Cochlear Stem Cells Research Articles

GelMA/PEDOT:PSS Composite Conductive Hydrogel-Based Generation and Protection of Cochlear Hair Cells through Multiple Signaling Pathways.

Recent advances in cochlear implantology are exemplified by novel functional strategies such as bimodal electroacoustic stimulation, in which the patient has intact low-frequency hearing and profound high-frequency hearing pre-operatively. Therefore, the synergistic restoration of dysfunctional cochlear hair cells and the protection of hair cells from ototoxic insults have become a persistent target pursued for this hybrid system. In this study, we developed a composite GelMA/PEDOT:PSS conductive hydrogel that is suitable as a coating for the cochlear implant electrode for the potential local delivery of otoregenerative and otoprotective drugs. Various material characterization methods (e.g., 1H NMR spectroscopy, FT-IR, EIS, and SEM), experimental models (e.g., murine cochlear organoid and aminoglycoside-induced ototoxic HEI-OC1 cellular model), and biological analyses (e.g., confocal laser scanning microscopy, real time qPCR, flow cytometry, and bioinformatic sequencing) were used. The results demonstrated decent material properties of the hydrogel, such as mechanical (e.g., high tensile stress and Young's modulus), electrochemical (e.g., low impedance and high conductivity), biocompatibility (e.g., satisfactory cochlear cell interaction and free of systemic toxicity), and biosafety (e.g., minimal hemolysis and cell death) features. In addition, the CDR medicinal cocktail sustainably released by the hydrogel not only promoted the expansion of the cochlear stem cells but also boosted the trans-differentiation from cochlear supporting cells into hair cells. Furthermore, hydrogel-based drug delivery protected the hair cells from oxidative stress and various forms of programmed cell death (e.g., apoptosis and ferroptosis). Finally, using large-scale sequencing, we enriched a complex network of signaling pathways that are potentially downstream to various metabolic processes and abundant metabolites. In conclusion, we present a conductive hydrogel-based local delivery of bifunctional drug cocktails, thereby serving as a potential solution to intracochlear therapy of bimodal auditory rehabilitation and diseases beyond.

Viral-mediated expression of c-Myc and cyclin A2 induces cochlear progenitor cell proliferation

Cochlear progenitor cells have a limited proliferative capability, which prevents their application in treating sensorineural hearing loss. In this study, we showed that the expression of c-Myc and cyclin A2 was down-regulated during the development of cochlear tissue and CPC differentiation. Over-expression of these two genes using adenovirus transduction, significantly affected the CPC cell cycle and promoted the CPC proliferation. We further demonstrated that this promotion involves the classic CKI-cyclin-CDK pathway. Our study suggests that genetically modified CPCs may be a promising cell source for cochlear stem cell transplantation that improves the efficacy of cell therapy.

Comparison of sphere-forming capabilities of the cochlear stem cells derived from apical, middle and basal turns of murine organ of Corti

The presence of stem cells in the organ of Corti raises the hope of regeneration of mammalian inner ear cells. However, little is known about the distribution of endogenous stem cells in the inner ear as well as their sphere-forming abilities. The aim of this study is to evaluate the stem cells derived from different turns of the organ of Corti and analyze the sphere-forming capabilities of these stem cells. We dissected and isolated cochleae from postnatal day 1, 7 and 14 mice to separate organ of Corti into apical, middle and basal turns for cell isolation. Our results show that different turns of the organ of Corti harbor distinctly different populations of stem cells. Apical turn-derived cells give rise to more sphere-forming cells than middle and basal turn-derived cells, and middle turn-derived cells give rise to sphere cells number significantly higher than basal turn. Our findings indicate that apical turn of young age murine organ of Corti is best suited for isolation of endogenous stem cells for regeneration of hearing loss.

