Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea
© Oiticica et al; licensee BioMed Central Ltd. 2010
Received: 2 May 2010
Accepted: 18 November 2010
Published: 18 November 2010
Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.
Organs of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα). Immunofluorescence assays were conducted for phenotype characterization.
The TGFα group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells.
Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.
The sense of hearing, one of the five primary senses, is mediated through a complex sensory system that allows the perception and reaction to a huge variety of sound stimuli. Hearing makes feasible individual interaction with the environment and is essential for communication. Typically, the auditory system comprises a highly specialized sensory epithelium, the organ of Corti. It contains mechanosensory hair cells as the primary transducers of auditory stimuli, and supporting cells that provide a structural and physiological supporting epithelium. One end of hair cells interacts with physical inputs and transmits these signals to the neural circuits, linked to the opposite end of the cell by a synapsis. Most types of congenital and acquired hearing loss arise from damage and irreversible loss of cochlear hair cells or their associated neurons.
A remarkable characteristic of highly differentiated and specialized mammalian cells, including cochlear sensory hair cells, is that after birth they are held in a post-mitotic state which contributes to their terminal differentiation and inability of repair. A complex network of cyclin-dependent kinases and negative cell cycle regulators are involved in blocking cell cycle reentry, progression and differentiation in mammalian inner ear, maintaining the cell cycle arrest[4–7]. However, it has been reported that supporting cell proliferation and hair cell regeneration spontaneously occurs in vitro after aminoglycoside ototoxicity in the vestibular sensory epithelia of adult mammals, including guinea pigs and humans[8, 9]. In these instances, new hair cells seem to originate from supporting cells that reenter the cell cycle and subsequently divide asymmetrically; or they may arise after transdifferentiation from supporting cells of the vestibular system, but not from cochlea[10, 11].
It is now known that mouse adult vestibular sensory epithelia and neonatal organ of Corti tissue harbor cells that, when subjected to suspension culturing, are able to generate floating clonal colonies, the so-called spheres[12, 13]. These spheres demonstrated capacity for self-renewal, and express inner ear precursor markers such as nestin and Sox2. However, the sphere formation ability of the dissociated mouse cochlea decreases during the second and third postnatal weeks, in a way substantially faster than the vestibular organ, which maintains its stem cell populations up to more advanced ages. These findings suggest that in the organ of Corti the stem cell properties become limited along the development. Standardization of procedures for cell culturing and characterization is a major step toward the study of cochlea progenitor cell differentiation and the definition of strategies for inner ear molecular, gene and cell therapy. However, the establishment of dissociated organ of Corti suspension culture is still challenging. Although the guinea pig has been widely adopted as an animal model for cochlea experimental surgery, it has not been demonstrated as an appropriate source of cells for suspension culturing of the organ of Corti. The aim of this study was to compare conditions and outcomes of suspension cultures of dissociated organ of Corti from neonatal mouse and guinea pig, and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.
The experimental protocol was previously approved by the Internal Review Board on Ethics in Animal Research from the Medical School and the Institute of Biosciences of the University of São Paulo. All experiments were conducted in accordance with the guidelines for the care and use of laboratory animals established by the American National Research Council. In this study, we used postnatal day 3 (P3) C57BL/6J mouse (Mus musculus) and guinea pig (Cavea porcellus), obtained from specialized breeders (Biotério de Camundongos Isogênicos do Instituto de Ciências Biomédicas, USP and Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia, CEDEME, UNIFESP, São Paulo, Brazil). Animals presenting acute or chronic ear infection or congenital malformations were excluded from the study. Animals were sacrificed in a carbon dioxide chamber.
Tissue isolation and dissociation
After bathing the animals in absolute ethanol, they were decapitated and had the temporal bones removed and maintained in Leibovitz's L-15 medium (Sigma-Aldrich, St Louis MO). Cochlear sensory epithelia containing the organ of Corti were surgically isolated using micro-mechanical dissection technique under a stereo-microscope (Tecnival, SQF-F); stria vascularis and spiral ganglion were removed. The epithelia containing the organ of Corti were isolated, transferred to a flask containing 1 mL of HBSS solution (Hank's Balanced Salt Solution, 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 5.6 mM glucose, 300 mM HEPES pH 7.4) and 0.05 U/mL elastase (Sigma-Aldrich, St Louis MO), and incubated for 15 minutes at 37°C. Further enzymatic dissociation of organ of Corti was achieved by adding CaCl2 to 3 mM and 600 U/mL collagenase type II (Invitrogen, Carlsbad CA) and incubating for extra 15 minutes at 37°C. Trypsin dissociation of tissue was sequentially performed with 0.05% Tryple (Invitrogen, Carlsbad CA) for 15 min. at 37°C. Tissue was precipitated by gravity within the microtube, and the supernatant was discarded by aspiration. After washing the sample twice with HBSS, cells were mechanically dissociated by passing through fire-polished Pasteur pipettes with decreasing calibers and filtered through a 100-μm cell strainer (BD Falcon™) to remove cell debris. Twenty μL of the supernatant were used for cell morphology observation and counting at an Axiovert 40C microscope (Zeiss, Germany). Cell suspension was centrifuged at 200 × g, 4°C, for five minutes. The supernatant was discarded and the cells were resuspended in complete medium.
