, lesions in the AE lead to permanent hearing loss. One possible therapeutic approach for restoring hearing may be implantation of exogenous cells such as stem cells (SC) into the cochlea. This therapy would consist of two major stages: (i) producing the cells and (ii) inserting them into the target tissue, the AE. Progress in the first stage has provided
several methods for producing new HCs from different types of SCs.1–4 Here, we start to address the second stage, inserting and integrating exogenous cells into the native AE tissue. Inserting and integrating SCs into the AE is an essential practical step for therapeutic use of these cells for hearing restoration. Integration of exogenous cells in an epithelial layer is a complex and challenging task. Like other epithelia, the AE is a highly organized layer of confluent cells that are connected to each other by apical junctional complexes. The supporting cells of the AE rest on the basilar membrane, a thick layer of connective tissue immediately beneath the basement membrane. Cells injected into the fluid (perilymph) in the space under the basilar membrane, the scala tympani (ST), can survive but do not cross the basilar membrane into the AE. It is therefore necessary to attempt integrating cells by injecting them into the lumen of the cochlear duct (endolymph of the scala media (SM)). However, this fluid compartment is not hospitable to cells, because the luminal fluid (endolymph) contains high levels of potassium.5 Consequently, cells injected into SM endolymph do not survive.6 It is therefore a major challenge to help exogenous cells survive in the SM until they integrate into the AE. After establishing conditions that allow cells to survive, the next challenge is to induce their integration into the AE. This is difficult because the apical junctions between the AE cells are extremely robust, consisting of intermittent adherens/tight junctions,7,8 which make the organ of Corti an especially difficult barrier to breach. These elaborate junctions persist in the deafened AE depleted of HCs.9 The combination of this strong epithelial barrier and the overlying toxic fluids presents special challenges for inserting cells into this tissue. Not only must the highly resistant intercellular junctions be broken to permit intercalation of exogenous cells but doing so may expose neighboring tissues to the toxic endolymph. To facilitate survival of transplanted cells until they are integrated, and their integration into the AE without exposing surviving native cells to hazards, it is necessary to perform several simultaneous manipulations of multiple cochlear components.
Correspondence: Yehoash Raphael, Department of Otolaryngology-Head and Neck Surgery, KHRI, The University of Michigan, Ann Arbor, MI, USA. E-mail: [email protected] Molecular Therapy vol. 22 no. 4, 873–880 apr. 2014
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One possibility to accomplish insertion and integration of exogenous cells into the AE is to change the conditions in the cochlea in a transient fashion, to reduce or eliminate the obstacles to survival and integration of implanted cells. Specifically, it would be necessary to “condition” the cochlea by eliminating the high potassium and the endocochlear potential (EP) and by opening the cell–cell junctions of the AE in a reversible way. Here, we present results of experiments designed to test whether such conditioning of the cochlea can facilitate survival of exogenous cells in SM and their integration into the AE. Because the apical junctions also play an essential role in survival of the spiral ganglion neurons (SGNs) by maintaining the luminal barrier, we also evaluated the effects of opening the AE junctions on survival of the SGN. To condition the cochlea, we flushed away the endolymph and replaced it with artificial perilymph, blocked ion pumps in the stria vascularis (SV) with furosemide and applied sodium caprate, known to disrupt tight junctions. We then injected tagged HeLa cells into the SM. HeLa cells were selected as a stable and robust type of cell to test the conditioning approach and establish proof for the principle that cells can survive in the SM of the conditioned cochlea. The data demonstrate that conditioning the cochlea can prepare it to support survival of transplanted HeLa cells for at least 7 days.
