Ning Cong

Survival and fate of transplanted embryonic neural stem cells by Atoh1 gene transfer in guinea pigs cochlea

Embryonic neural stem cells (NSCs) were isolated from the neuroepithelium of the dorsal telencephalon of embryonic rats and infected by Ad5-Atoh1-enhanced green fluorescent protein. These NSCs were then delivered into neurosphere culture medium or transplanted into the endolymphatic space of the normal guinea pig cochlea through cochleostomy. Embryonic NSC phenotype of these isolated cells was determined by immunohistochemical detection of cell-specific protein markers. Survival, location and hair cell (HC) differentiation of the implanted NSCs were determined by the expression of the report gene, enhanced green fluorescent protein, and a specific marker for HCs, Myosin VIIa. These implanted cells can survive in the endolymphatic space of the cochlea. Some of the surviving cells differentiated into HCs by Atoh1 gene transfer.

Therapeutic value of nerve growth factor in promoting neural stem cell survival and differentiation and protecting against neuronal hearing loss

Nerve growth factor (NGF) is a neurotrophic factor that modulates survival and differentiation of neural stem cells (NSCs). We investigated the function of NGF in promoting growth and neuronal differentiation of NSCs isolated from mouse cochlear tissue, as well as its protective properties against gentamicin (GMC) ototoxicity. NSCs were isolated from the cochlea of mice and cultured in vitro. Effect of NGF on survival, neurosphere formation, and differentiation of the NSCs, as well as neurite outgrowth and neural excitability in the subsequent in vitro neuronal network, was examined. Mechanotransduction capacity of intact cochlea and auditory brainstem response (ABR) threshold in mice were also measured following GMC treatment to evaluate protection using NGF against GMC-induced neuronal hearing loss. NGF improved survival, neurosphere formation, and neuronal differentiation of mouse cochlear NSCs in vitro, as well as promoted neurite outgrowth and neural excitability in the NSC-differentiated neuronal culture. In addition, NGF protected mechanotransduction capacity and restored ABR threshold in gentamicin ototoxicity mouse model. Our study supports a potential therapeutic value of NGF in promoting proliferation and differentiation of NSCs into functional neurons in vitro, supporting its protective role in the treatment of neuronal hearing loss.

Role of the planar cell polarity pathway in regulating ectopic hair cell-like cells induced by Math1 and testosterone treatment.

Planar cell polarity (PCP) signaling regulates cochlear extension and coordinates orientation of sensory hair cells in the inner ear. Retroviral-mediated introduction of the Math1 transcription factor leads to the transdifferentiation of some mature supporting cells into hair cells. Testosterone, a gonadal sex steroid hormone, is associated with neuroprotection and regeneration in Central Nervous System (CNS) development. Experiments were performed in vitro using Ad5-EGFP-Math1/Ad5-Math1 in neonatal mouse cochleas. Establishment of ectopic hair-cell like cell(HCLC) polarity in the lesser epithelial ridge (LER) with or without testosterone-3-(O-carboxymethyl) oxime bovine serum albumin (testosterone-BSA) treatment was investigated to determine the role of the PCP pathway in regulating ectopic regenerated (HCLCs) through induction by Math1 and testosterone treatment. After Math1 infection, new ectopic regenerated HCLCs were detected in the LER. After the HCLCs developed actin-rich stereocilia, the basal bodies moved from the center to the distal side. Moreover, the narrower, non-sensory LER region meant that the convergent extension (CE) was also established after transfection with Math1. After 9 days of in vitro testosterone-BSA treatment, more Edu(+), Sox2(+), and HCLC cells were observed in the LER with an accompanying downregulation of E-cadherin. Interestingly, the CE of the Ad5-EGFP-math1 treated LER is altered, but the intrinsic cellular polarity of the HCLCs is not obviously changed. In summary, our results indicate that PCP signaling is involved in the development of ectopic HCLCs and the CE of the ectopic sensory region is altered by testosterone-BSA through downregulation of cell-cell adhesion. Testosterone-BSA and Math1 treatment could promote an increase in HCLCs in the LER through proliferation and transdifferentiation.

