Investigation of neural stem cell-derived donor contribution in the inner ear following blastocyst injection

BACKGROUND: Utilising the enormous proliferation and multi-lineage differentiation potentials of somatic stem cells represents a possible therapeutical strategy for diseases of non-regenerative tissues like the inner ear. In the current study, the possibility of murine neural stem cells to contribute to the developing inner ear following blastocyst injection was investigated. METHODS: Fetal brain-derived neural stem cells from the embryonic day 14 cortex of male mice were isolated and expanded for four weeks in neurobasal media supplemented with bFGF and EGF. Neural stem cells of male animals were harvested, injected into blastocysts and the blastocysts were transferred into pseudo-pregnant foster animals. Each blastocyst was injected with 5-15 microspheres growing from single cell suspension from neurospheres dissociated the day before. The resulting mice were investigated six months POST PARTUM for the presence of donor cells. Brainstem evoked response audiometry (BERA) was performed in six animals. To visualize donor cells Lac-Z staining was performed on sliced cochleas of two animals. In addition, the cochleas of four female animals were isolated and genomic DNA of the entire cochlea was analyzed for donor contribution by Y-chromosome-specific PCR. RESULTS: All animals had normal thresholds in brainstem evoked response audiometry. The male-specific PCR product indicating the presence of male donor cells were detected in the cochleas of three of the four female animals investigated. In two animals, male donor cells were detected unilateral, in one animal bilateral. CONCLUSION: The results suggest that descendants of neural stem cells are detectable in the inner ear after injection into blastocysts and possess the ability to integrate into the developing inner ear without obvious loss in hearing function.     

Stem cell therapy for the inner ear: recent advances and future directions

In vertebrates, perception of sound, motion, and balance is mediated through mechanosensory hair cells located within the inner ear. In mammals, hair cells are only generated during a short period of embryonic development. As a result, loss of hair cells as a consequence of injury, disease, or genetic mutation, leads to permanent sensory deficits. At present, cochlear implantation is the only option for profound hearing loss. However, outcomes are still variable and even the best implant cannot provide the acuity of a biological ear. The recent emergence of stem cell technology has the potential to open new approaches for hair cell regeneration. The goal of this review is to summarize the current state of inner ear stem cell research from a viewpoint of its clinical application for inner ear disorders to illustrate how complementary studies have the potential to promote and refine stem cell therapies for inner ear diseases. The review initially discusses our current understanding of the genetic pathways that regulate hair cell formation from inner ear progenitors during normal development. Subsequent sections discuss the possible use of endogenous inner ear stem cells to induce repair as well as the initial studies aimed at transplanting stem cells into the ear.     

Migration of R28 Retinal Precursor Cells into Cochlear and Vestibular Organs

Introduction Permanent cochlear and vestibular damage can becaused by a number of factors including: ototoxic drugs,acoustic trauma, genetic disorders, aging, anoxia, viralinfection, bacterial infection and more (Ding and Salvi,2005; Ding et al, 1999, Sal…     

The potential role of endogenous stem cells in regeneration of the inner ear

Stem cells in various mammalian organs retain the capacity to renew themselves and may be able to restore damaged tissue. Their existence has been proven by genetic tracer studies that demonstrate their differentiation into multiple tissue types and by their ability to self-renew through proliferation. Stem cells from the adult nervous system proliferate to form clonal floating colonies called spheres in vitro, and recent studies have demonstrated sphere formation by cells in the cochlea in addition to the vestibular system and the auditory ganglia, indicating that these tissues contain cells with stem cell properties. The presence of stem cells in the inner ear raises the hope of regeneration of mammalian inner ear cells but is difficult to correlate with the lack of spontaneous regeneration seen in the inner ear after tissue damage. Loss of stem cells postnatally in the cochlea may correlate with the loss of regenerative capacity and may limit our ability to stimulate regeneration. Retention of sphere forming ability in adult vestibular tissues suggests that the limited capacity for repair may be attributed to the continued presence of progenitor cells. Future strategies for regeneration must consider the distribution of endogenous stem cells in the inner ear and whether the tissue retains cells with the capacity for regeneration.     

Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway

CONCLUSION: Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. OBJECTIVE: To gain understanding of the gene pathways involved in the development of the normal cochlea. MATERIALS AND METHODS: Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. RESULTS: Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.     

Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo

Aminoglycoside uptake in the inner ear remains poorly understood. We subcutaneously injected a fluorescently-conjugated aminoglycoside, gentamicin-Texas Red (GTTR), to investigate the in vivo uptake of GTTR in the inner ear of several vertebrates, and in various murine sensory cells using confocal microscopy. In bullfrogs, GTTR uptake was prominent in mature hair cells, but not in immature hair cells. Avian hair cells accrued GTTR more rapidly at the base of the basilar papilla. GTTR was associated with the hair bundle; and, in guinea pigs and mice, somatic GTTR fluorescence was initially diffuse before punctate (endosomal) fluorescence could be observed. A baso-apical gradient of intracellular GTTR uptake in guinea pig cochleae could only be detected at early time points (<3h). In 21-28 day mice, cochlear GTTR uptake was greatly reduced compared to guinea pigs, 6-day-old mice, or mice treated with ethacrynic acid. In mice, GTTR was also rapidly taken up, and retained, in the kidney, dorsal root and trigeminal ganglia. In linguinal and vibrissal tissues rapid GTTR uptake cleared over a period of several days. The preferential uptake of GTTR by mature saccular, and proximal hair cells resembles the pattern of aminoglycoside-induced hair cell death in bullfrogs and chicks. Differences in the degree of GTTR uptake in hair cells of different species suggests variation in serum levels, clearance rates from serum, and/or the developmental and functional integrity of the blood-labyrinth barrier. GTTR uptake by hair cells in vivo suggests that GTTR has potential to elucidate aminoglycoside transport mechanisms into the inner ear, and as a bio-tracer for in vivo pharmacokinetic studies.     

Charakterisierung von Stammzellen des neonatalen auditorischen Sinnesepithels

Background

In contrast to regenerating hair cell-bearing organs of nonmammalian vertebrates the adult mammalian organ of Corti appears to have lost its ability to maintain stem cells. The result is a lack of regenerative ability and irreversible hearing loss following auditory hair cell death. Unexpectedly, the neonatal auditory sensory epithelium has recently been shown to harbor cells with stem cell features. The origin of these cells within the cochlea’s sensory epithelium is unknown.

Material And Methods

We applied a modified neurosphere assay to identify stem cells within distinct subregions of the neonatal mouse auditory sensory epithelium. Sphere cells were characterized by multiple markers and morphologic techniques.

Results

Our data reveal that both the greater and the lesser epithelial ridge contribute to the sphere-forming stem cell population derived from the auditory sensory epithelium. These self-renewing sphere cells express a variety of markers for neural and otic progenitor cells and mature inner ear cell types.

Conclusion

Stem cells can be isolated from specific regions of the auditory sensory epithelium. The distinct features of these cells imply a potential application in the development of a cell replacement therapy to regenerate the damaged sensory epithelium.     

哇巴因对小鼠内耳神经胶质细胞的影响

目的 研究哇巴因对小鼠内耳神经胶质细胞的影响,为干细胞移植治疗感音神经性聋的研究奠定基础.方法 成年雌性SPF级CBA/J小鼠60只,随机分为实验组和对照组,每组30只;实验组动物接受哇巴因(3 mM)经圆窗渗透给药,对照组给予等量生理盐水,于给药后7、14和30天用免疫组织荧光染色法观察位于耳蜗螺旋神经节内的内耳神经胶质细胞(inner ear glial cells,IEGs)的变化.结果 给药后7、14、30天实验组耳蜗各回螺旋神经节内可见内耳神经胶质细胞存活,但数量减少,排列紊乱;与对照组相比,哇巴因给药后7天实验组耳蜗各回神经胶质细胞的数量及密度即显著减少,给药后14天及30天后胶质细胞数量进一步减少;给药后30天各回螺旋神经节内内耳神经胶质细胞数降至最低,与实验组给药后7天、14天及与同时间点对照组比较显著减少,差异有统计学意义(P<0.05).结论 哇巴因经圆窗渗透给药可直接造成CBA/J小鼠内耳螺旋神经节内神经胶质细胞的急性进行性损伤.     