RESULTS Cell survival in artificial cochlear fluids
To evaluate HeLa cell survival in the artificial cochlear fluids, HeLa cells grown in cultures were exposed to artificial endolymph, perilymph, or a 50:50 mixture of both. Culture plates were examined at 30 minutes, 1 hour, 3 hours, or 6 hours after the culture medium was replaced by artificial cochlear fluids. HeLa cells exposed to artificial endolymph promptly detached from the culture plate, assumed a spherical shape, and rapidly disintegrated. Floating debris from dead cells could be detected but intact floating cells were not seen. Reduction in cell density was evident as early as 1 hour (Figure 1d versus Figure 1a,e,f) and most cells died within 6 hours of being placed in the artificial endolymph (Figure 1j). Control
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In contrast, cells in artificial perilymph or in the 50:50 mixture survived longer than those in the endolymph only (Figure 1k and l). Quantitative analysis of cell survival over time determined that the number of surviving cells in the artificial endolymph was significantly decreased compared with cells exposed to artificial perilymph or the mixture of endolymph and perilymph (P < 0.01) (Figure 2).
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Figure 1 HeLa cell (mCherry fluorescence labeled) survival in culture dishes at 30 minutes. (a–c), 1 hour (d–f), 3 hours (g–i), or 6 hours (j–l) after dishes were filled with artificial endolymph (a, d, g, j), artificial perilymph (b, e, h, k), or mixture of endolymph and perilymph (c, f, i, l). In the artificial endolymph, HeLa cells begin to die within 30 minutes after exposure and most cells are dead within 6 hours. In the artificial perilymph and in the mixture of endolymph and perilymph, most HeLa cells survived at least 6 hours. Scale bar = 100 µm.
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Figure 2 Quantitative analysis of HeLa cell survival in the artificial cochlear fluids. At 1, 3 and 6 hours after artificial cochlear fluid exposure, survival of HeLa cells in endolymph is significantly reduced compared with that in perilymph or in a mixture of endolymph and perilymph. No differences in HeLa cell survival were observed between perilymph and the mixture of endolymph and perilymph at any time point. Statistical comparison was by t test; Asterisk indicates P < 0.01.
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Effect of sodium caprate on auditory epithelial junctions The concentration of sodium caprate selected to disrupt apical epithelial junctions, 10 mmol/l, was determined by preliminary experiments not reported here. To investigate the effect of sodium caprate on apical AE junctions in vivo, we injected sodium caprate dissolved in artificial perilymph into the SM of neomycin-deafened animals (7 days after inducing the ototoxic neomycin lesion). At 1 or 4 hours after the surgery, animals were sacrificed and whole mounts of the AE were stained with antibody against ZO-1 (a tight junction component) and prepared for epifluorescence. In untreated control ears, the AE was flat, with no remaining HCs or differentiated supporting cells. Cell–cell junctional areas in the AE appeared as a well-defined single line, suggesting tight junctions between cells were intact (Figure 3a). In ears injected with sodium caprate, the AE was also flat, but in contrast to controls, 1 hour after introducing sodium caprate into the SM, the staining pattern of ZO-1 in the AE included gaps and spaces between
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cells, and the overall appearance of the junctions was somewhat disrupted and less well defined. This suggested that sodium caprate induced deterioration of the apical junctions (Figure 3b). In tissues obtained at 4 hours after sodium caprate treatment, junctions appeared normal again, suggesting that after initially opening, the cell–cell contacts reorganized and resealed (Figure 3c).