Expression of Numb and Numb-like in the development of mammalian auditory sensory epithelium.

The Numb and Numb-like are evolutionarily conserved cell fate-determining factors in mammals. For the first time, we investigate the involvement of the Numb and Numb-like in the developing auditory sensory epithelium. We show that both of them are expressed in the rat auditory sensory epithelium, and the four isoforms of the Numb have dynamic expression patterns during cochlear development. At the early stage of the auditory epithelium development, they occur in all progenitor cells. At the late stage of cell differentiation, they are expressed mainly in the cytoplasm of apical cells and their locations are different. Furthermore, we find overexpression of the Numb or Numb-like, in cochlear whole mount cultures, can upregulate mRNA level of Rath1, which is important in the hair-cell development.

Three-Dimensional Reconstruction of C57BL/6 Mouse Inner Ear during Development

Purpose: To explore the three-dimensional (3-D) morphological characteristics of the complicated inner ear development of the C57BL/6 mouse. Methods: Specimens of C57BL/6 mouse embryos on days E7.5, E8.5, E9.5, E10.5, E11.5, E12.5 and E14.5 were collected and sectioned serially in this experiment. After hematoxylin and eosin staining, parts of the inner ear were pictured under the microscope with specific positional control. Using the protocol of 3D-DOCTOR software, we outlined the inner ear margin including the inner and extra face in different colors and put them into the 3-D program to reconstruct the 3-D model. Results: 3-D models of the E9.5, E10.5, E11.5, E12.5 and E14.5 inner ear were obtained and proved to be fine. Different parts of the inner ear were shown clearly in different colors in the 3-D model and significant morphological changes of the inner ear were shown during development between E9.5 and E14.5. Conclusion: The new technology of 3-D reconstruction is a useful and important tool to directly observe the complex development of the inner ear, and the development between E9.5 and E14.5 has been proved by the 3-D model to be the most complex and important period of the development of the C57BL/6 mouse inner ear.

Celastrol enhances Atoh1 expression in inner ear stem cells and promotes their differentiation into functional auditory neuronal-like cells

ABSTRACT We aimed to investigate the beneficial effect of Celastrol on inner ear stem cells and potential therapeutic value for hearing loss. The inner ear stem cells were isolated and characterized from utricular sensory epithelium of adult mice. The stemness was evaluated by sphere formation assay. The relative expressions of Atoh1, MAP-2 and Myosin VI were measured by RT-PCR and immunoblotting. The up-regulation of MAP-2 was also analysed with immunofluorescence. The in vitro neuronal excitability was interrogated by calcium oscillation. The electrophysiological property was determined by inward current recorded on patch clamp. Our results demonstrated that Celastrol treatment significantly improved the viability and proliferation of mouse inner ear stem cells, and facilitated sphere formation. Moreover, Celastrol stimulated differentiation of mouse inner ear stem cells to neuronal-like cells and enhanced neural excitability. Celastrol also enhanced neuronal-like cell identity in the inner ear stem cell derived neurons, as well as their electrophysiological function. Most notably, these effects were apparently associated with the upregulation of Atoh1 in response to Celastrol treatment. Celastrol showed beneficial effect on inner ear stem cells and held therapeutic promise against hearing loss.

Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds: supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein (EGFP) was used to track cell division during hair cell transformation. 5-Bromo-2′-deoxyuridine (BrdU) was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Junctional E-cadherin/p120-catenin Is Correlated with the Absence of Supporting Cells to Hair Cells Conversion in Postnatal Mice Cochleae

