Methods of generating human inner ear sensory epithelia and sensory neurons

a technology of sensory epithelia and inner ear, which is applied in the field of generating human inner ear sensory epithelia and sensory neurons, can solve the problems of no pharmacological, genetic, or cell therapy that can treat hearing loss

Inactive Publication Date: 2019-03-28
INDIANA UNIV RES & TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nearly half a billion people have hearing loss world-wide, yet there are no pharmacological, genetic, or cell therapies that can treat hearing loss.

Method used

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  • Methods of generating human inner ear sensory epithelia and sensory neurons
  • Methods of generating human inner ear sensory epithelia and sensory neurons
  • Methods of generating human inner ear sensory epithelia and sensory neurons

Examples

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example 1

Generation of Inner Ear Organoids with Functional Hair Cells from Human Pluripotent Stem Cells

[0054]The human inner ear contains approximately 20,000 sensory hair cells that detect sound and movement via mechanosensitive stereocilia bundles1. Genetic mutations or environmental insults, such as loud noises, can cause irreparable damage to these hair cells, leading to dizziness or hearing loss2,3. We previously demonstrated how to generate inner ear organoids from mouse pluripotent stem cells (PSCs) using timed manipulation of the FGF, TGFβ, BMP, and Wnt signaling pathways in a 3D culture system4-6. We have shown that mouse inner ear organoids contain sensory hair cells with similar structure and function to native vestibular hair cells in the mouse inner ear7. Moreover, our past findings bolstered a working model of otic induction signaling dynamics, wherein BMP signaling activation and TGFβ inhibition initially specifies non-neural ectoderm and subsequent BMP inhibition and FGF acti...

example 2

Modeling Otic Neurogenesis

[0112]In day-12 aggregates, we observed patches of PAX8+ PAX2+ epithelia reminiscent of otic placodes; therefore, we assayed for culture conditions promoting otic vesicle formation. Under control conditions (DMSO), we did not observe vesicle formation (data not shown). Since otic induction is dependent on Wnt signaling, we treated 12-day aggregates with a potent GSK3β inhibitor and known Wnt signaling agonist27, CHIR99021 (CHIR), between days 12-18. Under these conditions, PAX8+ PAX2+ SOX10+ vesicles evaginate from the outer epithelium (˜10-30 vesicles per aggregate; see FIGS. 4A-4R). Remarkably, Islet1+ (ISL1+) neuroblasts appear to delaminate from the otic vesicles (FIGS. 16A-16B). We also see neuroblasts in DMSO-treated aggregates and in the non-otic interior of CHIR-treated aggregates (FIG. 16A, arrowheads). Thus, we hypothesized that CHIR-treated aggregates yield a mixture of otic (i.e. vesicle-derived) and epibranchial (i.e. epithelium-derived) neuron...

example 3

Exemplary Protocol for In Vitro Production of Inner Ear Organoids

[0115]This example describes a protocol for inducing formation of non-neural ectoderm and inner ear sensory tissue from human pluripotent stem cells. As described in greater detail in the following paragraphs, pluripotent stem cells aggregates were cultured in a medium containing Matrigel, which is rich in basement membrane proteins, to induce ectoderm development and production of ectoderm epithelium on the aggregate surface. We then used a combined treatment of bone morphogenetic protein-4 (BMP4) and a transforming growth factor beta (TGFβ) inhibitor such as the small molecule SB-431542 (“SB”) to promote non-neural differentiation in the epithelium. To further initiate inner ear induction, BMP signaling was inhibited and fibroblast growth factor (FGF) signaling was activated using recombinant FGF-2 approximately 24 hours after the initial BMP4 and SB treatment. Remarkably, the combined treatment protocol initiated se...

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Abstract

Provided herein are methods for directing differentiation of human pluripotent stem cells into inner ear sensory epithelia and sensory neurons. More particularly, provided herein are methods for obtaining three-dimensional cultures comprising human pluripotent stem cell-derived pre-otic epithelium, otic vesicles, and inner ear sensory epithelia containing hair cells, sensory neurons, and supporting cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 244,568, filed Oct. 21, 2015, which is incorporated herein by reference as if set forth in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR[0002]DEVELOPMENT[0003]This invention was made with government support under DC015624, DC012617, and DC013294 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION[0004]Provided herein are methods for directing differentiation of human pluripotent stem cells into inner ear sensory epithelia and sensory neurons. More particularly, provided herein are methods for obtaining three-dimensional cultures comprising human pluripotent stem cell-derived pre-otic epithelium, otic vesicles, and inner ear sensory epithelia containing hair cells and supporting cells as well as sensory neurons innervating the sensory epithelia.BACKGROUND OF THE INVENTION[0005]Nea...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0793C12N5/0735
CPCC12N5/062C12N5/0606C12N2501/115C12N2501/15C12N2501/155C12N2501/415C12N2506/02C12N2533/52C12N2533/90C12N5/0607
Inventor KOEHLER, KARL R.HASHINO, ERI
Owner INDIANA UNIV RES & TECH CORP
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