Survival of transplanted cells in the SM To evaluate transplanted HeLa cell survival in vivo, mCherry fluorescence labeled HeLa cells were transplanted into the SM in untreated control and conditioned (intravenous furosemide injection and endolymph flushed with sodium caprate in artificial perilymph) animals. At 4 hours, 1 day, or 7 days after transplantation, animals were sacrificed, and cochlear tissues were examined under a fluorescence stereomicroscope. Upon dissection of the tissues, no signs of inflammatory response were noted in the cochlea. In untreated control ears, cochleae were devoid of red fluorescence signal (Figure 4a), suggesting that the implanted cells did not survive. However, in conditioned cochleae, bright red fluorescence signals were observed inside the cochlea as much as 7 days after HeLa cell transplantation (Figure 4b–d). To examine the areas with red fluorescence more closely, cochleae were dissected and stained to label tight junctions (ZO-1 antibody, green) and nuclei (Hoechst 33342, blue) in whole mounts. Under epifluorescence combined with phase contrast microscopy, transplanted HeLa cells were observed around the
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Figure 3 Whole-mounts of the cochlear sensory epithelium in neomycin-deafened control ear (untreated, a) and deafened ears obtained 1 hour (b), or 4 hours (c) after sodium caprate treatment, stained for ZO-1 (green) and photographed with epifluorescence. (a) In neomycin-deafened control ear without sodium caprate treatment, there are no gaps between cells in the auditory epithelium. (b) One hour after sodium caprate treatment, cell junctions have irregular contours and gaps (arrow-heads) suggesting disruption of apical junction complexes. (c) Four hours after sodium caprate treatment, a few gaps remain between cells, but most of the tissue appears to have reorganized to resemble the control ear without sodium caprate treatment. Scale bar = 30 µm.
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Figure 4 Fluorescence stereomicroscopy images of partially dissected cochleae that were implanted with HeLa cells (red) 4 hours after injection into unconditioned cochlea (a) or several time points after injection into cochleae conditioned with infusion of sodium caprate in artificial perilymph and systemic furosemide (b, 4 hours; c, 1 day; and d, 7 days). (a) Absence of red fluorescence at 4 hours after injection of cells into unconditioned cochlea suggests rapid demise of implanted cells. (b–d) Bright red fluorescence detected in conditioned ears indicates survival of implanted cells up to 7 days after transplantation. Scale bar = 500 µm.
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AE and in other areas adjacent to the epithelium as late as 7 days after injection into the SM (Figure 5a–c). Transplanted HeLa cells displayed intact nuclei (Figure 5b,d,f), suggesting that they were living cells. To determine if surviving HeLa cells were integrated into the AE, we looked for presence of labeled ZO-1 in junctions between the implanted cells and native cells in the AE. We found that some of the red cells displayed ZO-1 and others did not, suggesting that at least some of the transplanted cells formed junctional contacts with native cells (Figure 5b,d,f). To further assess the extent of integration of the implanted cells in the AE, we used confocal microscopy and compared the focal plane of the red cells to that of the native cochlear cells. We determined that some of the implanted cells appeared to adhere to the AE and reside above it (Figure 6a), whereas others appeared at the same focal plane as the native AE cells, suggesting that the exogenous cells have integrated into the tissue (Figure 6b,c). In some cases, we observed red cells positioned between green (ZO-1) labeled cells, based on 3D reconstruction of optical sections (Figure 6d1,d2), providing further evidence that exogenous cells have integrated into the AE.
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Effect of sodium caprate on spiral ganglion neuron survival Opening junctions in the AE may pose a risk to neuronal survival because potassium from SM can potentially reach the spiral ganglion and induce nerve degeneration. To investigate the effect of opening AE junctions on SGN survival in vivo, we investigated the worst case scenario, where junctions are opened but SV pumps were not poisoned by furosemide. To that end, we injected sodium caprate dissolved in artificial perilymph into the SM of left cochleae of normal control animals. We elected to use normal (nondeafened) animals because their population of SGNs is complete and comparable with many human patients who will be future candidates for SC therapy. Seven days after surgery, animals were sacrificed and plastic cross-sections of the cochlea were prepared. We compared SGN number between normal ears and sodium caprate–treated ears. Rosenthal’s canal appeared to contain a confluent population of neurons in first and second turns of untreated control (right) cochleae (Figure 7a,c) and also sodium caprate–treated ears (Figure 7b,d). Quantitative analysis revealed that SGN densities were decreased only in the upper basal turn of sodium caprate– treated ears, but not in other areas (Figure 7e). Thus, the overall pattern of the SGN density data indicates that transient opening of junctions in the AE did not induce a drastic SGN degeneration throughout the entire cochlea.
