# US 10,308,911 B2 · Methods for generating the inner ear and other cranial placode-derived tissues using pluripotent stem cells

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- Number: US 10,308,911 B2
- Document ID: US10308911B2
- Status: Granted
- Kind: B2
- Application number: 15/446,552
- Filed: 2017-03-01
- Published: 2017-08-24
- Granted: 2019-06-04
- Earliest claimed: 2012-05-04
- Assignees: Indiana University Research and Technology Corporation
- Applicants: Indiana University Research & Technology Corporation
- Inventors: Eri Hashino; Gerry Oxford; Karl R. Koehler
- Web page: https://patentgrep.com/patent/US10308911B2

## CPC

- C12N 5/0627: {Hair cells}
- C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain}
- G01N 33/5044: {involving specific cell types}
- G01N 33/5073: {Stem cells}
- C12N 2501/113: Acidic fibroblast growth factor (aFGF, FGF-1)
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2506/02: from embryonic cells
- C12N 2506/03: from non-embryonic pluripotent stem cells
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Disclosed herein are methods and compositions for generating cultures and isolated cell populations containing preplacodal ectoderms cells, otic placode cells, and inner ear sensory hair cells derived from pluripotent cells by modulating TGFβ, BMP, and FGF signaling pathways under defined culture conditions. Also described are methods for obtaining non-otic placodal tissues from pluripotent stem cells. Methods for identifying agents that induce or enhance differentiation and generation of hair cells are also disclosed. Methods for identifying cytoprotective agents for hair cells are also described.

## Claims

### Claim 1

A method for producing preplacodal ectoderm cells, the method comprising the steps of:
(i) culturing pluripotent stem cells selected from the group consisting of human pluripotent stem cells and mouse pluripotent stem cells for between 1 to 2 days under conditions that result in formation of embryoid bodies from the cultured pluripotent stem cells;
(ii) adding laminin and one or more extracellular matrix proteins selected from entactin, type IV collagen, fibronectin, vitronectin, and a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells to the cultured embryoid bodies formed in step (i);
(iii) culturing the embryoid bodies of step (ii) for about 2 days to about 3 days in the presence of BMP2, BMP4, or BMP7 and a TGFβ inhibitor to form non-neural ectoderm; and
(iv) culturing the non-neural ectoderm formed in (iii) for about 3 days to about 5 days in the absence of the BMP2, BMP4, or BMP7 and the TGFβ inhibitor, and in the presence of an exogenous FGF and a BMP inhibitor, in floating culture, to generate a cell population comprising preplacodal ectoderm cells, wherein human preplacodal ectoderm cells express one or more of SIX1/4, EYA1/2, IRX1/2/3, AP2, DLX3/5/6, GATA2/3, FOXI1/3 and E-cadherin or wherein mouse preplacodal ectoderm cells express at least one of Dlx3, Dlx5, Pax2, Pax8 and Eya1.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (iii) comprises culturing the embryoid bodies in the presence of BMP4.

### Claim 3 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are human pluripotent stem cells.

### Claim 4 (depends on 3)

The method of claim 3, wherein the BMP2, BMP4, or BMP7, and the TGFβ inhibitor are added to the cultured embryoid bodies on day 1 of culturing step (iii).

### Claim 5 (depends on 1)

The method of claim 1, wherein the one or more extracellular matrix proteins are added to the cultured embryoid bodies on day 1 of culturing step (iii).

### Claim 6 (depends on 1)

The method of claim 1, wherein the one or more extracellular matrix proteins comprise laminin or a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

### Claim 7 (depends on 6)

The method of claim 6, wherein the one or more extracellular matrix proteins comprise entactin.

