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Methods of generating human inner ear sensory epithelia and sensory neurons

一种上皮细胞、抑制剂的技术，应用在生物化学设备和方法、神经系统细胞、微生物等方向，能够解决没有听力损失等问题

Active Publication Date: 2018-07-17
INDIANA UNIV RES & TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Nearly 500 million people worldwide suffer from hearing loss, but there is no pharmacological, genetic or cellular therapy for 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

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 - Generation of Inner Ear Organoids with Functional Hair Cells from Human Pluripotent Stem Cells

[0057] The human inner ear contains approximately 20,000 sensory hair cells that detect sound and movement via mechanosensitive stereocilia bundles 1 . Genetic mutations or environmental damage (such as loud noise) can irreparably damage these hair cells, causing dizziness or hearing loss 2，3 . We previously showed how to generate inner ear organoids from mouse pluripotent stem cells (PSCs) using timed manipulation of FGF, TGFβ, BMP, and Wnt signaling pathways in a 3D culture system 4-6 . We have shown that mouse inner ear organoids contain sensory hair cells that are structurally and functionally similar to native vestibular hair cells in the mouse inner ear 7 . Furthermore, our past findings support a working model of otic-induced signaling dynamics in which activation of BMP signaling and inhibition of TGFβ initially specify non-neuroectoderm and subseq...

Embodiment 2

[0122] Example 2 - Modeling of ear neurogenesis

[0123] In aggregates at day 12, we observed sheets of Pax8+Pax2+ epithelium reminiscent of the ostium; we therefore determined culture conditions that promote otic vesicle formation. Under control conditions (DMSO), we did not observe vesicle formation (data not shown). As otic induction is dependent on Wnt signaling, we treated day 12 aggregates on days 12–18 with a potent GSK3β inhibitor and a known agonist of Wnt signaling27, CHIR99021 (CHIR). Under these conditions, PAX8+PAX2+SOX10+ vesicles are everted from the outer epithelium (approximately 10-30 vesicles per aggregate; see Figures 4A-4R ). Notably, Islet1+ (ISL1+) neuroblasts appeared to delaminate from the otic bulla (Fig. 16A-16B). We also saw neuroblasts in DMSO-treated aggregates and in the non-ear interior of CHIR-treated aggregates (Fig. 16A, arrows). We therefore hypothesized that CHIR-treated aggregates produced a mixture of otic (ie, vesicle-derived) and...

Embodiment 3

[0126] Example 3 - Exemplary protocol for in vitro generation of inner ear organoids

[0127] This example describes a protocol for the induction of non-neuroectoderm and inner ear sensory tissue from human pluripotent stem cells. As described in more detail in the following paragraphs, pluripotent stem cell aggregates were cultured in media containing Matrigel rich in basement membrane protein to induce ectoderm development and generate ectoderm epithelium on the surface of the aggregates. We then used combined treatment of bone morphogenetic protein-4 (BMP4) and transforming growth factor beta (TGFβ) inhibitors such as the small molecule SB-431542 ("SB") to promote non-neural differentiation in the epithelium. To further initiate inner ear induction, recombinant FGF-2 was used to inhibit BMP signaling and activate fibroblast growth factor (FGF) signaling approximately 24 hours after initial BMP4 and SB treatment. Notably, the combined treatment regimen initiated the self-...

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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

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 244,568, filed October 21, 2015, which is incorporated herein by reference as if set forth in its entirety. [0003] Statement Regarding Federally Funded Research or Development [0004] This invention was made with government support under grants DC015624, DC012617, and DC013294 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] Provided herein are methods for directing the differentiation of human pluripotent stem cells into inner ear sensory epithelium and sensory neurons. More particularly, provided herein are methods for obtaining a three-dimensional culture comprising: a human pluripotent stem cell-derived preauricular cell epithelium, an ear vesicle, and an inner ear sensory epithelium containing hair cells and supporting cells, and sensory neurons that innervate the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/0793C12N5/0797C12N5/22
CPCC12N2501/115C12N2501/15C12N2501/155C12N2501/415C12N2506/02C12N2533/52C12N2533/90C12N5/062C12N5/0607C12N5/0606
Inventor 卡尔·R·克勒埃里·哈希诺
Owner INDIANA UNIV RES & TECH CORP
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