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Figure 5 Whole-mounts of the AE obtained at different time points after scala media cell transplantation into conditioned cochleae, imaged with epifluorescence of mCherry labeled HeLa cells combined with a phase contrast image (a, c, e) or epifluorescence labeling ZO-1 (green) and nuclei (blue) (b, d, f). The medial aspect of the whole-mounts (modiolar side) faces the bottom in each image. Tissues were obtained 4 hours (a, b), 1 day (c, d) or 7 days (e, f) after HeLa cell injection. (a, c, e) Numerous transplanted HeLa cells were observed in close association with the AE. (b, d, f) The red HeLa cells display intact nuclei and some of them displayed ZO-1 (arrowhead), suggesting that living HeLa cells have a junctional contact with native AE cells. Scale bar = 30 µm. AE, auditory epithelium.
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Figure 6 Stacks of confocal optical sections of whole-mounts of the AE 4 hours after HeLa cell (red) transplantation into scala media of conditioned cochlea stained for ZO-1 (green). Images (sequential focal planes of the same stack) were obtained from top (luminal) to bottom of the AE (a, b, c) or, in another stack (d), cross-sectional images were reconstructed (d1 and d2). (a) The red HeLa cells appear to be attached to the luminal aspect of the AE (the endolymphatic side). (b, c) Some of the red cells appear at the same focal plane as AE cells, suggesting red cells have integrated into the AE. (d) In another area, red cells are seen between ZO-1 positive cells (green label) in reconstructed optical sections (d1, d2, arrow-head), suggesting red cells have integrated into the AE. Scale bar = 30 µm. AE, auditory epithelium.
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in the middle ear which partly covered the otic capsule. This fluid was noted in deafened animals regardless of the conditioning and inoculation protocols, indicating that it resulted from the injection of neomycin. The fluids of the cochlea appeared clear. The recovery of EP following furosemide injection is an important parameter for better understanding the conditions to which inoculated cells can adapt and for determining the functionality of the SV after the cochlear conditioning protocol. To that end, we measured the EP in two animals that received furosemide. In one animal EP was recorded 1 week after furosemide injection and in another 10 days after the injection. EP was 50 and 79 mV, respectively, demonstrating that it can recover following the diuretic treatment.
DISCUSSION
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Figure 7 Comparison of SGN density in normal and conditioned cochleae. Light micrographs of cross-sections through Rosenthal’s canal in the first (a,b) and second turn (c and d) of a normal ear (a and c), or 7 days after sodium caprate in artificial perilymph injection into scala media (b and d), and density of SGNs (e). (a–e) Rosenthal’s canal in sodium caprate in artificial perilymph-injected ears exhibits a confluent population of neurons similar to normal ears. Scale bar = 50 µm. (e) Quantitative analysis indicates SGN density is not significantly reduced following treatment with sodium caprate dissolved in artificial perilymph (black bars) compared with normal ears (gray bars) except in the upper basal region. Statistical comparison was by t test; asterisk indicates P < 0.05. (L = Lower and U = Upper). SGN, spiral ganglion neurons.
