# US 9,750,768 B2 · Methods for purifying midbrain dopaminergic neural progenitor cells

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

- Number: US 9,750,768 B2
- Document ID: US9750768B2
- Status: Granted
- Kind: B2
- Application number: 14/118,982
- Filed: 2012-05-18
- Published: 2014-07-17
- Granted: 2017-09-05
- Earliest claimed: 2011-05-20
- Assignees: The McLean Hospital Corporation
- Applicants: Kwang-Soo Kim; Sangmi Chung
- Inventors: Kwang-Soo Kim; Sangmi Chung
- Web page: https://patentgrep.com/patent/US9750768B2

## CPC

- 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
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/40: against enzymes
- C12N 5/0623: {Stem cells}
- C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2501/60: Transcription factors
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are methods of isolating a novel cell population of midbrain dopaminergic neuronal progenitor cells derived from stem cells using a novel combination markers. The cell population may be used for cell therapies for the treatment of Parkinson's disease and as substrates in pharmacological assays.

## Claims

### Claim 1

A method for purifying human midbrain dopaminergic neural progenitor cells comprising:
(a) providing a human neural progenitor cell population comprising midbrain dopaminergic (mDA) neural progenitor cells in cell culture medium;
(b) isolating human neural progenitor cells that express Corin and Frizzled-5 (Fzd5) from the cell population of step (a),
wherein the isolating excludes cells that express Corin but which do not express Fzd5; and
wherein isolating comprises contacting the mDA neural progenitor cells with i) an antibody specific for Corin and ii) an antibody specific for Fzd5; and selecting human neural progenitor cells which are bound by the antibodies of i) and ii),
thereby purifying human mDA neural progenitor cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the neural progenitor cell population is produced by a method comprising:
(i) providing a cell population comprising pluripotent cells in cell culture medium; and
(ii) differentiating at least some of the pluripotent cells into neural progenitor cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the neural progenitor cell population is produced by a method comprising:
(i) culturing a population of pluripotent cells in the presence of leukocyte inhibitory factor (LIF) and serum;
(ii) culturing the cells produced in step (i) in the absence of LIF;
(iii) culturing the cells produced in step (ii) in the absence of serum, and in the presence of insulin, transferin, selenium, and fibronectin; and
(iv) isolating nestin-positive cells produced in step (iii) and culturing the nestin-positive cells in the presence of laminin and one or more growth factors selected from fibroblast growth factor 8 (FGF8) and basic fibroblast growth factor (bFGF), to produce a population of neural progenitor cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the neural progenitor cell population is produced by a method comprising:
(i) culturing a population of pluripotent cells in the presence of one or more growth factors selected from basic fibroblast growth factor (bFGF) and fibroblast growth factor 8 (FGF8);
(ii) culturing the cells produced in step (i) in the presence of sonic hedgehog (SHH) protein and in the absence of serum; and
(iii) culturing the cells obtained in step (ii) in the presence of bFGF to produce a population of neural progenitor cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the neural progenitor cell population is produced by a method comprising:
(i) culturing a population of pluripotent cells in the presence of one or more growth factors selected from fibroblast growth factor 8 (FGF8), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF);
(ii) culturing the cells produced in step (i) in the absence of bFGF; and
(iii) culturing the cells produced in step (ii) in the presence of bFGF to produce a population of neural progenitor cells.

### Claim 6 (depends on 1)

The method of claim 1, wherein the neural progenitor cell population is a substantially homogenous cell population of Nestin-positive cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mDA neural progenitor cells produced in step (b) further express one or more of the markers selected from the group consisting of: forkhead box A2 (FoxA2); orthodenticle homeobox 2 (Otx2); LIM homeobox transcription factor 1, alpha (Lmx1a); LIM homeobox transcription factor 1, beta (Lmx1b); Glast; Vimentin; Nestin; glial fibrillary acidic protein (GFAP); and beta-tubulin.

### Claim 8 (depends on 1)

The method of claim 1, wherein the mDA neural progenitor cells are further differentiated into a cell population of neuronal differentiated (ND) cells by culturing the mDA neural progenitor cells in the absence of growth factors selected from the group consisting of fibroblast growth factor 8 (FGF8) and basic fibroblast growth factor (bFGF).

### Claim 9 (depends on 8)

The method of claim 8, further comprising removing epidermal growth factor (EGF) from the culture medium to produce a population of ND cells.

### Claim 10 (depends on 8)

The method of claim 8, wherein the ND cells express:
(a) one or more markers selected from the group consisting of tyrosine hydroxylase, dopamine active transporter, and dopamine decarboxylase; and
(b) one or more markers selected from the group consisting of orthodenticle homeobox 2 (Otx2); paired-like homeodomain 3 (Pitx3); LIM homeobox transcription factor 1, alpha (Lmx1a); LIM homeobox transcription factor 1, beta (Lmx1b); forkhead box A2 (FoxA2); engrailed homeobox 1 (En-1); and nuclear receptor subfamily 4, group A, member 2 (Nurr1).