Stefan Heller Ph.D.

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Integrative Analysis

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Integrative Analysis
Seth Ament, Ph.D. (co-chair), University of Maryland
Ronna Hertzano, M.D., Ph.D. (co-chair), National Institute on Deafness and Other Communication Disorders
Albert Edge, Ph.D., Mass Eye & Ear
Stefan Heller, Ph.D., Stanford University
David Raible, Ph.D., University of Washington
Jennifer Stone, Ph.D., University of Washington

This group will take the lead on data curation and analysis. A dedicated full-time HRP analyst is working across groups to help collect and process data, thereby facilitating a broader analysis of cell states and trajectories across species. The group will start by annotating hair cell types from all species so that anyone in the field can assess what kind of hair cell their regeneration approaches may produce, while also easing identification of common hair cell genes, which will help the Cross-Species Epigenetics group. Analysis of the hair cells produced in mouse organoids will be performed as an example. The Ament lab will leverage their expertise in bioinformatics, while the Hertzano lab will continue to oversee upkeep of gEAR, with a goal of making it even easier for HRP members to post their new data and for others in the community to analyze those data. The Edge lab will take the lead on the development of organoids as a screening platform for the future. The Heller, Hertzano, Raible, and Stone labs will validate markers by in situ hybridization across species, and all working group members will help direct the analysis.

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Cross-Species Epigenetics

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Cross-Species Epigenetics
Tatjana Piotrowski, Ph.D. (chair), Stowers Institute for Medical Research
Alain Dabdoub, Ph.D., Sunnybrook Research Institute
Andy Groves, Ph.D., Baylor College of Medicine
Stefan Heller, Ph.D., Stanford University
The Lab of Neil Segil, Ph.D., University of Southern California
Litao Tao, Ph.D., Creighton University

This group will complete the collection of transcriptomic and epigenetic data from systems that regenerate (neonatal mouse, zebrafish, chick) and those that do not (mature mouse and human). In addition, they will begin to perform cross-species comparisons of the behavior of a shared set of hair cell loci across species. Collection of chick data is spearheaded by the Heller lab; the Groves, Segil, and Tao labs are responsible for mouse data; the Piotrowski lab leads work on zebrafish; and the Dabdoub lab will add data from humans. High-quality single-cell RNA-seq, ATAC-seq, and SHARE-seq data from multiple timepoints and conditions will be generated by all member labs.

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla
Stefan Heller, Ph.D., Stanford University

Birds robustly regenerate their cochlear hair cells through the conversion of dormant supporting cells into new hair cells. Our project uses selective, high-sensitivity methods to reveal the molecular changes in supporting cells after their activation by, for example, ototoxic drugs that cause hair cell death. By examining the responses of many single cells, we have begun to identify triggers that initiate, execute, sustain, and ultimately terminate the regenerative process. Recent experiments have confirmed what we already knew, that the two halves of the chicken cochlea (neural and abneural) predominantly use different regeneration mechanisms. Using bioinformatics methods to process the resulting data, this year we are focusing on analyzing chicken cochlea cell populations isolated at various time points during hair cell regeneration and specifically characterizing the two distinct responses. By examining regeneration in an animal that replaces hair cells after damage, we will be able to find triggers that may be activated in mammals to reverse hair cell loss.

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla
Stefan Heller, Ph.D., Stanford University

Birds regenerate cochlear hair cells by activating dormant supporting cells. This project builds on innovative methods and findings to study how supporting cells are activated when ototoxic drugs cause hair cell death. The project uses single cell analysis (during which we study the complement of genes that are active in many individual cells) to identify triggers that initiate, execute, sustain, and ultimately terminate the regenerative process. Using bioinformatics methods to process the resulting data, we will focus this year on the analysis of the chicken cochlea cell populations isolated at various time points during hair cell regeneration, which will reveal the molecular steps that occur during hair cell regeneration. We have already identified a first candidate gene signaling pathway that may regulate the regenerative process in the chicken cochlea, and we will be confirming that this pathway plays a role in hair cell regeneration. Interestingly, this pathway is not active during inner ear development, which sets it apart from other pathways linked to chicken (and zebrafish) hair cell regeneration; the other pathways are involved in cell development and may not represent a unique regenerative trigger. Finding triggers that specifically control regeneration may be an important stepping stone on the path to developing cures for hair cell loss in mice and, eventually, humans.

Transcriptome Changes in Single Chick Cells

Transcriptome changes in single chick cells
Stefan Heller, Ph.D., Stanford University

Chicks regenerate hair cells in auditory and vestibular organs after damage, making them a valuable animal model to study the signals controlling hair cell regeneration. This project aims to identify changes in gene expression after hair cell loss in the chick cochlea and vestibular system. The collected data will be comprehensive because it will cover all detectable expressed genes. Subsequent data analysis will focus on establishing a sequence of gene expression changes that we hypothesize will correlate with important steps of the hair cell regeneration process. These steps include the signals that initiate, execute, sustain, and ultimately terminate the regenerative process. Comparison among the different organs and across species through collaborations with other HRP investigators will allow us to draw conclusions about species-specific specialized mechanisms as well as more general processes that control hair cell regeneration.