General response to conditioning and cell inoculation procedures There were no mortalities in animals used in this set of experiments. Morbidity was limited to signs that are typical after neomycin ototoxicity, including unsteadiness and occasional head tilt. These resolved within a few days and did not worsen after the inoculation of the exogenous cells. Upon dissection of the temporal bone, some of the animals exhibited a whitish fluid Molecular Therapy vol. 22 no. 4 apr. 2014
The goal of this project was to devise means of enhancing survival and integration of cells injected into the hostile fluid environment of the SM. Our in vitro experiments with synthetic endolymph and perilymph confirm that endolymph is toxic and that dilution with an equal amount of perilymph is sufficient to greatly enhance cell survival for at least 6 hours. Based on this result, we developed a transplant protocol that included replacement of endolymph with artificial perilymph and furosemide injection to temporarily inhibit SV pumps. In addition, we sought to enhance integration of the transplanted cells into the deafened AE by using sodium caprate to temporarily weaken cell junctions in the host tissue. Results of our in vivo experiments in guinea pigs demonstrate that the combination of treatments did increase survival of HeLa cells in the cochlea relative to untreated control ears. Moreover, the surviving cells were frequently observed to be embedded within the native cell layer, not merely attached to its surface. We also show that injury to SGN is restricted to the basal turn, that EP can recover to near normal levels and that no other pathology could be observed after administration of sodium caprate in normal ears. The survival of exogenous cells injected into the SM after conditioning the cochlea is an all-or-none outcome, since nonconditioned cochleae contain no surviving exogenous cells. It therefore appears that the three-component conditioning accounts for the survival of the implanted cells. Nevertheless, the exact contribution and impact of each of the three conditioning components (fluid flush, furosemide, and sodium caprate) needs to be evaluated and considered independently. In addition, indirect consequences of the conditioning procedures need to be considered. For instance, it is possible that the procedures to flush the endolymph and inject the cells resulted in injury to Reissner’s membrane, creating a less toxic fluid solution due to mixing of endolymph and perilymph. It is also possible that one of the two procedures for reducing potassium levels, by itself, would have sufficed to promote cell survival over the first critical hours after injection. While these details will need to be resolved in future studies, the results presented here serve to demonstrate the general concept that manipulation of conditions in SM can enhance survival of transplanted cells. Considering the transient effects of the conditioning, it is unclear why cells could survive in the SM for 7 days. The transient nature of furosemide effects have been shown to last for hours10,11 877
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but certainly not for 7 days, which is the longest survival time we examined. In addition, we have shown here that the EP in normal animals that receive the conditioning treatment can recover. Thus, once the effects of furosemide subside and the SV regains its function, potassium levels in the SM begin to recover and return to normal within hours11–13 as does the EP.13–15 Our finding that implanted cells survive despite the return of a potassium rich fluid around them suggests that gradual return to normal conditions in SM is compatible with survival of transplanted cells. It is unclear whether this survival is possible because it is gradual, allowing the cells to slowly adapt to the extracellular potassium, or because cells integrate into the AE, leaving only their apical membrane exposed to endolymph, similar to the native cochlear cells. Additional work in cultures may be able to shed light on the ability of cells to survive in SM-like conditions that are introduced slowly, over hours. Such studies should also characterize the ability of cells to gradually change channels on their plasma membrane and become less susceptible to the high potassium. Alternatives to SM as target for cell transplantation should be considered. For instance, ST is an easier target to reach surgically, especially in the human ear. Previous attempts to inject exogenous cells into the perilymph showed some success and an increased survival and differentiation of transplanted cells in damaged inner ears, compared with uninjured controls.16,17 However, cells did not find their way into SM. It appears that the basilar membrane forms a barrier that, while permeable to small molecules, prevents migration of cells from ST to the AE. Procedures for a transient degradation of the basilar membrane may facilitate targeting cells into the AE after a perilymph inoculation. Once the procedure of cell transplantation is extended to successfully include SCs, it will be necessary to consider practical applicability to the human ear. This may present a challenge because access to the SM in human ears is complex, and the ability to replace the entire endolymph with low potassium fluid like perilymph is not readily available. A potential way to overcome this difficulty may be to induce a longer block of the SV that will last several hours before cells are injected allowing potassium to be naturally depleted prior to surgery. While our data present a concept for enhancing cell survival and integration, further details and optimization steps are needed. We selected sodium caprate for transient disruption of the junctional complexes based on the ability of this compound to increase intestinal absorption of drugs and therapeutic oligonucleotides in various mammals via apical junction disruption.18,19 Previous studies have shown that sodium caprate increases paracellular permeability by triggering a phospholipase C-dependent release of intracellular calcium, which results in withdrawal of actin from peri-apical adherens junction rings, reduction of occludin and ZO-1 density in tight junctions, and increased space between cells at the tight junctions.20,21 The effect of sodium caprate on cell–cell junction permeability is also dose-dependent and time-reversible. We showed with ZO-1 staining that after sodium caprate treatment, the junctions between cells in the AE appeared somewhat disrupted and reorganized 4 hours after. The indication for junction disruption was based on epifluorescence observation, where cells appeared to have an irregular pattern of junctional lines when stained for ZO-1, in contrast to well-defined lines in 878