Comparing the cultivated cochlear cells derived from neonatal and adult mouse

BackgroundPrevious reports showed the presence of limited numbers of stem cells in neonatal murine cochlear sensory epithelia and these cells are progressively lost during the postnatal development. The goal of this study was to investigate whether stem cells can be derived from mature mouse cochleae under suspension culture conditions, and to analyze the expression of the stem cell and inner ear progenitor cell markers in cells dissociated from neonatal and adult mouse organs of Corti.MethodsOrgans of Corti were dissected from postnatal day 1 (P1) or postnatal day 60 (P60) mouse. The dissociated cells were cultivated under suspension cultures conditions. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were conducted for phenotype characterization.ResultsThe number of cochlear stem cells (otospheres) yielded from P1 organ of Corti was significantly higher than that of the P60 organ of Corti. RT-PCR analyses showed that the stem markers, such as nanog, sox2, klf4, and nestin can be found to be distributed similarly in the cells derived from both of organisms, but the inner ear developmental/progenitor cell markers showed lower expression in P60 organ of Corti compared to P1. Immunocytochemistry results also revealed the evidence that P60 otospheres lacking of differentiation potential in vitro, which opposed to the strong differentiation potential of otospheres at P1 stage.ConclusionsOur findings suggest that the loss of numbers and features of stem cells in the adult organ of Corti is associated with the substantial down-regulation of inner ear progenitor key-markers during maturation of the cells in organ of Corti.

Loss of osteoprotegerin expression in the inner ear causes degeneration of the cochlear nerve and sensorineural hearing loss

Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations and variations in the OPG gene cause many human diseases that are characterized by not only skeletal abnormalities but also poorly understood hearing loss: Paget's disease, osteoporosis, and celiac disease. To gain insight into the mechanisms of hearing loss in OPG deficiency, we studied OPG knockout (Opg−/−) mice. We show that they develop sensorineural hearing loss, in addition to conductive hearing loss due to abnormal middle-ear bones. OPG deficiency caused demyelination and degeneration of the cochlear nerve in vivo. It also activated ERK, sensitized spiral ganglion cells (SGC) to apoptosis, and inhibited proliferation and survival of cochlear stem cells in vitro, which could be rescued by treatment with exogenous OPG, an ERK inhibitor, or bisphosphonate. Our results demonstrate a novel role for OPG in the regulation of SGC survival, and suggest a mechanism for sensorineural hearing loss in OPG deficiency.

Nestin-expressing cells in the developing, mature and noise-exposed cochlear epithelium

The auditory sensory epithelium in non-mammalian vertebrates can replace lost hair cells by transdifferentiation of supporting cells, but this regenerative ability is lost in the mammalian cochlea. Future cell-based treatment of hearing loss may depend on stem cell transplantation or on transdifferentiation of endogenous cells in the cochlea. For both approaches, identification of cells with stem cell features within the mature cochlea may be useful. Here we use a Nestin-β-gal mouse to examine the presence of Nestin positive cells in the mature auditory epithelium, and determine how overstimulation of the ear impacts these cells. Nestin positive cells were found in the apical turn of the cochlea lateral to the outer hair cell area. This pattern of expression persisted into mature age. The area of Nestin positive cells was increased after the noise lesion. This increase in area coincided with an increase in expression of the Nestin mRNA. The data suggest that cells with potential stem cell features remain in the mature mammalian cochlea, restricted to the apical turn, and that an additional set of signals is necessary to trigger their contribution to cell replacement therapy in the ear. As such, this population of cells could serve to generate cochlear stem cells for research and potential therapy, and may be a target for treatments based on induced transdifferentiation of endogenous cochlear cells.

The Past and Present of the Research on Cochlear Stem Cell

The Past and Present of the Research on Cochlear Stem Cell

Hypoxia enhances the stemness markers of cochlear stem/progenitor cells and expands sphere formation through activation of hypoxia-inducible factor-1alpha

Unlike neural stem cells that maintain populations in the adult brains of both rodents and humans, cochlear stem cells appear to diminish in number after birth and may become quiescent in adult mammalian cochleae. Hypoxia has been observed to promote an undifferentiated cell state in various stem cell populations; however, little is known about such an effect on cochlear stem/progenitor cells (SPCs). The aims of this study were to assess the effect of hypoxia on cochlear SPCs and to examine the impact of hypoxia-inducible factor-1alpha ( Hif-1a) on regulating such an effect. Our data demonstrate that hypoxic culturing for 24 h significantly increased sphere formation and viability of cochlear SPCs compared with those cultured under normoxic conditions. Concurrent with these proliferation promotion effects are changes in the expression of multiple stemness and cell-cycle quiescent associated gene targets, including Abcg2, nestin, p27 Kip1 and Vegf. Knockdown of Hif-1a expression by small-interfering RNA inhibited hypoxia-induced cochlear SPC expansion and resulted in downregulation of Vegf, Abcg2, and nestin and upregulation of p27 Kip1 gene expression. These results suggest that Hif-1a plays an important role in the stimulation of the proliferation of cochlear SPCs, which confers a great benefit of expanding cochlear SPCs via hypoxic conditions.

