# US 2019/0169576 A1 · Generation of midbrain-specific organoids from human pluripotent stem cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0169576 A1
- Document ID: US20190169576A1
- Status: Published application
- Kind: A1
- Application number: 16/085,553
- Filed: 2017-03-14
- Published: 2019-06-06
- Earliest claimed: 2016-03-14
- Assignees: Agency for Science, Technology and Research; National University of Singapore
- Applicants: Agency for Science, Technology and Research; National University of Singapore
- Inventors: Junghyun JO; Huck Hui NG; Hyunsoo Shawn JE
- Web page: https://patentgrep.com/patent/US20190169576A1

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0619: {Neurons}
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/33: Insulin
- C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/02: from embryonic cells
- C12N 2513/00: 3D culture
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/56: Fibrin; Thrombin
- C12N 2533/72: Chitin, chitosan
- C12N 2533/74: Alginate
- C12N 2533/76: Agarose, agar-agar
- C12N 2533/78: Cellulose
- 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)

## Abstract

The present disclosure provides a method of deriving and maintaining a midbrain-like organoid in culture, comprising (a) culturing pluripotent stem cells to obtain neuronal lineage embryoid bodies; (b) culturing the neuronal lineage embryoid bodies from (a) to obtain midbrain regionalized tissues; (c) embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix to obtain neuroepithelial tissues; and (c) culturing the neuroepithelial tissues from (c) to obtain a midbrain-like organoid. Also disclosed herein are culture media suitable for deriving and maintaining neuronal lineage embryoid bodies comprising (a) TGF-β Inhibitor and/or SMAD2/3 inhibitors; and (b) WNT-signaling activator; culture media suitable for deriving and maintaining midbrain regionalized tissues comprising (a) TGF-β Inhibitor and/or SMAD2/3 inhibitors; (b) WNT-signaling activator; (c) hedgehog signaling protein; and (d) fibroblast growth factor; culture media suitable for deriving and maintaining neuroepithelial tissues comprising (a) hedgehog signaling protein; and (b) fibroblast growth factor and culture media suitable for deriving and maintaining a midbrain-like organoid comprising (a) neurotrophin factor; (b) ascorbic acid; and (c) activator of cAMP-dependent pathway.

## Claims

### Claim at position 1

1-57. (canceled)

### Claim 58

A method of deriving and maintaining a midbrain-like organoid comprising:
(a) culturing pluripotent stem cells to obtain neuronal lineage embryoid bodies;
(b) culturing the neuronal lineage embryoid bodies from (a) to obtain midbrain regionalized tissues;
(c) embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix to obtain developing neuroepithelial tissues; and
(d) culturing the neuroepithelial tissues from (c) to obtain a midbrain-like organoid.

### Claim 59 (depends on 58)

The method of claim 58, wherein the pluripotent stem cells in operation (a) are cultured in a first cell culture medium, wherein the first cell culture medium comprises:
(i) TGF-β inhibitor and/or SMAD2/3 inhibitor; and
(ii) WNT-signaling activator;
optionally wherein the neuronal lineage embryoid bodies of operation (b) are cultured in a second cell culture medium, wherein the second cell culture medium comprises:
(iii) TGF-β inhibitor and/or SMAD2/3 inhibitor;
(iv) WNT-signaling activator;
(v) hedgehog signaling protein; and
(vi) fibroblast growth factor;
optionally wherein the midbrain regionalized tissues of operation (c) are cultured in a third cell culture medium, wherein the third cell culture medium comprises:
(vii) hedgehog signaling protein; and
(viii) fibroblast growth factor.

### Claim 60 (depends on 58)

The method of claim 58, wherein the extracellular matrix promotes cell differentiation and/or can maintain three-dimensional culture and/or promotes the development of complex tissue and is made of a material selected from the group consisting of matrigel, gelatin, methylcellulose, collagen, alginate, alginate beads, agarose, fibrin, fibrin glue, fibrinogen, blood plasma fibrin beads, whole plasma or components thereof, laminins, fibronectins, proteoglycans, HSP, chitosan, heparin, other synthetic polymer or polymer scaffolds and solid support materials.

### Claim 61 (depends on 58)

The method of claim 58, wherein the neuroepithelial tissues of operation (d) are cultured in a fourth cell culture medium, wherein the fourth cell culture medium comprises:
(a) neurotrophic factor;
(b) ascorbic acid; and
(c) activator of cAMP-dependent pathways.

### Claim 62 (depends on 59)

The method of claim 59, wherein the TGF-β inhibitor and/or SMAD2/3 inhibitor is selected from the group consisting of 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide (SB 431542), (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide (ROCK inhibitor Y27632), and derivatives thereof, optionally wherein the WNT-signaling activator is a GSK3 inhibitor,
optionally wherein the GSK3 inhibitor is CHIR-99021 6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile, or derivatives thereof.

### Claim 63 (depends on 59)

The method of claim 59, wherein the hedgehog signaling protein is selected from the group consisting of: Desert hedgehog homolog (DHH), Indian hedgehog homolog (IHH) and Sonic hedgehog (SHH), optionally wherein the fibroblast growth factor is FGF8.