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normal junctions. The actual morphology of the cell-to-cell contact area after sodium caprate application will need to be studied in more detail using transmission electron microscopy. The procedure we used to disrupt apical junctions in the AE might induce negative consequences for neuronal survival because leakage of potassium from the SM could induce degeneration of SGNs. We therefore tested the effects of transient junctional disruption with sodium caprate on SGN survival. We determined that the density of SGNs in the area near the injection was reduced compared with normal ears, but overall data suggest that this transient junctional opening in the AE may not induce SGN degeneration throughout the cochlea. The results we present demonstrate that transient conditioning of the deaf cochlea facilitates survival and integration of transplanted cells. This is the first step toward introducing cells for therapy in a deaf ear. HeLa cells may be considered for future therapies that provide secreted molecules like neurotrophins. However, to replace missing supporting cells or HCs, it will be necessary to further develop the technology to include actual SCs and to design a method to guide their differentiation after they are integrated in the tissue. In addition, it will be necessary to “home” the transplanted cells towards the appropriate location within the AE, along the length or width of the cochlea. For HC transplantation, it will also be important to enhance formation of functional synapses with SGNs. This task might be resolved by gene therapy with the neurotrophic factors such as neurotrophin 3 and brainderived neurotrophic factor.9 Our data on implantation and integration of exogenous cells in the AE may also be applicable for SC therapy in other epithelial tissues that require therapies based on cell replacement. While our data are first to show survival of implanted cells in the SM, the results have to be extended from use of HeLa cells, as done here, to actual SCs. To achieve this aim, it will be important to consider whether injected cells will be undifferentiated SCs that will be guided to differentiate to the needed cell types after their integration, or differentiated SCs. Each of these two approaches poses challenges. In addition, when designing the cells to be transplanted, one could also consider producing cells that are more tolerant of the high potassium in SM. When potassium-tolerating cells are available, perhaps fewer steps will be needed for conditioning the cochlea. In conclusion, our data show that without specific steps to facilitate survival and integration of implanted cells in the SM compartment of the cochlea, exogenous cells can be expected to degenerate rapidly after transplantation. However, with specially designed measures to condition the cochlea, the recipient tissue can be transiently modified to enhance survival and integration of exogenous cells after injection into SM. Extending the conditioning to also support integration of actual SCs may necessitate additional steps.
MATERIALS AND METHODS
Cells and culture in artificial cochlear fluids. HeLa cells were selected for these experiments because they are robust and stable, yet without conditioning they cannot survive in SM. The HeLa cells we used were stably transfected with mCherry for red fluorescence. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS, Mediatech Inc.,
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Manassas, VA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml puromycin in a humidified chamber with 5% CO2 at 37 °C. The cells (4 × 105) were seeded in 35 mm cell culture microplates and grown to confluence. To evaluate HeLa cell survival in the artificial cochlear fluids, culture media was removed and replaced with artificial endolymph (KCl 140 mmol/l and KHCO3 25 mmol/l), artificial perilymph (NaCl 145 mmol/l, KCl 2.7 mmol/l, MgSO4 2.0 mmol/l, CaCl2 1.2 mmol/l and HEPES C8H18N2O4S 5.0 mmol/l) or a 50:50 mixture of the two. At 30 min, 1, 3, or 6 hours after exposure to the artificial cochlear fluids, the culture microplates were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS and then covered with round cover glasses using CrystalMount. The fixed cells were examined using a Leitz Fluovert FS fluorescence microscope with a digital monochrome Spot camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Animals. All animal experiments were approved by the University of
Michigan, University Committee on the Use and Care of Animals and performed using accepted veterinary standards. We used young adult pigmented guinea pigs from our breeding colony at the University of Michigan. At the beginning of the experiment, all animals had normal Preyer’s reflex and weighed 300–350 g. A total of 48 guinea pigs were enrolled this study. Forty two animals were deafened bilaterally with neomycin (see below): left ears were used for the experimental group and right ears were used for control. The other six animals were not deafened and were used to evaluate effect of junctional disruption with sodium caprate on SGN survival (N = 4) or to measure EP following furosemide injection (N = 2).
