Regulation of cell fate in the sensory epithelia of the inner ear

The sensory epithelia of the inner ear contain mechanosensory hair cells and non-sensory supporting cells. Both classes of cell are heterogeneous, with phenotypes varying both between and within epithelia. The specification of individual cells as distinct types of hair cell or supporting cell is regulated through intra- and extracellular signalling pathways that have been poorly understood. However, new methodologies have resulted in significant steps forward in our understanding of the molecular pathways that direct cells towards these cell fates.     

In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme

The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.     

Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.     

The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination

During embryonic development of the inner ear, the sensory primordium that gives rise to the organ of Corti from within the cochlear epithelium is patterned into a stereotyped array of inner and outer sensory hair cells separated from each other by non-sensory supporting cells. Math1, a close homolog of the Drosophila proneural gene atonal, has been found to be both necessary and sufficient for the production of hair cells in the mouse inner ear. Our results indicate that Math1 is not required to establish the postmitotic sensory primordium from which the cells of the organ of Corti arise, but instead is limited to a role in the selection and/or differentiation of sensory hair cells from within the established primordium. This is based on the observation that Math1 is only expressed after the appearance of a zone of non-proliferating cells that delineates the sensory primordium within the cochlear anlage. The expression of Math1 is limited to a subpopulation of cells within the sensory primordium that appear to differentiate exclusively into hair cells as the sensory epithelium matures and elongates through a process that probably involves radial intercalation of cells. Furthermore, mutation of Math1 does not affect the establishment of this postmitotic sensory primordium, even though the subsequent generation of hair cells is blocked in these mutants. Finally, in Math1 mutant embryos, a subpopulation of the cells within the sensory epithelium undergo apoptosis in a temporal gradient similar to the basal-to-apical gradient of hair cell differentiation that occurs in the cochlea of wild-type animals.     

Hair cells without supporting cells: further studies in the ear of the zebrafish mind bomb mutant

Each sensory hair cell in the ear is normally surrounded by supporting cells, which separate it from the next hair cell. In the mind bomb mutant, as a result of a failure of lateral inhibition, cells that would normally become supporting cells differentiate as hair cells instead, creating sensory patches that consist of hair cells only. This provides a unique opportunity to pinpoint the functions for which supporting cells are required in normal hair cell development. We find that hair cells in the mutant develop an essentially normal cytoskeleton, with a correctly structured hair bundle and well-defined planar polarity, and form apical junctional complexes with one another in standard epithelial fashion. They fail, however, to form a basal lamina or to adhere properly to the adjacent non-sensory epithelial cells, which overgrow them. The hair cells are eventually expelled from the ear epithelium into the underlying mesenchyme, losing their hair bundles in the process. It is not clear whether they undergo apoptosis: many cells staining strongly with the TUNEL procedure are seen but do not appear apoptotic by other criteria. Supporting cells, therefore, are needed to hold hair cells in the otic epithelium and, perhaps, to keep them alive, but are not needed for the construction of normal hair bundles or to give the hair bundles a predictable polarity. Moreover, supporting cells are not absolutely required as a source of materials for otoliths, which, though small and deformed, still develop in their absence.     

Human Sensory Neurons Derived from Induced Pluripotent Stem Cells Support Varicella-Zoster Virus Infection

After primary infection, varicella-zoster virus (VZV) establishes latency in neurons of the dorsal root and trigeminal ganglia. Many questions concerning the mechanism of VZV pathogenesis remain unanswered, due in part to the strict host tropism and inconsistent availability of human tissue obtained from autopsies and abortions. The recent development of induced pluripotent stem (iPS) cells provides great potential for the study of many diseases. We previously generated human iPS cells from skin fibroblasts by introducing four reprogramming genes with non-integrating adenovirus. In this study, we developed a novel protocol to generate sensory neurons from iPS cells. Human iPS cells were exposed to small molecule inhibitors for 10 days, which efficiently converted pluripotent cells into neural progenitor cells (NPCs). The NPCs were then exposed for two weeks to growth factors required for their conversion to sensory neurons. The iPS cell-derived sensory neurons were characterized by immunocytochemistry, flow cytometry, RT-qPCR, and electrophysiology. After differentiation, approximately 80% of the total cell population expressed the neuron-specific protein, βIII-tubulin. Importantly, 15% of the total cell population co-expressed the markers Brn3a and peripherin, indicating that these cells are sensory neurons. These sensory neurons could be infected by both VZV and herpes simplex virus (HSV), a related alphaherpesvirus. Since limited neuronal populations are capable of supporting the entire VZV and HSV life cycles, our iPS-derived sensory neuron model may prove useful for studying alphaherpesvirus latency and reactivation.     

