# US 11,898,163 B2 · Method for producing dopaminergic neuron progenitor cell

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

- Number: US 11,898,163 B2
- Document ID: US11898163B2
- Status: Granted
- Kind: B2
- Application number: 16/095,092
- Filed: 2017-04-24
- Published: 2019-04-18
- Granted: 2024-02-13
- Earliest claimed: 2016-04-22
- Assignees: Kyoto University; Sumitomo Pharma Co., Ltd.
- Applicants: Kyoto University; Sumitomo Pharma Co., Ltd.
- Inventors: Jun Takahashi; Daisuke Doi; Kenji Yoshida; Atsushi Kuwahara; Masayo Takahashi
- Web page: https://patentgrep.com/patent/US11898163B2

## CPC

- C12N 5/0619: {Neurons}
- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61P 25/16: Anti-Parkinson drugs
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 2500/38: Vitamins
- 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 2501/13: 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 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/45: from artificially induced 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

A cell population comprising Corin- and/or Lrtm1-positive cells was produced by the following steps (1) and (2), from which Corin positive and/or Lrtm1 positive cells are collected using a substance that binds to Corin and/or a substance that binds to Lrtm1, and dopaminergic neuron progenitor cells are produced by performing suspension culture of the Corin positive and/or Lrtm1 positive cells in a culture solution containing one or more nutritional factors: (1) a step of performing adhesion culture of pluripotent stem cells in a medium for maintaining undifferentiated state containing a Sonic hedgehog (SHH) signal stimulant, and an undifferentiated state-maintaining factor in the absence of feeder cells but in the presence of an extracellular matrix, and (2) a step of culturing the cell population obtained in the step (1) in a culture solution containing one or more differentiation-inducing factors.

## Claims

### Claim 1

A method for producing a cell population comprising Corin positive and/or Lrtm1 positive cells capable of differentiating into dopaminergic neuron progenitor cells, said method comprising:
(i) culturing human pluripotent stein cells (PSC) on a Laminin-511 E8 coated plate in a medium comprising a Sonic hedgehog (SHH) signal stimulant and bFGF for 6 to 48 hours to precondition and maintain the stem cells in an undifferentiated state,
(ii) culturing the pluripotent stem cells obtained in step (i) the Laminin-511 E8 in a medium containing a BMP inhibitor and a TGF-beta inhibitor for at least one day;
(iii) replacing the medium from (ii) with a medium containing a BMP inhibitor, a TGF-beta inhibitor, a SHH signal-stimulating agent, and FGF8 and further culturing the cells for at least one day;
(iv) replacing the medium from (iii) with a medium containing a BMP inhibitor, a TGF-beta inhibitor, a SHH signal-stimulating agent, FGF8, and a GSK3β inhibitor and further culturing the cells for at least one day;
(v) replacing the medium from (iv) with a medium containing a BMP inhibitor and a GSK3β inhibitor and further culturing the cells for at least one day to produce a population of cells containing higher percentage of Corin- and/or leucine-rich repeats and transmembrane domains 1 (Lrtm1)-positive cells as compared to a population of Corin- and/or Lrtm1 positive cells produced from PSC that are not pre-conditioned in a medium comprising SHH signal-stimulating agent and bFGF, and;
wherein steps (ii)-(v) are performed for 10-21 days.

### Claim 2 (depends on 1)

The method of claim 1 further comprising:
(vii) collecting Corin- and/or leucine-rich repeats and transmembrane domains 1 (Lrtm1)-positive cells from the cells obtained in (vi) using an antibody which binds to Corin and/or an antibody which binds to Lrtm1; and
(viii) culturing the cells obtained in (vii) in suspension in a medium containing a neurotrophic factor for at least 7 days to produce dopaminergic neuron progenitor cells.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the SHH signal stimulant is SAG (N-methyl-N′-(3-pyridinylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), shh protein or a fragment thereof, Purmorphamine, or a combination thereof.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the medium for maintaining undifferentiated state in (i) further comprises a TGFβ inhibitor or a BMP inhibitor.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the TGFβ inhibitor is A83-01, and the BMP inhibitor is LDN 193189.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the pluripotent stem cells are cultured for a period of about 18 to 28 hours in (i).

### Claim 7 (depends on 1)

The method according to claim 1, wherein the medium in (ii) further comprises a ROCK inhibitor.

### Claim 8

A method for producing dopaminergic neuron progenitor cells comprising:
(i) culturing human pluripotent stem cells (PSC) on a Laminin-511 E8 coated plate in a medium comprising a Sonic hedgehog (SHH) signal stimulating agent and bFGF for 6 to 48 hours to precondition and maintain the stem cells in an undifferentiated state,
(ii) culturing the pluripotent stern cells obtained in step (i) on the Laminin-511 E8 in a medium containing a BMP inhibitor and a TGF-beta inhibitor for at least one day:
(iii) replacing the medium from (ii) with a medium containing a BMP inhibitor, a TGF-beta inhibitor, a SHH signal-stimulating agent, and FGF8 and further culturing the cells for at least one day:
(iv) replacing the medium from (iii) with a medium containing a BMP inhibitor, a TGF-beta inhibitor, a SHH signal-stimulating agent, FGF8, and a GSK3β inhibitor and further culturing the cells for at least one day;
(v) replacing the medium from (iv) with a medium containing a BMP inhibitor and a GSK3β inhibitor and further culturing the cells for at least one day to produce a population of cells containing higher percentage of Corin- and/or leucine-rich repeats and transmembrane domains 1 (Lrtm1)-positive cells as compared to a population of Corin- and/or Lrtm1 positive cells produced from PSC that are not pre-conditioned in a medium comprising SHH signal-stimulating agent and bFGF; wherein steps (ii)-(v) are performed for 10-21 days; and
(vi) collecting Corin positive and/or Lrtm1 positive cells and culturing the Corin positive and/or Lrtm1 positive cells in a suspension culture in a medium comprising one or more neurotrophic factors to produce dopaminergic neuron progenitor cells.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the substance that binds to Corin or the substance that binds to Lrtm1 is an antibody or aptamer that binds to Corin or Lrtm1.

### Claim 10 (depends on 8)

The method according to claim 8, wherein the neurotrophic factors are BDNF and GDNF.

### Claim 11 (depends on 8)

The method according to claim 8, wherein the medium comprising a neurotrophic factor further comprises B27 Supplement, ascorbic acid, and a cAMP analog.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the cAMP analogue is Dibutyryl cyclic AMP.