Surgical procedure for deafening, conditioning, cell transplantation and EP measurement. Before surgery, the animals were anesthetized with ket-
amine HCl 40 mg/kg (Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) and xylazine 10 mg/kg (AnaSed, Shenandoash, IA) and 0.5 ml of 1% lidocaine HCl was injected subcutaneously in the postauricular area for local anesthesia. The animals were placed in a prone position on a thermoregulated heated pad. The temporal bone was exposed via a postauricular incision and opened to visualize the round window membrane. A small cochleostomy was made in the bone near the round window with a sharp pick. Using a microcannula connected to the tip of a 30 gauge needle and Hamilton syringe, 10 µl of 5% neomycin sulphate solution (Neo-Rx, Pharma-Tek, in sterile distilled water) were injected into the ST through the cochleostomy using an infusion pump for 2 minutes. After removal of the microcannula, the cochleostomy and bulla wall were sealed with tissue adhesive (Vetbond, 3M) and carboxylate cement (Durelon, ESPE). The skin incision was closed in two layers. At 7 days after the deafening surgery, animals were given a second surgery for cell transplantation. Under anesthesia (as above), the animals were placed in a supine position on a thermoregulated heated pad. Furosemide 60 mg/kg (Hospira Inc., Lake forest, IL) was injected intravenously and the temporal bone bulla was exposed via a ventral approach. After removal of the ventral part of the bulla, the medial aspect of the cochlea could be visualized from apex to base. The pigment in the SV was used as a landmark for accessing the SM. The otic capsule was
perforated and opened at two locations, (in the second and third turns) to access the SM. 2 µl of 10 mmol/l sodium caprate (Sodium decanoate, Sigma–Aldrich Co., St. Louis, MO) dissolved in artificial perilymph was injected into the SM through the second turn cochleostomy using an infusion pump for 2 minutes while excess fluid was permitted to escape through the third turn opening. Then, 30 minutes later, a bolus of dissociated HeLa cells with Cherry red fluorescence tags (21 × 106 cells/ml) was injected through the second turn cochleostomy. The cochleostomies and bulla wall were sealed in the same manner as above. In 20 animals, mCherry fluorescence labeled HeLa cells were transplanted into the SM after conditioning (intravenous furosemide injection and flushing endolymph with sodium caprate in artificial perilymph) and in eight animals, HeLa cells were transplanted into the SM without conditioning. Another 14 animals were deafened and conditioned but not given HeLa cells; these animals were used to assess the effect of conditioning on intercellular junctions. In addition, four animals were given sodium caprate but not cells or furosemide and were not flushed, and these were used to assess the effect of junction disruption on survival of SGN. The time-lines for these experiments are shown in Figure 8. EP was measured as described.22 Tissue preparation and immunohistochemistry. To assess the intercellular junctions of the AE, animals were sacrificed 1 or 4 hours after injection of furosemide intravenously and sodium caprate into SM. Following decapitation under deep anesthesia, temporal bones were removed and the fluid spaces of the inner ear perfused with 4% paraformaldehyde in PBS for 1 hour at room temperature. After removal of cochlear bony walls and lateral wall tissues, cochlear tissues were prepared for immunostaining. Tissues were permeabilized with 0.3% Triton X-100 (Sigma–Aldrich Co., St. Louise, MO) for 10 minutes and rinsed in PBS and were incubated in a solution of 5% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) in PBS for 1 hour to reduce nonspecific antibody binding. After rinsing in PBS, tissues were incubated with monoclonal anti-ZO-1 antibody (Zymed Laboratories, Inc., San