Notch inhibition is known to generate supernumerary hair cells (HCs) at the expense of supporting cells (SCs) in the mammalian inner ear. However, inhibition of Notch activity becomes progressively less effective at inducing SC-to-HC conversion in the postnatal cochlea and balance organs as the animal ages. It has been suggested that the SC-to-HC conversion capacity is inversely correlated with E-cadherin accumulation in postnatal mammalian utricles. However, whether E-cadherin localization is linked to the SC-to-HC conversion capacity in the mammalian inner ear is poorly understood. In the present study, we treated cochleae from postnatal day 0 (P0) with the Notch signaling inhibitor DAPT and observed apparent SC-to-HC conversion along with E-cadherin/p120ctn disruption in the sensory region. In addition, the SC-to-HC conversion capacity and E-cadherin/p120ctn disorganization were robust in the apex but decreased toward the base. We further demonstrated that the ability to regenerate HCs and the disruption of E-cadherin/p120ctn concomitantly decreased with age and ceased at P7, even after extended DAPT treatments. This timing is consistent with E-cadherin/p120ctn accumulation in the postnatal cochleae. These results suggest that the decreasing capacity of SCs to transdifferentiate into HCs correlates with E-cadherin/p120ctn localization in the postnatal cochleae, which might account for the absence of SC-to-HC conversion in the mammalian cochlea.

The Role of the Notch Signal Pathway in Mucosal Cell Metaplasia in Mouse Acute Otitis Media

Otitis media (OM) is a major cause of morbidity in pediatric and adult patients. This inflammatory condition is characterized by mucous cell hyperplasia that is thought to produce mucins from the middle ear mucosa. We are interested in the role of Notch signalling pathway in this inflammatory process. Using an acute otitis media (AOM) mouse model through injection of Streptococcus Pneumoniae into the middle ear, histopathologic examination and quantitative RT-PCR, acute inflammation with the thickness of mucosa, Goblet cell hyperplasia, and cilia loss were determined and gene expression related to the Notch signaling pathway were evaluated. Upregulation of the mucous cell markers, Argr2 and Muc5AC, and downregulation of the cilia cell marker, Foxj1 and Dnai2, were observed in AOM. In addition, genes encoding Notch receptors and ligands (Notch1, Notch2, Notch3, Notch4 and Dll1) and the Notch target genes (Hes1, Hes5, Hey1, NRARP) in AOM decreased significantly. The expression of the Notch1 and Jagged1 also showed down-regulation throughout the mouse middle ear epithelium. Taken together, this study suggests that downregulation of the Notch signaling pathway is involved in the mucosa hyperplasia during AOM.

Contents Vol. 71, 2009

R.L. Alford, Houston, Tex. M. Anniko, Uppsala D.D. Backous, Seattle, Wash. Y.A. Bayazit, Ankara H.H. Birdsall, Houston, Tex. P.J. Bradley, Nottingham J. Califano, Baltimore, Md. P.F. Castellanos, Bimingham, Ala. C. Cernea, São Paulo F.-L. Chi, Shanghai A. Chiu, Philadelphia, Pa. N. Cohen, Philadelphia, Pa. M.D. Eisen, Hartford, Conn. E. Ferekidis, Athens A. Ferlito, Udine R.L. Ferris, Pittsburgh, Pa. L.L. Gleich, Cincinnati, Ohio D.-M. Han, Beijing J.P. Harris, San Diego, Calif. R. Häusler, Bern I. Hochmair, Innsbruck K. Hörmann, Mannheim W. Hosemann, Greifswald K.-B. Hüttenbrink, Köln S. Iurato, Bari A. Kakigi, Tokyo B.N. Landis, Geneva T. Linder, Luzern W.J. Mann, Mainz J.J. Manni, Maastricht J.B. Nadol, Jr., Boston, Mass. J.N. Palmer, Philadelphia, Pa. G.J. Petruzzelli, Chicago, Ill. R. Probst, Zürich A. Rinaldo, Udine R.J. Ruben, Bronx, N.Y. I. Salahuddin, Karachi A. Schrott-Fischer, Innsbruck A. Shiotani, Saitama G.A. Tavartkiladze, Moscow T.N. Teknos, Columbus, Ohio K. Tomoda, Osaka R.P. Tufano, Baltimore, Md. R.T. Younis, Miami, Fla. P. Zbären, Bern Journal for Oto-Rhino-Laryngology, Head and Neck Sugery