Suspension cell culture of dissociated organ of Corti
To obtain suspension cultures, 104 cells were plated into a well of a 96-well dish previously coated with poly-HEME (Sigma-Aldrich, St Louis MO) to prevent cell attachment. Cultures were maintained in a defined medium composed of DMEM-F12 (1:1), supplemented with 1X B27, 1X N2, 1X glutamine, 1X insulin, transferrin and selenium (ITS, all from Invitrogen, Carlsbad CA), ampicillin at 0,3 μg/mL (Teuto Brazilian Laboratory, Brazil), 20 ng/mL human epidermal growth factor (EGF), and either 10 ng/mL basic fibroblast growth factor (bFGF) or 20 ng/mL transforming growth factor alpha (TGFα, Invitrogen), at 37°C and 5% CO2. Fifty percent of the culture medium was replaced every 48 hours.
Establishment of subcultures
The primary sphere cultures were maintained for seven days in vitro (DIV); while for first (P1) and second (P2) passages cells were cultured for five and three DIV, respectively. Passages were performed by adding Tryple (Invitrogen) to each well at a ratio of 1:1, at 37°C and 5% CO2,, for ten minutes, followed by mechanical dissociation with Pasteur pipettes. After spinning the cell suspension at 200 × g, 4°C, for four minutes, cells were resuspended with complete medium, counted, and plated at 104 cells per well.
For analysis of cell differentiation, otospheres were transferred into poly-L-ornithine (0.1 mg/mL) and fibronectin (5 ug/mL) treated eight-well culture slides (BD Falcon™) and allowed to attach for 24 hours in wells filled with defined medium without growth factors. After the cells were attached, we replaced eighty percent of the medium DMEM-F12 (1:1) and repeated this procedure every second or third day. Differentiated cells were analyzed after seven DIV by indirect immunofluorescence.
Indirect immunofluorescence and phenotypic sphere characterization
For sphere analyses and characterization by indirect immunofluorescence, P1 or P2 cells were transferred to coverslips within wells of a 24-well dish, previously coated with 30 μg/mL poly-D-lysine (Sigma) and 2 μg/mL laminin (Sigma). After plating, dishes were maintained for two hours, at 37°C and 5% CO2, and centrifuged at 200 × g, at 4°C, for two minutes. The remaining medium was removed and sphere attachment to the coverslips was monitored microscopically. Cells were fixed in 4% paraformaldehide in HBSS for one hour at 37°C, rinsed in HBSS, and permeabilized in 0,3% triton X-100 for 20 minutes at room temperature. Cells were blocked in 10% goat serum (Santa Cruz Biotechnologies, Santa Cruz CA) and incubated with primary antibodies diluted in 3% bovine serum albumin (BSA, Invitrogen) in HBSS, for one hour at room temperature. Primary antibody dilutions were 1:100 for monoclonal anti-nestin (Chemicon), 1:100 for monoclonal anti-sox2 (Chemicon) or 1:50 for polyclonal anti-sox2 (Santa Cruz), 1:50 for polyclonal anti-myosinVIIa (Affinity BioReagents, ABR), 1:50 for polyclonal anti-jagged1 (Santa Cruz), 1:50 for monoclonal anti-p27kip1 (Abcam), 1:50 for polyclonal anti-jagged2 (Santa Cruz). Cells were rinsed in HBSS and incubated with secondary antibodies, diluted in HBSS-BSA, for one hour at room temperature: Cy3-conjugated anti-mouse (1:1000, Invitrogen), Alexa Fluor 488-conjugated anti-mouse, anti-goat and anti-rabbit (1:400, Invitrogen), Alexa Fluor 546-conjugated anti-goat and anti-rabbit (1:400, Invitrogen). Samples were mounted in ProLong Gold Antifade reagent (Invitrogen) containing DAPI (4',6-diamidine-2-phenyl indol) for nuclear identification. Images were acquired by fluorescence microscopy (Axioplan, Carl Zeiss, Germany) using a software to collect digital images (Isis Fish Imaging Meta System), and confocal microscopy (LSM410 or LSM510, Carl Zeiss, Germany), as indicated.