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection

CONCLUSIONS: In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. OBJECTIVE: To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. MATERIAL AND METHODS: This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. RESULTS: mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.     

Viral vector tropism for supporting cells in the developing murine cochlea

Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear.     

Developmentally-regulated coexpression of vimentin and cytokeratins in the rat inner ear.

In the present study the expression of vimentin-type intermediate filament proteins and cytokeratins was studied immunohistochemically in the rat inner ear from 12 days postconception up to 40 days after birth. With the use of a broad spectrum monoclonal antibody, cytokeratin expression was found to be present in the whole epithelial lining except for the sensory cells, throughout all the developmental stages examined. Vimentin was detected in the mesenchymal cells, the mesenchyme-derived tissues and the intermediate cells of the stria vascularis, confirming their origin from melanocyte precursor cells. In addition, the coexpression of vimentin and cytokeratins in the epithelial lining of the membranous inner ear was found to be developmentally regulated. During the final stages of differentiation, vimentin expression disappeared from the majority of the cell types. In the mature cochlea the coexpression of vimentin and cytokeratins was still found in the supporting cells of the organ of Corti, in the cells of Claudius and in external sulcus cells. As far as we could conclude from this study, the sensory cells showed only vimentin expression but not cytokeratin expression. A possible relationship between vimentin expression in adult epithelial cells of the inner ear and a specialised function of these cells is discussed.     

Transplantation of neural stem cells into explants of rat inner ear

Damage and loss of hair cells in the inner ear is the most frequent cause of hearing loss and balance disorders. Mammalian hair cells do not regenerate in the conventional ways. To regenerate the hair cell in the mammalian inner ear we transplanted neural stem cells into explants of rat inner ear. The stem cells integrated successfully into the sensory epithelium of the vestibular organs, but not into the organ of Corti. This method is useful to investigate efficient ways to transplant stem cells into the inner ear.     

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection

Conclusions. In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. Objective. To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. Material and methods. This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. Results. mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.     

Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway

Conclusion. Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. Objective. To gain understanding of the gene pathways involved in the development of the normal cochlea. Materials and methods. Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. Results. Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.     

Cisplatin: evaluation of its ototoxic potential

The ototoxic potential of cisplatin was analyzed in an organ culture model exposing the hair cells and other inner ear structures to cisplatin doses from 0.1 to 10 micrograms/ml. Selective hair cell degeneration was obvious at concentrations of 0.1 microgram/ml. Incubation with 1 microgram/ml caused morphologic damage in the supporting cells in both the cochlear and vestibular parts of the labyrinth. Exposure to 10 micrograms/ml during five days caused a total collapse of the membranous labyrinth. The morphologic degeneration pattern at the ultrastructural level is nonspecific, except that nuclear chromatin was either swollen and disintegrated or considerably condensed. Based on inner ear concentrations equivalent to aminoglycoside antibiotics in the range of 0.1 to 10 micrograms/ml, cisplatin is, in the in vitro model used in this study, the most ototoxic drug known. However, because of its single dose administration and long intervals between administration, clinical ototoxicity is less pronounced than that from aminoglycoside antibiotics.     

Feasibility of treating hearing disorders with stem cells: update

The recent isolation of adult stem cells from the mouse utricle that have the capacity to differentiate into cells from all three germ layers--and more importantly, into inner ear hair cells--offers a viable option for the treatment of hearing loss. The finding that embryonic stem cells are also capable of differentiating into hair cells further expands the possibility that we may someday develop restorative treatment of sensorineural hearing loss.     

Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.     