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Establishing the Human Utricle from Surgical Patients as a Translational in Vitro Model for Hair Cell Regeneration

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Establishing the human utricle from surgical patients as a translational in vitro model for hair cell regeneration
Alain Dabdoub, Ph.D. University of Toronto
Stefan Heller, Ph.D. Stanford University
Michael Lovett, Ph.D. Imperial College London

These investigators will work with human utricles harvested during surgery to examine whether the response of human inner ear tissue to damage is similar to that of our mammalian model, the mouse. They will culture human utricles for extended lengths of time following damage with aminoglycoside drugs and determine whether the utricles show any proliferation of their supporting cells (making new supporting cells) or regeneration of their hair cells (making new hair cells). Mouse utricles have a limited ability to show proliferation and regeneration, and it is important to determine whether they are a good model for humans. The investigators also intend to examine gene expression in these human utricles to determine the molecular similarity of the cells of interest to their human counterpart cells.

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Transcriptomics-based Analysis of Hair Cell Regeneration in Non-Mammals

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Transcriptomics-based analysis of hair cell regeneration in non-mammals
Stefan Heller, Ph.D. Stanford University
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research
Jennifer Stone, Ph.D. University of Washington
Mark Warchol, Ph.D. Washington University in St. Louis
Michael Lovett, Ph.D. Imperial College London

In this third-year project, we will disrupt hair cells, then analyze how gene expression changes in single cells from species that show robust hair cell regeneration. Heller’s lab will examine the consequences of aminoglycoside damage at the single-cell level in the chick utricle, while the Piotrowski lab will examine fish lateral-line cells. In another component, the project will add two time points to the chick cochlea and utricle bulk RNA-seq datasets that were generated by Lovett and Warchol and which are extremely valuable datasets for the HRP consortium.

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Single-cell RNA-seq Expression Analysis of Homeostatic Zebrafish Neuromasts

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Single-cell RNA-seq expression analysis of homeostatic zebrafish neuromasts
Stefan Heller, Ph.D. Stanford University School of Medicine
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research

The zebrafish is one of the primary models of the HRP, as hair cells in its lateral line organ show robust regeneration. Because of its superficial location in the skin, we can watch the regeneration process at the single-cell level using microscopy. We discovered that even within a regenerating cell population, individual cells are not synchronized in their behavior and gene expression. Therefore, conventional
gene expression analyses of the entire cell population provide only averages of gene expression, masking the cell heterogeneity.This project will use single-cell expression analysis techniques to determine how many supporting cell types exist and in future experiments how they respond to damage with high precision.

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Single-cell transcriptional profiling of chicken utricle and Basilar Papilla Sensory Epithelium Cells After Aminoglycoside-induced Hair Cell loss

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Single-cell transcriptional profiling of chicken utricle and basilar papilla sensory epithelium cells after aminoglycoside-induced hair cell loss
Stefan Heller, Ph.D. Stanford University School of Medicine
Jennifer S. Stone, Ph.D. University of Washington
Michael Lovett, Ph.D. Imperial College London
Mark Warchol, Ph.D. Washington University School of Medicine

The broad premise of the HRP is to identify molecules that could control hair cell regeneration. To do this, we are studying cell types and regenerative processes in multiple contexts and species, then integrating these data together to identify mechanisms that could potentially be turned on in the mouse cochlea to drive transdifferentiation (activating the correct set of hair cell–promoting genes in supporting cells). The role of this systems biology project is to provide the necessary data integration “glue,” binding together the results from the data generation projects. We will combine much of the data that is being generated by the HRP to advance our knowledge of hair cells, supporting cells, conversion of one cell type to another, and the potential for regeneration. By modeling all of the available HRP data, we will identify regulatory molecules that may contribute to regeneration.

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Standardized Mouse Model for Hearing Loss Studies

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Standardized mouse model for hearing loss studies
Albert Edge, Ph.D. Massachusetts Eye and Ear Infirmary, Harvard Medical School
Stefan Heller, Ph.D. Stanford University School of Medicine

Testing how the mouse inner ear responds to hair-cell damage will be greatly facilitated by the mouse model developed in this project. Using these mice, the HRP will be able to rapidly kill hair cells very selectively, determine which cells were supporting cells prior to hair cell death, and to visualize newly formed hair cells. We expect that these mice will be useful in a variety of phase I, II, and III experiments.

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Single Cell RT-PCR-based Expression Analysis of Homeostatic and Regenerating Zebrafish Neuromasts

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Single cell RT-PCR-based expression analysis of homeostatic and regenerating zebrafish neuromasts
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research
Stefan Heller, Ph.D. Stanford University School of Medicine

The zebrafish is one of the primary models of the HRP; hair cells in its lateral line organ show robust regeneration, the accessibility of this organ means that we can watch the whole process take place using microscopy. However, the lack of complete synchronization of this process means that when we are looking at molecular changes, they are smeared out in time. This project will use single-cell transcript analysis techniques to order supporting cell responses to damage and determine the order of expression of transcripts with high precision.

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Single Cell Transcriptional Profiling of Chicken Utricle and Basilar Papilla Sensory Epithelium Cells After Aminoglycoside-Induced Hair Cell Loss

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Single cell transcriptional profiling of chicken utricle and basilar papilla sensory epithelium cells after aminoglycoside-induced hair cell loss
Stefan Heller, Ph.D. Stanford University School of Medicine
Mark Warchol, Ph.D. Washington University School of Medicine
Jennifer S. Stone, Ph.D. University of Washington
Michael Lovett, Ph.D. Imperial College London

While the HRP has excellent datasets showing the response of chicken auditory and vestibular cells to damage, the experiments examine all cells (including both hair cells and supporting cells) and are not temporally precise. In these experiments, the investigators will damage hair cells, then examine the molecular responses of hundreds of individual cells. Bioinformatics techniques will allow them to order those cells along a timeline that will reveal the molecular changes that unfold during hair cell regeneration.

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