### Claim 64 (depends on 61)

The method of claim 61, wherein the neurotrophic factor is selected from the group consisting of neurotrophin, glial cell-line derived neurotrophic factor family ligand (GFL), and neuropoietic cytokine, optionally wherein the neurotrophin is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4), optionally wherein the GFL is selected from the group consisting of glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN).

### Claim 65 (depends on 61)

The method of claim 61, wherein the activator of cAMP-dependent pathways is dibutyryl-cAMP(dbCAMP).

### Claim 66 (depends on 59)

The method of claim 59, wherein the first cell culture medium further comprises a basal cell growth medium at a percentage of 40% to 50%, a basal embryonic neuronal cell growth medium at a percentage of 40% to 50%, a glutamine supplement at a percentage of 0.5% to 3%, a non-essential amino acid (NEAA) supplement at a percentage of 0.5% to 3%, antibiotics at a percentage of 0.5% to 3%, a reducing agent at a percentage of 0.05% to 0.5%, a supplement for expansion of undifferentiated cells, at a percentage of 0.5% to 3%, a supplement for the maintenance of neurons, at a percentage of 0.5% to 5% and a supplement for cell proliferation, at a concentration of 0.1 to 5 μg/ml, optionally wherein the second cell culture medium further comprises a basal cell growth medium at a percentage of 40% to 50%, a basal embryonic neuronal cell growth medium at a percentage of 40% to 50%, a glutamine supplement at a percentage of 0.5% to 3%, a non-essential amino acid (NEAA) supplement at a percentage of 0.5% to 3%, antibiotics at a percentage of 0.5% to 3%, a reducing agent at a percentage of 0.05% to 0.5%, a supplement for expansion of undifferentiated cells, at a percentage of 0.5% to 3%, a supplement for the maintenance of neurons, at a percentage of 0.5% to 5% and a supplement for cell proliferation, at a concentration of 0.1 to 5 μg/ml, optionally wherein the third cell culture medium further comprises a basal embryonic neuronal cell growth medium at a percentage of 90% to 95%, a glutamine supplement at a percentage of 0.5% to 3%, a non-essential amino acid (NEAA) supplement at a percentage of 0.5% to 3%, antibiotics at a percentage of 0.5% to 3%, a reducing agent at a percentage of 0.05% to 0.5%, a supplement for expansion of undifferentiated cells, at a percentage of 0.5% to 3%, a supplement for the maintenance of neurons, at a percentage of 0.5% to 5%, a growth supplement at a concentration of 0.5 to 10 μg/ml, and a growth enhancer of stem cells at the concentration of 50 to 500 ng/ml.

### Claim 67 (depends on 61)

The method of claim 61, wherein the fourth cell culture medium further comprises a basal embryonic neuronal cell growth medium at a percentage of 90% to 95%, a glutamine supplement at a percentage of 0.5% to 3%, a non-essential amino acid (NEAA) supplement at a percentage of 0.5% to 3%, antibiotics at a percentage of 0.5% to 3%, a reducing agent at a percentage of 0.05% to 0.5%, a supplement for expansion of undifferentiated cells, at a percentage of 0.5% to 3%, a supplement for the maintenance of neurons, at a percentage of 0.5% to 5%, a growth supplement at a concentration of 0.5 to 10 μg/ml, and a growth enhancer of stem cells at the concentration of 50 to 500 ng/ml.

### Claim 68 (depends on 66)

The method of claim 66, wherein the basal cell growth medium is Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient F-12.

### Claim 69 (depends on 66)

The method of claim 66, wherein the basal embryonic neuronal cell growth medium is Neurobasal medium, 0 optionally wherein the glutamine supplement is GlutaMAX (L-alanyl-L-glutamine dipeptide), optionally wherein the antibiotics are penicillin/streptomycin, optionally wherein the reducing agent is R-mercaptoethanol, optionally wherein the supplement for expansion of undifferentiated cells is N-2 Supplement, optionally wherein the supplement for the maintenance of neurons is B27 without vitamin-A.

### Claim 70 (depends on 66)

The method of claim 66, wherein the supplement for cell proliferation is heparin, optionally wherein the growth supplement is insulin, optionally wherein the growth enhancer of stem cells is laminin.

### Claim 71 (depends on 58)

The method of claim 58, wherein the method is a three-dimensional method.

### Claim 72 (depends on 58)

The method of claim 58, wherein culturing under (a) is for 3 to 5 days, optionally wherein culturing under (b) is for 3 to 5 days, optionally wherein culturing under (c) is for 1 to 2 days.

### Claim 73

An isolated midbrain-like organoid obtained by a method of deriving and maintaining a midbrain-like organoid comprising:
(a) culturing pluripotent stem cells to obtain neuronal lineage embryoid bodies;
(b) culturing the neuronal lineage embryoid bodies from (a) to obtain midbrain regionalized tissues;
(c) embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix to obtain developing neuroepithelial tissues; and
(d) culturing the neuroepithelial tissues from (c) to obtain a midbrain-like organoid.

### Claim 74 (depends on 73)

The isolated midbrain-like organoid of claim 73 comprising midbrain specific cells.