Study groups and variables
Mouse and guinea pig organ of Corti suspension cultures were maintained overall for 15 DIV with EGF, and either bFGF or TGFα, for initial comparative analyses. Quantitative analysis was performed through direct counting the spheres from 20 consecutive microscope fields for each coverslip. For each growth factor treatment, bFGF or TGFα, two variables were examined: the number of spheres per coverslip and the number of cells in each sphere, each of them determined by confocal counting of DAPI-positive nuclei. These variables were compared between mouse and guinea pig cultures. We also observed the overall distribution of nestin and sox2.
The results were expressed as the mean ± standard deviation of the percentage of labeled cells in each growth factor treatment condition, EGF plus bFGF or EGF plus TGFα. The continuous variables were compared by Student's t-test. The level of statistical significance was set at p ≤ 0.05. Statistical analysis was performed using the GraphPad Instat program.
Comparison of otosphere size parameters between treatment groups and species
EGF + bFGF
EGF + TGFα
Number of spheres per coverslip
9 ± 1
23.3 ± 8.5
18.5 ± 11
11.5 ± 4.9
Number of cells in each sphere
16.3 ± 4.1
37.6 ± 23.5
32.6 ± 30.5
12.5 ± 5.8
When we analyzed the sphere number between organisms, we observed no difference in sphere number between mouse (18.5 ± 11) and guinea pig (11.5 ± 4.9) cultures (p = 0.458, Student's t-test). On the other hand, mouse cultures (32.6 ± 30.5) yielded a higher number of cells per spheres than guinea pig cultures (12.5 ± 5.8, p = 0.041, Student's t-test). We concluded therefore that TGFα in the presence of EGF increases the number of spheres in cultures of dissociated organ of Corti, when compared to bFGF. Our data also shows that at the neonatal period mouse cochlea yields more cells per sphere than the guinea pig ones.
Conditions for in vitro differentiation of otospheres into hair cells or supporting cells have been reported. We cultured P1/P2 otospheres under adherent conditions in medium composition favoring differentiation into hair and supporting cells. We demonstrate the presence of cells expressing markers for either supporting (p27kip1 and jagged1) or hair cells (myoVIIa and jagged2) from mouse otospheres (Figure 4). As no adherence could be obtained for guinea pig otosphere, we could not observe cell differentiation. This may be explained by the low number of cells observed for guinea pig otosphere comparatively to the mouse.
Progenitor cells have been shown to be present in vertebrate sensory epithelia, based on a number of evidences: (1) sphere formation was demonstrated from inner ear sensory epithelia of birds[23, 24], fish, neonatal rat cochlea, postnatal mouse cochlea and vestibular system[12, 13], and adult human and guinea pig spiral ganglion; (2) spheres were shown to be clonal and capable of self renewal[12, 13]; and (3) spheres were able to differentiate into cell types corresponding to all three germ layers, ectoderm, endoderm, and mesoderm, indicating that these are pluripotent stem cells. Cells in the spheres could differentiate into hair cells and neurons with inner ear cell properties[13, 29]. This raises the possibility that, if properly stimulated, they can be induced to differentiate in vivo as the basis for future therapies, including replacement of cells in the inner ear.
More recent data from mammals suggests that supporting cells or a subset of supporting cells can act as precursors for hair cells, and several studies suggest that supporting cells have stem cell characteristics. Those properties may vary among the different supporting cell types, which have distinct morphologies and gene expression profiles[14, 18, 30, 31]. Stem cell markers such as Sox2, Nestin, Musahi, Notch, Prox1, Islet1 were demonstrated to be expressed in postnatal supporting cells[32–37].