Inner ear pathology associated with Reye's syndrome

Severe pathological changes were observed in the inner ear tissues of a 2-month-old patient who died of Reye's syndrome after 5 days of hospitalization. In the organ of Corti, the inner hair cells appeared to be more severely damaged than the outer hair cells. Various degrees of degeneration were observed in all non-sensory epithelial cells lining the cochlear duct. In most turns of the cochlear duct, Reissner's membrane was ruptured and/or collapsed onto the organ of Corti. Likewise, both sensory and non-sensory cells of the vestibular end organs were markedly degenerated. These observations suggest that the inner ear tissues are acutely affected in patients with Reye's syndrome, and that the changes may cause impairment of hearing and/or equilibrium in patients who recover.     

Monoclonal antibodies to inner ear antigens: II. Antigens expressed in sensory cell stereocilia.

To develop biological reagents for investigating structure-function relationships in the organ of Corti, we have raised monoclonal antibodies, (MAb) to inner ear tissues. Our first series of antibodies prepared after intrasplenic immunization of mice with guinea pig tissues, identified antigens restricted to supporting cell structures, but no hair cell specific antibodies were developed [Zajic et al., Hear. Res. 52, 59-72, 1991]. In this report we describe the isolation, binding specificity and initial characterization of the stereocilia-binding monoclonal antibodies, KHRI-4, and KHRI-5. Mice were immunized with avian, amphibian and mammalian sensory hair cell-containing tissues and antibodies were screened for selective binding to cochlear extracts in ELISA. In the inner ear, KHRI-4 and KHRI-5 bind specifically to stereocilia in both avian and mammalian cochlear and vestibular tissue preparations using immunofluorescence and immunoperoxidase assays. In other tissues only certain cells of mesothelial origin, such as smooth muscle in gut and the arteriolar vasculature, were stained by KHRI-4 indicating that the antigenic structure defined by this antibody has limited distribution. KHRI-5 binding could be detected in other tissues only at high antibody concentrations suggesting that the gene product identified by this antibody is also weakly expressed in other cell lineages. Western blot analysis showed that KHRI-4 and -5 detect different protein complexes. KHRI-4 identifies an antigenic structure common to gut, cochlea, vestibular tissue and cultured fibroblasts consisting of a approximately 195 and a 230 kDa heterodimer designated p195/230. KHRI-5 binds to a prominent approximately 200-210 kDa band in Western blots of cochlear tissues, gut and fibroblasts. In immunoprecipitation experiments, KHRI-5 precipitated three proteins of Mr approximately 200-210, 230 and 260 kDa indicating that the approximately 200-210 kDa protein carrying the epitope for this antibody is a member of a heterotrimer complex. Our results show that these protein complexes are structural components of stereocilia and that the same proteins are arrayed in conjunction with the actin stress fibers of cultured mesothelial cells. Thus, they are likely to be important for maintaining the actin structure of stereocilia essential to transduction in sensory hair cells.     

豚鼠内耳磁微粒栓塞缺血模型初探

OBJECTIVE: To set up an inner ear ischemic model in guinea pig with ferromagnetic embolism. METHODS: A magnet was fitted in the external auditory canal and carbonyl iron filings (1%, 1 ml/kg) was injected into jugular, then the inner ear vessels were obstructed by ferromagnetic spheres. Cochlear blood flow (CBF) and number of red blood cells in the stria vessels were used to detect the model's ischemia of cochleae. The slice of temple bone and basal membrane stained by silver nitrate were used for inner ear's histopathological observations. RESULTS: The iron spheres were amassed in the one and two-day-later's model of inner ear vessels, which resulted in embolism. The number of red blood cells in the stria vessels decreased and then recovered to normal level after 4 days, but the CBF decreased to 50% +/- 10% of basic level immediately and recovered to 99% +/- 41% 4 days later. Scattered lesion of out hair cell cilium could be seen in cochleae in eight-day-later's model, and degenerations in different degree were found in vascular stria. CONCLUSION: The methods of inner ear ischemic model with ferromagnetic embolism could be practical and the decrease of CBF was reversible, so it may be an ideal model for studying some ischemic inner ear diseases and evaluating the effects of therapeutic drugs.