In vitro long-term development of cultured inner ear stem cells of newborn rat

The adult mammalian auditory receptor lacks any ability to repair and/or regenerate after injury. However, the late developing cochlea still contains some stem-cell-like elements that might be used to regenerate damaged neurons and/or cells of the organ of Corti. Before their use in any application, stem cell numbers need to be amplified because they are usually rare in late developing and adult tissues. The numerous re-explant cultures required for the progressive amplification process can result in a spontaneous differentiation process. This aspect has been implicated in the tumorigenicity of stem cells when transplanted into a tissue. The aim of this study has been to determine whether cochlear stem cells can proliferate and differentiate spontaneously in long-term cultures without the addition of any factor that might influence these processes. Cochlear stem cells, which express nestin protein, were cultured in monolayers and fed with DMEM containing 5% FBS. They quickly organized themselves into typical spheres exhibiting a high proliferation rate, self-renewal property, and differentiation ability. Secondary cultures of these stem cell spheres spontaneously differentiated into neuroectodermal-like cells. The expression of nestin, glial-fibrillary-acidic protein, vimentin, and neurofilaments was evaluated to identify early differentiation. Nestin expression appeared in primary and secondary cultures. Other markers were also identified in differentiating cells. Further research might demonstrate the spontaneous differentiation of cochlear stem cells and their teratogenic probability when they are used for transplantation.

Stem cells and cell lines from the human auditory organ: applications, hurdles and bottlenecks in the development of regenerative therapies for deafness

The development of any stem-cell-based therapy (and a potential one for deafness is no exception) relies on the generation of the necessary tools: 'cell drugs' that can be safely manufactured for their clinical application. An increasing body of work has focussed on the identification, in animal models, of potential stem cell sources that could have an application for regenerative therapy in the auditory organ. A still more circumscribed effort--owing to ethical and technical difficulties--aims to obtain the actual potential therapeutic candidates (i.e. stem cells of human origin). A recently isolated population of human fetal auditory stem cells could become an ideal model for some of the challenges lying ahead regarding cochlear stem cell purification, expansion and maintenance.

Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiation

Cyclin A2 plays a major role in cell cycle modulation. Cyclin A2 level has not been determined in the inner ear yet. RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry were used to measure cyclin A2 expression in rat cochlea tissues of different ages, isolated cochlear stem cells and stem cell-derived differentiated cells. The results indicated that cyclin A2 level in cochlea tissues decreased gradually from newborn to adult. Furthermore, cyclin A2 level fell down after differentiation of cochlear stem cells. It was suggested that cyclin A2 might be involved in the modulation of rat cochlea development and cochlear stem cell differentiation.

Identification and Characterization of Mouse Cochlear Stem Cells

Genetic, noise- and drug-induced loss of hair cells in the mouse and human cochlea leads to permanent hearing loss due to lack of regeneration of hair cells, which may be due to reduced numbers or loss of the regenerative ability of stem cells in the adult cochlea. We hypothesized that the mouse neonate cochlea harbors stem cells capable of differentiating into hair cells. Cells from the primary neonate cochlear culture began to proliferate and formed floating spheres after 14 days in vitro (DIV). By comparison, spheres from the primary culture of the cortex were observed after 7 DIV. Cochlear sphere cells could be passaged and the new spheres were observed after 7 DIV. Cochlear sphere cells were capable of differentiating into astrocytes and oligodendrocytes, but not neurons under the conditions tested. Cochlear sphere cells expressed Sox2 and Myo7a, but failed to show markers that are expressed exclusively in mature cochlear tissue, while cells from cortex spheres expressed Sox2 and Otx2, but not Myo7a. Our results show that cochleae from neonatal mice harbor cells capable of forming spheres and cells from these spheres appear to be better endowed to become hair cells.