Stefan Heller

Stanford Medicine, Stanford, California, United States

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Publications (85)557.04 Total impact

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    ABSTRACT: The organ of Corti harbors highly specialized sensory hair cells and surrounding supporting cells that are essential for the sense of hearing. Here, we report a single cell gene expression data analysis and visualization strategy that allows for the construction of a quantitative spatial map of the neonatal organ of Corti along its major anatomical axes. The map displays gene expression levels of 192 genes for all organ of Corti cell types ordered along the apex-to-base axis of the cochlea. Statistical interrogation of cell-type-specific gene expression patterns along the longitudinal gradient revealed features of apical supporting cells indicative of a propensity for proliferative hair cell regeneration. This includes reduced expression of Notch effectors, receptivity for canonical Wnt signaling, and prominent expression of early cell-cycle genes. Cochlear hair cells displayed expression gradients of genes indicative of cellular differentiation and the establishment of the tonotopic axis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 05/2015; 2(9). DOI:10.1016/j.celrep.2015.04.062 · 7.21 Impact Factor
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    ABSTRACT: Sensorineural hearing loss is most commonly caused by the death of hair cells in the organ of Corti, and once lost, mammalian hair cells do not regenerate. In contrast, other vertebrates such as birds can regenerate hair cells by stimulating division and differentiation of neighboring supporting cells. We currently know little of the genetic networks which become active in supporting cells when hair cells die and that are activated in experimental models of hair cell regeneration. Several studies have shown that neonatal mammalian cochlear supporting cells are able to trans-differentiate into hair cells when cultured in conditions in which the Notch signaling pathway is blocked. We now show that the ability of cochlear supporting cells to trans-differentiate declines precipitously after birth, such that supporting cells from six-day-old mouse cochlea are entirely unresponsive to a blockade of the Notch pathway. We show that this trend is seen regardless of whether the Notch pathway is blocked with gamma secretase inhibitors, or by antibodies against the Notch1 receptor, suggesting that the action of gamma secretase inhibitors on neonatal supporting cells is likely to be by inhibiting Notch receptor cleavage. The loss of responsiveness to inhibition of the Notch pathway in the first postnatal week is due in part to a down-regulation of Notch receptors and ligands, and we show that this down-regulation persists in the adult animal, even under conditions of noise damage. Our data suggest that the Notch pathway is used to establish the repeating pattern of hair cells and supporting cells in the organ of Corti, but is not required to maintain this cellular mosaic once the production of hair cells and supporting cells is completed. Our results have implications for the proposed used of Notch pathway inhibitors in hearing restoration therapies.
    Frontiers in Cellular Neuroscience 03/2015; 9:110. DOI:10.3389/fncel.2015.00110 · 4.18 Impact Factor
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    ABSTRACT: Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells.
    Frontiers in Cellular Neuroscience 03/2015; 9:79. DOI:10.3389/fncel.2015.00079 · 4.18 Impact Factor
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    ABSTRACT: Single-cell gene expression analysis has contributed to a better understanding of the transcriptional heterogeneity in a variety of model systems, including those used in research in developmental, cancer and stem cell biology. Nowadays, technological advances facilitate the generation of large gene expression data sets in high-throughput format. Strategies are needed to pertinently visualize this information in a tissue structure-related context, so as to improve data analysis and aid the drawing of meaningful conclusions. Here we describe an approach that uses spatial properties of the tissue source to enable the reconstruction of hollow sphere-shaped tissues and organs from single-cell gene expression data in 3D space. To demonstrate our method, we used cells of the mouse otocyst and the renal vesicle as examples. This protocol presents a straightforward computational expression analysis workflow, and it is implemented on the MATLAB and R statistical computing and graphics software platforms. Hands-on time for typical experiments can be <1 h using a standard desktop PC or Mac.
    Nature Protocols 03/2015; 10(3):459-474. DOI:10.1038/nprot.2015.022 · 7.78 Impact Factor
  • Robert Durruthy-Durruthy, Stefan Heller
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    ABSTRACT: Single cell trajectory analysis is a computational approach that orders cells along a pseudotime axis. This temporal modeling approach allows the characterization of transitional processes such as lineage development, response to insult, and tissue regeneration. The concept can also be applied to resolve spatial organization of cells within the originating tissue. Known as temporal and spatial transcriptomics, respectively, these methods belong to the most powerful analytical techniques for quantitative gene expression data currently available. Here, we discuss three different approaches: principal component analysis, the 'Monocle' algorithm, and self-organizing maps. We use a previously published qRT-PCR dataset of single neuroblast cells isolated from the developing mouse inner ear to highlight the basic features of the three methods and their individual limitations, as well as the distinct advantages that make them useful for research on the inner ear. The complex developmental morphogenesis of the inner ear and its specific challenges such as the paucity of cells as well as important open questions such as sensory hair cell regeneration render this organ a prime target for single cell trajectory analysis strategies.
    Cell and Tissue Research 12/2014; DOI:10.1007/s00441-014-2079-2 · 3.33 Impact Factor
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    ABSTRACT: Background: Cisplatin is a widely-used chemotherapeutic agent that can also cause ototoxic injury. One potential treatment for cisplatin-induced hearing loss involves the activation of endogenous inner ear stem cells, which may then produce replacement hair cells. In this series of experiments, we examined the effects of cisplatin exposure on both hair cells and resident stem cells of the mouse inner ear.Results: Treatment for 24 hours with 10 µM cisplatin caused significant loss of hair cells in the mouse utricle, but such damage was not evident until four days after the cisplatin exposure. In addition to killing hair cells, cisplatin treatment also disrupted the actin cytoskeleton in remaining supporting cells, and lead to increased histone H2AX phosphorylation within the sensory epithelia. Finally, treatment with 10 µM cisplatin appeared to have direct toxic effects on resident stem cells in the mouse utricle. Exposure to cisplatin blocked the proliferation of isolated stem cells and prevented sphere formation when those cells were maintained in suspension culture.Conclusion: The results suggest that inner ear stem cells may be injured during cisplatin ototoxicity, thus limiting their ability to mediate sensory repair. Developmental Dynamics, 2014. © 2014 Wiley Periodicals, Inc.
    Developmental Dynamics 10/2014; 243(10). DOI:10.1002/dvdy.24150 · 2.67 Impact Factor
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    ABSTRACT: The otocyst harbors progenitors for most cell types of the mature inner ear. Developmental lineage analyses and gene expression studies suggest that distinct progenitor populations are compartmentalized to discrete axial domains in the early otocyst. Here, we conducted highly parallel quantitative RT-PCR measurements on 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling-pathway-associated transcripts, and novel otic-specific genes. By applying multivariate cluster, principal component, and network analyses to the data matrix, we were able to readily distinguish the delaminating neuroblasts and to describe progressive states of gene expression in this population at single-cell resolution. It further established a three-dimensional model of the otocyst in which each individual cell can be precisely mapped into spatial expression domains. Our bioinformatic modeling revealed spatial dynamics of different signaling pathways active during early neuroblast development and prosensory domain specification.
    Cell 04/2014; 157(4). DOI:10.1016/j.cell.2014.03.036 · 33.12 Impact Factor
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    ABSTRACT: Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, yet the physiological role of α-tubulin K40 acetylation remains elusive. While genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell-substrate adhesion were significantly compromised in cultured αTat1(-/-) fibroblasts. First, αTat1(-/-) fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1(-/-) cells failed to activate Hippo signaling in response to increased cell density and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1(-/-) cells contained very few focal adhesions and their ability to adhere to growth surfaces was greatly impaired. While the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion, restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Since α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway.
    Molecular biology of the cell 04/2014; DOI:10.1091/mbc.E13-10-0609 · 5.98 Impact Factor
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    ABSTRACT: In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to human embryonic stem cells into cells of the otic lineage using chemically-defined attached-substrate conditions. Generation of human otic progenitor cells was dependent on FGF signaling and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic embryonic stem cell reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.
    Stem cells and development 02/2014; DOI:10.1089/scd.2014.0033 · 4.20 Impact Factor
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    ABSTRACT: In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFβ/BMP signaling, in which low levels of TGFβ/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/β-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.
    Proceedings of the National Academy of Sciences 08/2013; DOI:10.1073/pnas.1307376110 · 9.81 Impact Factor
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    ABSTRACT: Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.
    PLoS ONE 06/2013; 8(6):e66026. DOI:10.1371/journal.pone.0066026 · 3.53 Impact Factor
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    ABSTRACT: Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.
    Development 03/2013; 140(6):1196-206. DOI:10.1242/dev.087528 · 6.27 Impact Factor
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    ABSTRACT: TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.
    PLoS ONE 02/2013; 8(2):e58174. DOI:10.1371/journal.pone.0058174 · 3.53 Impact Factor
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    ABSTRACT: Mammalian FCHSD1 and FCHSD2 are homologous proteins containing an amino-terminal F-BAR domain and two SH3 domains near their carboxyl-termini. We report here that FCHSD1 and FCHSD2 are expressed in mouse cochlear sensory hair cells. FCHSD1 mainly localizes to the cuticular plate, whereas FCHSD2 mainly localizes along the stereocilia in a punctuate pattern. Nervous Wreck (Nwk), the Drosophila ortholog of FCHSD1 and FCHSD2, has been shown to bind Wsp and play an important role in F-actin assembly. We show that, like its Drosophila counterpart, FCHSD2 interacts with WASP and N-WASP, the mammalian orthologs of Drosophila Wsp, and stimulates F-actin assembly in vitro. In contrast, FCHSD1 doesn't bind WASP or N-WASP, and can't stimulate F-actin assembly when tested in vitro. We found, however, that FCHSD1 binds via its F-BAR domain to the SH3 domain of Sorting Nexin 9 (SNX9), a well characterized BAR protein that has been shown to promote WASP-Arp2/3-dependent F-actin polymerization. FCHSD1 greatly enhances SNX9's WASP-Arp2/3-dependent F-actin polymerization activity. In hair cells, SNX9 was detected in the cuticular plate, where it colocalizes with FCHSD1. Our results suggest that FCHSD1 and FCHSD2 could modulate F-actin assembly or maintenance in hair cell stereocilia and cuticular plate.
    PLoS ONE 02/2013; 8(2):e56516. DOI:10.1371/journal.pone.0056516 · 3.53 Impact Factor
  • Stefan Heller
    Hearing research 12/2012; DOI:10.1016/j.heares.2012.12.009 · 2.85 Impact Factor
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    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2012; 32(41):14053-14057. DOI:10.1523/JNEUROSCI.3336-12.2012 · 6.75 Impact Factor
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    ABSTRACT: The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels.
    Journal of Biological Chemistry 06/2012; 287(27):22701-8. DOI:10.1074/jbc.M112.368876 · 4.60 Impact Factor
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    Mohammad Ronaghi, Marjan Nasr, Stefan Heller
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    ABSTRACT: Hearing loss, caused by irreversible loss of cochlear sensory hair cells, affects millions of patients worldwide. In this concise review, we examine the conundrum of inner ear stem cells, which obviously are present in the inner ear sensory epithelia of nonmammalian vertebrates, giving these ears the ability to functionally recover even from repetitive ototoxic insults. Despite the inability of the mammalian inner ear to regenerate lost hair cells, there is evidence for cells with regenerative capacity because stem cells can be isolated from vestibular sensory epithelia and from the neonatal cochlea. Challenges and recent progress toward identification of the intrinsic and extrinsic signaling pathways that could be used to re-establish stemness in the mammalian organ of Corti are discussed.
    Stem Cells 01/2012; 30(1):69-74. DOI:10.1002/stem.785 · 7.70 Impact Factor
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    ABSTRACT: In patients with severe to profound hearing loss, cochlear implants (CIs) are currently the only therapeutic option when the amplification with conventional hearing aids does no longer lead to a useful hearing experience. Despite its great success, there are patients in which benefit from these devices is rather limited. One reason may be a poor neuron-device interaction, where the electric fields generated by the electrode array excite a wide range of tonotopically organized spiral ganglion neurons at the cost of spatial resolution. Coating of CI electrodes to provide a welcoming environment combined with suitable surface chemistry (e.g. with neurotrophic factors) has been suggested to create a closer bioelectrical interface between the electrode array and the target tissue, which might lead to better spatial resolution, better frequency discrimination, and ultimately may improve speech perception in patients. Here we investigate the use of a collagen surface with a cholesteric banding structure, whose orientation can be systemically controlled as a guiding structure for neurite outgrowth. We demonstrate that spiral ganglion neurons survive on collagen-coated surfaces and display a directed neurite growth influenced by the direction of collagen fibril deposition. The majority of neurites grow parallel to the orientation direction of the collagen. We suggest collagen coating as a possible future option in CI technology to direct neurite outgrowth and improve hearing results for affected patients.
    Archives of Oto-Rhino-Laryngology 09/2011; 269(4):1111-6. DOI:10.1007/s00405-011-1775-8 · 1.61 Impact Factor
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    ABSTRACT: The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few immigrating neural crest-derived cells. The otocyst plays a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory and prosensory domains, giving rise to individual vestibular and auditory organs and their associated ganglia. The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known. The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method. Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors. We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as SOX4, SOX5, SOX7, SOX8, SOX11, and SOX18, which previously were not known to be expressed in the developing inner ear. Despite its limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies.
    Journal of the Association for Research in Otolaryngology 08/2011; 12(6):697-710. DOI:10.1007/s10162-011-0286-z · 2.55 Impact Factor

Publication Stats

4k Citations
557.04 Total Impact Points

Institutions

  • 2007–2015
    • Stanford Medicine
      • • Department of Otolaryngology
      • • Department of Molecular and Cellular Physiology
      Stanford, California, United States
  • 2014
    • Icahn School of Medicine at Mount Sinai
      Borough of Manhattan, New York, United States
  • 2008–2014
    • Stanford University
      • • Department of Molecular and Cellular Physiology
      • • Department of Otolaryngology
      Palo Alto, California, United States
    • Inselspital, Universitätsspital Bern
      Berna, Bern, Switzerland
  • 2013
    • Shandong University
      • School of Life Science
      Chi-nan-shih, Shandong Sheng, China
  • 2012
    • Ludwig-Maximilian-University of Munich
      • Department of Pharmacy
      München, Bavaria, Germany
  • 2010
    • University Hospital Frankfurt
      Frankfurt, Hesse, Germany
  • 2002–2006
    • Harvard Medical School
      • Department of Otology and Laryngology
      Boston, MA, United States
  • 2004–2005
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2003
    • Massachusetts Eye and Ear Infirmary
      Boston, Massachusetts, United States
  • 1998–2003
    • The Rockefeller University
      • Laboratory of Sensory Neuroscience
      New York City, NY, United States