Nestin is an intermediate filament protein expressed by stem and progenitor cells early in development, and throughout the early postnatal period in the central and peripheral nervous systems, being considered a neural stem cell marker. It has been previously described in the organ of Corti of both developing and mature cochlea, suggesting the presence of immature precursor cells in the inner ear[14, 33, 38]. Nestin-positive cells expanded in culture from proliferating and floating spherical colonies have been shown to incorporate bromodesoyuridine into the DNA indicating their proliferation ability. In addition, they retain the ability to differentiate into cells displaying morphological features and expression of markers of hair cells and supporting cells[14, 39]. Sox2, a transcription factor, is another marker of the inner ear prosensory domain. In developing central and peripheral nervous systems, Sox2 expression is associated with progenitor and stem cell populations and with the sensory progenitors of the cochlea. Sox2 is widely expressed in the otocyst, but as the inner ear develops and proneural cells delaminate, its expression becomes restricted to prosensory domains. In experiments using fluorescent activated cell sorting (FACS) for isolation and purification of inner ear progenitor cells, from embryonic and postnatal cochlea, it was demonstrated that this specific population expresses cochlear sensory precursor markers as Sox2 and Nestin, and can differentiate in vitro into cells expressing markers of hair cells and supporting cells in vitro[18, 31].
Culturing organ of Corti progenitor cells under nonadherent conditions is challenging, because in vitro cell density and proliferation are low. Several growth factors may promote the proliferation of vestibular sensory epithelial cells after damage, including EGF, bFGF, TGFα, insulin-like growth factor 1 (IGF1), and others[41–43]. A nonadherent culture typical for mouse organ of Corti, established at postnatal day three, with approximately 104 cells at seeding, contains 4 ± 2.08 spheres after six DIV without further growth factor supplementation[21, 44]. According to Zine et al, after six DIV there were significantly more spheres formed, 41.25 ± 3.50 spheres, when the same amount of dissociated cells was maintained in EGF plus TGFα supplemented medium. After the sixth DIV 50% of sphere cells presented Abcg2 staining, an epithelial progenitor cell marker. The effects of these two growth factors on sphere formation are consistent with the results of our experiments, and with previous studies that have implicated the EGF and TGFα growth factor family in in vitro proliferation within sensory regions of mature utricles and organ of Corti explants[43–45]. Li et al observed that a combination of EGF plus IGF1 had a partially addictive effect resulting in a higher incidence of sphere formation, 68 ± 24 spheres per 105 plated cells, compared with single supplements, either EGF, bFGF or IGF1 alone, which provided 40 spheres per 105 plated cells. Kuntz and Oesterle showed through autoradiographic techniques after tritiated thymidine labeling that simultaneous infusion of TGFα and insulin directly into the inner ear of adult rats stimulated DNA synthesis in the vestibular sensory receptor epithelium, with the production of new supporting cells and putative hair cells; however the infusion of insulin alone or TGFα alone failed to stimulate significant DNA synthesis. Yamashita and Oesterle tested the effects of several growth factors on progenitor cell division in cultured mouse vestibular sensory epithelia and observed that cell proliferation was induced by TGFα in a dose-dependent manner, and by EGF when supplemented with insulin, but not by EGF alone. Zheng et al examined the possible influence of 30 growth factors on the proliferation of rat utricular epithelial cells in culture and found that IGF1, TGFα and EGF stimulated cell proliferation. Our experiments show that culture medium supplemented with TGFα has an additional effect on the number of forming spheres, 2.5 times higher when compared with bFGF group, in agreement with some observations of other authors. No significant difference was observed on cells numbers per sphere; however, there was a tendency toward higher values in the TGFα group. We were unable to demonstrate direct proliferative activity by BrdU labeling due to unspecific signals in immunofluorescence assays (data not shown). On the other hand, we registered during the culturing period the size expansion of otospheres from both organisms, which is suggestive of cell proliferation (Figure 3). In conclusion, our findings suggest that the combination of EGF and TGFα in the culture medium is a good alternative for otosphere production due to its higher rate of sphere formation.
Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Culture medium supplemented with EGF plus TGFα yielded a higher number of spheres than medium containing EGF plus bFGF for both animals. Compared to culturing of dissociated guinea pig organ of Corti, mouse cultures yielded a higher number of cells per sphere. This lower number of cells for guinea pig spheres may relate to its lack of differentiation in vitro, as opposed to the strong differentiation potential observed in vitro for mouse otospheres.
FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)
CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico)
We gratefully acknowledge financial support from CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasília, Brazil) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, São Paulo, Brazil), including their research centers RNTC (Rede Nacional de Terapia Celular), INCT (Instituto Nacional de Ciência e Tecnologia) and CEPID (Centros de Pesquisa, Inovação e Difusão).
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