### Claim 8

A method for producing otic placode cells, the method comprising the steps of:
(i) culturing pluripotent stem cells selected from the group consisting of human pluripotent stem cells and mouse pluripotent stem cells for between 1 to 2 days under conditions that result in formation of embryoid bodies from the cultured pluripotent stem cells;
(ii) adding laminin and one or more extracellular matrix proteins selected from entactin, type IV collagen, fibronectin, vitronectin, and a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells to the embryoid bodies formed in step (i);
(iii) culturing the embryoid bodies of step (ii) for about 2 days to about 3 days in the presence of BMP2, BMP4, or BMP7, and a TGFβ inhibitor to form non-neural ectoderm;
(iv) culturing the non-neural ectoderm formed in (iii) for about 3 days to about 5 days in the absence of a BMP and the TGFβ inhibitor, and in the presence of an exogenous FGF and a BMP inhibitor, in floating culture, to generate a cell population comprising preplacodal ectoderm cells, wherein human preplacodal ectoderm cells express one or more of SIX1/4, EYA1/2, IRX1/2/3, AP2, DLX3/5/6, GATA2/3, FOXI1/3 and E-cadherin, or wherein mouse preplacodal ectoderm cells express at least one of Dlx3, Dlx5, Pax2, Pax8 and Eya1; and
(v) culturing the preplacodal ectoderm cells formed in (iv), in floating culture, in the absence of the exogenous FGF and BMP inhibitor to obtain a cell population comprising otic placode cells, wherein human otic placode cells express one or more of Pax2, Pax8 and ECAD, or wherein mouse otic placode cells express one or more of Pax2, Pax8 and Sox2.

### Claim 9 (depends on 8)

The method of claim 8, wherein the preplacodal ectoderm cells in the floating culture of (v) are cultured in the presence of an activator of Wnt/β-catenin signaling.

### Claim 10 (depends on 9)

The method of claim 9, wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.

### Claim 11 (depends on 8)

The method of claim 8, wherein the exogenous FGF is any of FGF1-FGF23.

### Claim 12 (depends on 8)

The method of claim 8, wherein the pluripotent stem cells are human pluripotent stem cells.

### Claim 13 (depends on 12)

The method of claim 12, wherein the BMP2, BMP4, or BMP7 and the TGFβ inhibitor are added on day 1 of culturing step (iii), and the exogenous FGF and the BMP inhibitor are added on day 1 of culturing step (iv).

### Claim 14

A method for producing inner ear sensory hair cells, the method comprising the steps of:
(i) culturing pluripotent stem cells selected from the group consisting of human pluripotent stem cells and mouse pluripotent stem cells for between 1 to 2 days under conditions that result in formation of embryoid bodies from the cultured pluripotent stem cells;
(ii) adding laminin and one or more extracellular matrix proteins selected from entactin, type IV collagen, fibronectin, vitronectin, and a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells to the embryoid bodies formed in step (i);
(iii) culturing the embryoid bodies of step (ii) for about 2 days to about 3 days in the presence of BMP2, BMP4, or BMP7 and a TGFβ inhibitor to form non-neural ectoderm;
(iv) culturing the non-neural ectoderm formed in (iii) for about 3 days to about 5 days in the absence of a BMP and the TGFβ inhibitor, and in the presence of an exogenous FGF and a BMP inhibitor, in floating culture, to generate preplacodal ectoderm, wherein human preplacodal ectoderm cells express one or more of SIX1/4, EYA1/2, IRX1/2/3, AP2, DLX3/5/6, GATA2/3, FOXI1/3 and E-cadherin, or wherein mouse preplacodal ectoderm cells express at least one of Dlx3, Dlx5, Pax2, Pax8 and Eya1;
(v) culturing the preplacodal ectoderm cells formed in (iv), in floating culture, in the absence of the exogenous FGF and BMP inhibitor to obtain otic placode cells; and
(vi) culturing the otic placode cells formed in (v) in floating culture in N2 medium comprising one or more extracellular matrix proteins for a time sufficient to form inner ear sensory hair cells, wherein human hair cells express MYO7A, BRN3C, and ATOH1, or wherein mouse hair cells express MYO7A.

### Claim 15 (depends on 14)

The method of claim 14, wherein the exogenous FGF is FGF2.

### Claim 16 (depends on 14)

The method of claim 14, further comprising culturing the preplacodal ectoderm in (v) in the presence of an activator of Wnt/β-catenin signaling.

### Claim 17 (depends on 16)

The method of claim 16, wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.

### Claim 18 (depends on 14)

The method of claim 14, wherein the inner ear sensory hair cells comprise Type II vestibular hair cells.

### Claim 19 (depends on 14)

The method of claim 14, wherein the pluripotent stem cells are human pluripotent stem cells.