Cochlear stem/progenitor cells from a postnatal cochlea respond to Jagged1 and demonstrate that notch signaling promotes sphere formation and sensory potential

Hair cells and supporting cells of the mammalian cochlea terminally differentiate during development. Recent in vitro evidence suggests the presence of hair cell progenitors in the postnatal cochlea. Phenotypic properties of these cells and factors that promote their ability to generate spheres in aggregate cultures have not been reported. We define an in vitro system that allows stem/progenitor cells harvested from the early postnatal cochlea to develop into spheres. These spheres contain Abcg2, Jagged1 and Notch1 positive progenitor cells that can divide and generate new hair cell-like cells, i.e. immunopositive for specific hair cell markers, including Myosin VI, Myosin VIIa, Math1 and ability to uptake FM1-43.We demonstrate that reducing Notch signaling with a gamma secretase inhibitor decreases the number of spheres generated following treatment of the stem/progenitor cell cultures. Additionally, activation of Notch by an exogenous soluble form of a Notch ligand, i.e. Jagged1 protein, promotes sphere formation and the sensory potential of cochlear stem/progenitor cells. Our findings suggest that Notch1/Jagged1 signaling plays a role in maintaining a population of Abcg2 sensory stem/progenitor cells in the postnatal cochlea.     

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea

Correct patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses. We also explored the cytoskeletal organization of developing sensory and non-sensory cells, and used pharmacological modulation of cytoskeletal elements to show that the developmental increase of hair cell stiffness is a direct result of actin filaments, whereas the development of supporting cell surface mechanical properties depends on the extent of microtubule acetylation. Finally, this study found that the fibroblast growth factor signaling pathway is necessary for the developmental time course of cell surface mechanical properties, in part owing to the effects on microtubule structure.     

A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea

Recent studies have demonstrated that the Notch signaling pathway regulates the differentiation of sensory hair cells in the vertebrate inner ear [1] [2] [3] [4] [5] [6] [7] [8] [9]. We have shown previously that in mice homozygous for a targeted null mutation of the Jagged2 (Jag2) gene, which encodes a Notch ligand, supernumerary hair cells differentiate in the cochlea of the inner ear [7]. Other components of the Notch pathway, including the Lunatic fringe (Lfng) gene, are also expressed during differentiation of the inner ear in mice [6] [7] [8] [9] [10]. In contrast to the Jag2 gene, which is expressed in hair cells, the Lfng gene is expressed in non-sensory supporting cells in the mouse cochlea [10]. Here we demonstrate that a mutation in the Lfng gene partially suppresses the effects of the Jag2 mutation on hair cell development. In mice homozygous for targeted mutations of both Jag2 and Lfng, the generation of supernumerary hair cells in the inner hair cell row is suppressed, while supernumerary hair cells in the outer hair cell rows are unaffected. We also demonstrate that supernumerary hair cells are generated in mice heterozygous for a Notch1 mutation. We suggest a model for the action of the Notch signaling pathway in regulating hair cell differentiation in the cochlear sensory epithelium.     

A Novel Minimally-Invasive Method to Sample Human Endothelial Cells for Molecular Profiling

ObjectiveThe endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.MethodsNine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34 / CD105 / CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b / CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR).ResultsA median of 4,212 (IQR: 2161 – 6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001).ConclusionThis state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.     

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133? Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133⁺ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133⁺ and CD133⁻ colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10⁺ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133⁺ and CD133⁻ subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133⁺ cells showed significantly greater tumor growth than CD133⁻ cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133⁺ cells exhibited significantly more invasive behaviors than CD133⁻ cells (P<0.001), especially in cocultures with CD10⁺ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10⁺ fibroblasts enhanced the tumor growth of CD133⁺ cells significantly more than CD10⁻ fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133⁺ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10⁺ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.     

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44⁺/CD24^low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44⁺/CD24^low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.     

<Emphasis Type="Italic">Lmx1a</Emphasis> is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a ^dr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a “cochlear-gravistatic” endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants. This work was supported by grants from the NCRR/COBRE (P20 RR 018788; D.H.N.) and NIH (RO1 DC 005590; B.F.). Parts of this investigation were conducted in a facility constructed with support of a Research Facilities Improvement Program Grant from the National Center for Research Resources, National Institutes of Health. We acknowledge the use of the confocal microscope facility of the NCCB, supported by EPSCoR EPS-0346476 (CFD 47.076), and of the University of Nebraska microarray facility, supported by NCRR/COBRE.     

Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells

Purpose

To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear.

Methods

An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre.

Results

Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype.

Conclusions

Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.     

Coenzyme Q10 Protects Hair Cells against Aminoglycoside

It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10) is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group). In the neomycin group, utricles were cultured with neomycin (1 mM) to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30–0.3 µM). Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear.