2001 — 2014 |
Heller, Stefan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Inner Ear Function
Project Summary / Abstract The sensory receptors of the inner ear, the hair cells, mediate our senses of hearing and balance because of their ability to convert mechanical forces into electrical signals. This process happens in the mechanosensitive organelle, the hair bundle, which protrudes from the cell's apical surface. The hair bundle is composed of a few tens to hundreds of actin-rich stereocilia that are arranged in a hexagonal array. Mechanical deflection of the bundle toward the tallest stereocilia leads to shearing motions between adjacent stereocilia that are exerted by tip links, connectors between the tips of shorter stereocilia with its taller neighbor. The consequential increase of mechanical tension in the transduction apparatus increases the open probability of mechanically gated ion channels located at or near the tips of stereocilia, resulting in an influx of cations that depolarizes the hair cell, thereby generating a receptor potential. The understanding of hair bundle development, maintenance, and ultimately the molecular basis of its function, is fundamental for inner ear biology and also for our understanding of human hearing loss and disequilibrium. One aim of this proposal is to investigate the molecular basis of stereocilia height regulation by investigating the consequences of loss of the gene encoding twinfilin 2, a protein that is located near the tips of stereocilia. For this purpose, we will employ transgenic mouse technology, quantitative 3-dimensional microscopic analyses using confocal and electron microscopy, as well as electrophysiology. A second aim focuses on the role of a group of TRP proteins including members of the TRPML and TRPV subfamily that appear to be able to form heteromeric ion channels in hair cells. We hypothesize testing whether these heteromeric channels are functional, and whether they play a role in hair cell function. Particularly, we are interested in exploring a role of TRPML/TRPV heteromers in mechanoelectrical transduction. Analyses include biochemical, electrophysiological, and cell- based assays, as well as transgenic mice, histology, whole animal auditory function tests, and electrophysiology.
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2003 — 2013 |
Heller, Stefan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Inner Ear Progenitor Cells
DESCRIPTION (provided by applicant): Inner ear hair cell loss and lack of hair cell regeneration are the major cause of permanent hearing loss. In this application, it is proposed to test whether mouse embryonic stem cell-generated hair cell- like cells are functional in the sense that they display mechanotranduction currents, appropriate basolateral currents, as well as the ability to form appropriate synaptic connections. It is further planned to develop an in utero cell-replacement treatment to determine correlation between occurrence of graft-derived hair cells and regionalized restoration of the organ of Corti. Physiologically, we expect that such a restoration may lead to alleviation of hair cell loss and hearing impairment in a mouse model. A second Aim addresses the guidance of embryonic stem cells toward hair cell-like cells. It is proposed to devise a protocol of defined inductive steps, which will enable researchers to efficiently generate progenitor cells from embryonic stem cells that are competent to develop along the otic lineage. A third Aim proposes to identify a non-FGF-based activity that is involved in otic induction, which is released from a region of the chicken embryo that is adjacent to the otic placode. PUBLIC HEALTH RELEVANCE Toward finding a treatment for hearing loss, it is proposed to continue research on converting mouse embryonic stem cells into hair cells. First, it is planned to assess whether stem cell-generated hair cell-like cells display the same functional properties as wild type hair cells, particularly cochlear hair cells. Building on this, it is planned to use embryonic stem cell-derived inner ear progenitor cells to treat a mouse model of hereditary deafness and to test whether hearing loss can be alleviated with a cellular treatment. In a parallel Aim, it is proposed to increase the efficacy by which embryonic stem cells can be "primed" to become responsive to inner ear inducing signals. This will be done by mimicking the inductive steps that lead to establishment of the inner ear during embryonic development. In the third Aim, it is planned to identify a signaling molecule from chicken embryonic tissue that appears to be conducting such a "priming", which makes progenitor or stem cells responsive to FGF-based inner ear inducing activity.
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2005 — 2006 |
Heller, Stefan |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Human Inner Ear Stem Cells
[unreadable] DESCRIPTION (provided by applicant): The enormous clinical promise of stem cells makes these cells prime candidates to investigate their potential for the restoration of inner ear damage. Inner ear progenitor cells have recently been generated from adult murine inner ear stem cells and it has been shown that these progenitor cells can give rise to hair cells and neurons in vitro. Transplantation of murine inner ear progenitor cells into the developing inner ear resulted in the generation of hair cells in vivo. In pilot experiments using adult human inner ear tissue samples obtained in labyrinthectomy surgery, we found evidence for the presence of adult stem cells in the human inner ear. In vitro, these stem cells could be propagated and differentiated into cells that express marker proteins indicative of hair cells. This preliminary evidence suggests that human hair cells can be generated from adult human inner ear stem cells. The research proposed in this Feasibility Pilot Study focuses on establishing the most seminal requirements for the utilization of human adult inner ear stem cells in future research and in clinical applications. In Aim 1, we will characterize the potential of human inner ear stem cells to give rise to inner ear progenitor cells that can differentiate into hair cells. In Aim 2, we will investigate the long-term propagation capacities of human adult stem cells. Here we plan to develop a method that allows us to expand the stem cell population with the goal of establishing long-term storage of human inner ear stem cells in liquid nitrogen. Expansion and long-term storage are the principal requirements for further research on human adult inner ear stem cells and, potentially, for the future development of an inner ear stem cell donor bank. Subsequent research aimed to translate stem cell technology toward human therapy will only be possible by first establishing reliable and efficient methodology to isolate, expand, propagate, and store human inner ear stem cells, the goals proposed in this Exploratory Research Grant application. [unreadable] [unreadable]
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2007 |
Heller, Stefan |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Identification of Modulators of the Trpml3 and Trpn1 Ion Channels
[unreadable] DESCRIPTION (provided by applicant): The main goal of this project lies in identifying agonists for two transient receptor potential (TRP) ion channels that are potentially involved in inner ear mechanosensation. One of the major obstacles of inner ear biology is the lack of specific pharmacological tools to investigate the process of mechanotransduction of inner ear sensory hair cells. Although the vertebrate hair cell transduction channel remains a mystery, a number of TRPs are connected to a potential physiological function in the inner ear. Particularly TRPML3 and TRPN1, the focus of this application, have been shown to be important for hearing and balance in mice and zebrafish. Identification of agonists and, in future studies prospective antagonists, would not only advance inner ear and sensory neurobiology, but could also provide us with potential drug candidates for the treatment of vertigo and tinnitus. [unreadable] [unreadable] [unreadable]
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2009 — 2016 |
Heller, Stefan |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Administrative Core
The administrative shell has three specific aims. First, it focuses on cost-effective management of the funded grant. Second, it coordinates Core Center activities, and third, it gathers usage data of core equipment, user-related information and maintains databases to help the Core Center PI and the Core co-directors with long-term planning. Cost-effective management includes tracking of maintenance and repair costs for core equipment, monitoring the time effort of core personnel, finding best pricing for supplies, and maintaining records of all Core Center expenses. Coordination of activities includes scheduling of the monthly seminar / training sessions, periodic meetings of the core co-directors and core personnel, and assisting with daily operations management. Usage data is being obtained from equipment logbooks and computer-based tracking logs and is maintained as Excel spreadsheet. A user/usage database and training database of core user laboratory members is maintained to provide data for periodic evaluations of core usage. Periodic meetings of Core Center PI and co-directors will be coordinated and provided with data to help with planning decisions aimed to constantly improving the services. The Core Center PI is responsible for ail described activities of the administrative shell with the help of an administrative assistant.
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2009 — 2013 |
Heller, Stefan Popelka, Gerald R. (co-PI) [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Auditory Function Core
Existing and planned experimental work in several NIDCD ROI-funded laboratories requires sophlsficated measures of auditory function in small mammals. The breadth of these auditory funcfion measures ranges from simple high-throughput screening up to full comprehensive, frequency-specific measures concerning the middle ear, outer hair cell funcfion, and neural funcfion. These measures can be tailored for simple screening to frequency-specific threshold estimates. Measures of middle ear function will be added. The Core Auditory Function facility will ensure that these complex auditory measures are performed efficiently and accurately with the appropriate apparatus, under standardized, and calibrated condifions, and managed with a comprehensive database that stores, manages and retrieves individual and aggregate experimental auditory data. The Auditory Function Core consists of a Central Auditory Laboratory, three individual auditory measurement facilities, and a noise damaging facility, all in close proximity. The facilifies have convenfional apparatus and many custom components including high-frequency transducers for specific species. Roufine measures include distortion product otoacousfic emissions and frequency-specific auditory measures at the level ofthe brainstem tailored to the specific small mammals being measured, currently mice and guinea pigs. The three measurement stafions include sound and electrically shielded rooms, and specialized systems (Tucker Davis Technologies, Intelligent Hearing Systems) that include passive attenuators, filters, signal averaging algorithms, stimulus creation, etc. A fourth station will be used for generafing and controlling acousfic signals that are used for experimentally-controlled auditory damage. Our approach is to develop consistent, redundant, modular systems that provide all of these measures or subsets of these measures in a standardized, well-characterized manner;to plan for equipment and personnel redundancy so that no experiment would ever be compromised in the face ofthe usual transient breakdowns;and to develop a comprehensive server-based database so that every scientist can locate and download any result at any fime using a consistent interface.
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2009 — 2013 |
Heller, Stefan |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core Center-Nidcd Research Core Center P30
DESCRIPTION (provided by applicant): The main goals of the Stanford OHNS Core Center are to serve 1) as a hub for knowledge and technology, 2) to stimulate and support collaborative research, and 3) to provide access to high quality state-of-the-art technology via cost efficient shared utilization of Core Center equipment among R01-funded laboratories. Administration of the Core Center will be provided by the Principal Investigator and two Research Core Directors in conjunction with an administrative assistant. The Core's administration is responsible for daily operations, but also for long-term planning with the overall objective to ensure that the main goals of the Core Center are being met. Two Research Cores are proposed: Core I - Imaging Core, consists of four independent imaging systems, each one individually dedicated for specific tasks including routine confocal and fluorescence imaging, two photon imaging, live cell confocal imaging, and high-speed live cell confocal imaging. A full time imaging specialist will support Core users with expert advice, training, and supervision. Core II - Auditory Function Core consists of dedicated apparatus development space, two sound shielded rooms, two adjacent rooms for animal preparations and recovery, and three comprehensive auditory physiology systems. These systems are dedicated to measures of auditory function in vivo in small mammals including tympanometry, otoacoustic emissions and auditory evoked potentials from surface or subdural electrodes. A part time auditory function specialist will support core users with expertise, training, and supervision. The imaging and auditory function equipment is already being used by a group of researchers that are faculty members in Otolaryngology - Head & Neck Surgery, Neurobiology, Molecular & Cellular Physiology, as well as Bioengineering. There already is an urgent need for support personnel and an overall core organization, which will ensure efficient shared use of the existing equipment by a highly collaborative user base that is unified by a common interest in auditory and vestibular neurobiology.
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2009 — 2013 |
Heller, Stefan Ricci, Anthony J (co-PI) [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Imaging Core
The imaging core has four independent workstafions (Image Stafions 1-4), each incorporafing overlapping but independent imaging technologies. Three of four can be used for live cell imaging and two of four will have complementary electrophysiological recording stafions that allow the combinafion of powerful imaging and electrophysiological technologies. From an imaging perspective, the core facilities can accommodate multi-fluorophore immunocytochemical high-resolufion 3-dimensional imaging while also serving high speed physiological imaging. Addifionally, there are two dedicated computers with high end graphics boards, ample memory and processing power that can be used for image analysis offline from the imaging stafions. From conventional fluorescence and confocal imaging to high speed swept field confocal and two-photon imaging and uncaging, the core facility will serve to bring together ROI-funded invesfigators with a broad range of interests and expertise. The core will facilitate the use of this equipment so that translafional uses by the clinical faculty can be increased while at the same fime having the versafility to include users with common interests from mulfiple departments that share goals that are in agreement with the mission statement of the NIDCD. Clear examples of each of these uses exist already with the two-photon system being used to develop whole animal cochlea imaging that hopes to be ufilized in cochlear implant placements, while both the high speed swept field confocal system and the two photon system are presently being used to invesfigate synchronous firing in the cortex. This core grant proposal will allow us to expand the number of users and thus serve as a means to increase collaborafion across both basic science and clinical departments
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2009 — 2010 |
Heller, Stefan |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Otic Progenitors Via Somatic Cell Nuclear Reprogramming
DESCRIPTION (provided by applicant): In this exploratory research grant application, it is proposed to investigate the applicability of somatic cell nuclear reprogramming to generate otic progenitor cells. It is planned to explore whether induced pluripotent stem cells can be directed to differentiate into inner ear cell types (Specific Aim 1) and in a second set of experiments, it is proposed to investigate conversion of somatic cells directly into progenitors capable of differentiating into hair cell marker expressing cells (Specific Aim 3). An in-depth characterization of the generated cell types is proposed using gene chip technology and comparison of the transcriptional profile of purified embryonic and induced pluripotent stem cell-generated inner ear progenitors with the transcriptional profile of otic placode cells purified from transgenic mice with fluorescent otic placode cells (Specific Aim 2). Knowledge about the potential roles of specific genes gained with somatic cell reprogramming and stem cell guidance will be directly applicable and highly useful for studies focusing on hair cell regeneration in the damaged mammalian organ of Corti as well as for developmental inner ear biology. Moreover, the proposed experiments are highly relevant for potential treatment, where autologous sources of replacement cells are exceedingly desirable to avoid having to immunosuppress the recipient patient. Successful demonstration of applicability of somatic cell nuclear reprogramming to murine inner ear cell regeneration will provide the conceptual basis for future expansion of these experiments to patient-derived somatic cells such as human fibroblasts. Toward finding a treatment for hearing loss, it is proposed to explore the potential of induced pluripotent stem cells that can be generated from somatic cells such as fibroblasts. Specifically it will be tested whether induced pluripotent stem cells can be directed to differentiate into otic cell types, which could be used in future studies with animal models to replace lost cochlear hair cells. This is a highly relevant endeavor because utilization of autologous sources for replacement cells is desirable because these cells could be used without having to immuno-suppress the patient. The research is exploratory as it uses very recently developed novel somatic cell reprogramming technology, which has not been applied to inner ear research. Likewise, it proposes to explore a novel concept to directly convert somatic cells, such as fibroblasts, into inner ear progenitor cells. This direct conversion strategy does not convert the cells completely back into a pluripotent state, but rather into a more restricted state that is potentially already committed to the otic lineage. This novel strategy might bear distinct advantages over the "conventional" strategy, which first converts cells back into an embryonic stem cell state followed by a forward guidance toward a specific organ fate. If successful, this method might as well be applicable to other organs beside the inner ear.
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2012 — 2016 |
Heller, Stefan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Patient Derived Sensory Hair- and Supporting Cell-Like Cells
DESCRIPTION (provided by applicant): The overriding aim of this grant proposal is to translate technology developed with murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to the clinical setting. More specifically, it is proposed to generate and characterize human inner ear sensory hair cell-like cells and supporting cell-like cells from ESCs and iPSCs. In Aim 1, human ESC-derived hair cell-like cells are being characterized immunocytochemically, morphologically, and functionally. In Aim 2, it is proposed to generate and characterize hair cell-like cells from human iPSCs, specifically from patients carrying mutations in the MYO15A gene. It is expected that the consequences of the mutant MYO15A gene are reflected in the cellular phenotype of hair cell-like cells generated from hearing loss patients. Further proposed is a rescue experiment with wild type MYO15A to restore the cellular phenotype in patient-derived hair cell-like cells. Finally, Aim 3 focuses on characterization of gap junctions in supporting cell-like cells derived from DFNB1 patients homozygous for the common GJB2 35delG mutation. It is proposed to compare cellular properties of 35delG homozygotes with severe-to-profound hearing loss to 35delG homozygotes with mild-to-moderate hearing loss. Measures include gap junction physiology and whole transcriptome analysis, which will be combined with genome wide association studies in a large cohort of DFNB1 patients. The goal of this endeavor is the identification of clinically relevant genetic modifiers, which is a first step toward developing strategies to ameliorate the typical severe phenotype associated with the 35delG/35delG genotype in DFNB1 patients.
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2014 — 2016 |
Heller, Stefan |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Stanford Ohns Core Center |
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2016 — 2020 |
Heller, Stefan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Response of Cochlear Hair Cells to Pathological Changes in the Auditory System
Project Summary / Abstract The mouse cochlea harbors only 800 inner hair cells and about three times as many outer hair cells. This low number of cells has hampered progress specifically in the study of the molecular biology of inner ear cells. The use of single hair cell gene expression profiling has the potential to turn this disadvantage into a major advantage because it allows for oversampling the organ by generating high resolution quantitative gene expression maps for thousands of inner and outer hair cells of the organ of Corti, which is the first Aim of this grant application. It is anticipated that these maps, based on single cell RNA-Seq data will miss little information when compared for example with other sensory systems such as the retina, where analysis of a few thousand photoreceptor cells would represent <0.05% of the sensory cell population. It is anticipated that the maps will reveal how gradients of gene expression contribute to functional tonotopy in inner and outer hair cells. Moreover, the generated maps will serve as base line for identifying distinct changes in cochlear hair cells after noise-induced temporary threshold shift. Here, it is anticipated that the analysis will reveal functional modules of co-regulated hair bundle genes as well as candidate gene regulatory networks for susceptibility to noise-induced hearing loss. A second series of experiments aims to identify the molecular mechanisms by which non-traumatic sound exposure temporarily reduces susceptibility for permanent noise induced threshold shift. This research has the potential of elucidating genes and mechanisms involved in susceptibility to noise. Finally, it is proposed to investigate at the molecular level compensatory changes in inner and outer hair cells in response to sustained changes in cochlear integration such as lack of functional afferent or efferent innervation or lack of connection between outer hair cells and the tectorial membrane. Beside identification of co-regulated gene groups for example involved in cochlear amplification, it is expected that this research will reveal how hair cells are affected by pathological situations that do not trigger immediate hair cell loss.
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2021 |
Heller, Stefan |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
High Efficient Aav-Transducible Transgenic Quails
Project Summary / Abstract The main goal of this research proposal is to generate a transgenic quail line that ubiquitously expresses the human receptor for adeno-associated virus (AAV). Quails and chickens cannot be e?ciently transduced with AAV, which precludes the use of this class of popular viruses in these animals. Expressing the AAV receptor in avian cells, however, signi?cantly increases the transduction e?cacy for AAV. The ?rst aim of the proposed research is to generate a transgenic quail line that can be tested for e?cient virus transduction of the inner ear in vivo, which is the goal of aim two of the proposal. The ?nal aim is to demonstrate that the newly generated transgenic AAV receptor-expressing quail line can be used for AAV-mediated CRISPR-Cas9 genome editing in the inner ear. The successful completion of this research project will provide a new animal model for studying avian hair cell regeneration through gene manipulation in vivo. Inner ear sensory hair cells do not naturally regenerate in mammals but they do in birds, which emphasizes the importance of conducting genetic manipulations in birds. Knowledge gained with this new experimental model system, such as the signi?cance of speci?c signaling pathways and key genes during avian hair cell regeneration, shall be translatable to mammals, and ultimately can provide insight into curative treatments for sensorineural hearing loss.
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