Francisco, CA) at 1:100 dilution overnight at 4 °C. Specific binding of this antibody in the guinea pig cochlea has previously been determined.23 After rinsing in PBS for 10 minutes, tissues were incubated with AlexaFluor 488 goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR) at 1:200 dilution for 1 hour at room temperature. After rinsing in PBS, the specimens were further dissected to separate individual cochlear turns and mounted on glass slides using CrystalMount (Biomeda, Foster City, CA). The specimens were observed using a Leica DMRB epifluorescence microscope (Leica, Eaton, PA) with a digital monochrome Spot camera. To assess survival of implanted HeLa cells in the cochlea, animals were sacrificed 4 hours, 1 day, or 7 days after transplantation. After fixation and partial removal of the cochlear bony walls and lateral wall tissues, cochleae were examined using a Leica M205 FA automated fluorescence stereomicroscope (Leica, Eaton, PA). Dissection was then completed and the tissues were stained as above except that a DNA intercalating fluorescent probe (Hoechst 33342, Molecular Probes, Eugene, OR) diluted 10 µg/ml in PBS was added to stain nuclei. The whole mount specimens
Deafening
Conditioning and cell transplantation
Day 0
Day 7
Day 8
Day 14
Sacrifice
Sacrifice
Sacrifice
Figure 8 Schematic time-line of in vivo cell transplantation experiment. At 7 days after deafening, the cochlea was conditioned (intravenous furosemide injection and endolymph flushed with sodium caprate dissolved in artificial perilymph). Thirty minutes after conditioning, HeLa cells (mCherry fluorescence labeled) were transplanted into scala media. Animals were killed to evaluate presence and integration of transplanted HeLa cells at day 8 or 14.
Molecular Therapy vol. 22 no. 4 apr. 2014
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Exogenous Cell Survival in Conditioned Cochlea
were observed using a Leica DMRB epifluorescence microscope with a digital monochrome Spot camera or a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany). Tissue preparation for plastic cross-sections. SGN survival after junctional disruption of AE was evaluated in four animals receiving only sodium caprate and sacrificed (as above) 1 week later. After fixation, the cochleae were decalcified in 3% EDTA in PBS with 0.25% glutaraldehyde (typically 3 weeks), dehydrated in ethanol and embedded in JB-4 resin (Electron Microscopy Sciences, Hatfield, PA). Blocks were sectioned for light microscopy at a thickness of 3 µm on a Leica Ultracut R microtome. Sections were stained with toluidine blue and photographed using a Leica DMRB upright photomicroscope. Image processing and statistical analysis. Adjustment of image contrast,
superimposition of images, and colorization of monochrome fluorescence images were performed using Adobe Photoshop (version 7.0). To evaluate HeLa cell survival in the artificial cochlear fluids, images of five random areas were selected for each culture microplate. The number of HeLa cells exhibiting bright red fluorescence was counted. We compared the differences of average number of surviving HeLa cells in each artificial cochlear fluid using the Student’s t test at each time point. To evaluate SGN survival after sodium caprate application, images of five cross-sections were randomly selected out of the total of ~40 sections obtained for each cochlea. SGNs that exhibited a clear nucleus and cytoplasm were counted by an observer blinded to experimental group. The area of Rosenthal’s canal was measured using tpsDig2 software (F. James Rohlf, Ecology & Evolution, SUNY at Stony Brook) and the number of SGN per 10,000 µm2 was calculated. We compared the difference of average SGN density between normal and sodium caprate-treated ears using Student’s t test for paired samples, with Bonferroni adjustment for multiple comparisons. Corrected P values
