# US 2020/0399601 A1 · Differentiation of macrophages from pluripotent stem cells

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- Number: US 2020/0399601 A1
- Document ID: US20200399601A1
- Status: Published application
- Kind: A1
- Application number: 16/916,096
- Filed: 2020-06-29
- Published: 2020-12-24
- Earliest claimed: 2015-01-16
- Assignees: Agency for Science, Technology and Research
- Applicants: Agency for Science, Technology and Research
- Inventors: Hwei-In Shawn LIM; Tara Huber; Florent Ginhoux
- Web page: https://patentgrep.com/patent/US20200399601A1

## CPC

- G01N 33/5055: {involving macrophages}
- A61K 40/17: Monocytes; Macrophages
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/24: Antigen-presenting cells [APC]
- A61K 40/416: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance}
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- C12N 5/0645: {Macrophages, e.g. Kuepfer cells in the liver; Monocytes}
- G01N 33/5058: {Neurological cells}
- G01N 33/5073: {Stem cells}
- A61K 2239/31: characterized by the route of administration
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF)
- C12N 2501/2303: Interleukin-3 (IL-3)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- 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)
- 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
- 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 invention relates to a method of culturing primitive macrophages from stem cells. Specifically, the method comprises contacting and incubating stem cells with a serum-free culture media comprising a GSK3 inhibitor to differentiate stem cells into cell of the mesoderm lineage, contacting and incubating cells of the mesoderm lineage with a culture media comprising DKK1 to differentiate the cells into the hematopoietic lineage, maturing the cells of the hematopoietic lineage and contacting and incubating these cells with a culture media comprising M-CSF to drive differentiation into primitive-like macrophages. The invention also relates to a primitive-like macrophage, use of the primitive-like macrophage and a kit when used in the method of the invention.

## Claims

### Claim at position 1

1-37. (canceled)

### Claim 38

A method for producing cells that express a microglial marker from human pluripotent stem cells, wherein the microglial marker is IBA1, the method comprising:
(i) generating primitive macrophages from said human stem cells, wherein the primitive macrophages are generated by:
(a) contacting and incubating said stem cells with a serum-free culture media comprising VEGF, BMP4 and a GSK3 inhibitor under normoxic conditions such that said stem cells differentiate into cells of the mesoderm lineage;
(b) contacting and incubating said cells of the mesoderm lineage under hypoxic conditions with a culture media comprising SCF, FGF-2, IL-3, IL-6, VEGF and DKK1 such that the cells of the mesoderm lineage differentiate into cells of the hematopoietic cell lineage;
(c) culturing said cells of the hematopoietic cell lineage with a culture media comprising SCF, FGF-2, IL-3 and IL-6 under normoxic conditions such that the cells of the hematopoietic cell lineage mature;
(d) contacting and incubating said mature cells of the hematopoietic cell lineage with a culture media comprising M-CSF under normoxic conditions such that said hematopoietic cells differentiate into primitive macrophages that express CD163, CD11b, HLA-DR, IBA-1, CX3CR1 and CD14;
(ii) generating neurons from said human stem cells, wherein the neurons are generated by:
(e) contacting and incubating said stem cells with neural progenitor cell (NPC) medium for up to 7 days such that said stem cells differentiate to neural precursor cells;
(f) contacting and incubating the neural precursor cells from operation (e) with a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor;
(g) contacting and incubating the cells from operation (f) with NPC media comprising epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF);
(h) contacting and incubating the cells from operation (g) with neuronal differentiation media such that the cells from operation (g) generate neurons; and
(iii) contacting and incubating the primitive macrophages from (i) with the neurons from (ii) for up to 21 days such that the neurons from (ii) generate the cells expressing the microglial marker.

### Claim 39 (depends on 38)

The method of claim 38, wherein the neural precursor cells from operation (e) are contacted and incubated with 10 μM ROCK inhibitor.

### Claim 40 (depends on 38)

The method of claim 38, wherein the cells from operation (f) are contacted and incubated with NPC media comprising 20 ng/ml EGF and 20 ng/ml bFGF.

### Claim 41 (depends on 38)

The method of claim 38, wherein the cells expressing the microglial marker are used in a method for (i) developing in vitro disease models, wherein the disease is a neurodegenerative disease; or (ii) for screening compounds to treat a disease, wherein the disease is a neurodegenerative disease.

### Claim 42

A cell expressing a microglial marker IBA1 obtained by a method comprising:
(i) generating primitive macrophages from said human stem cells, wherein the primitive macrophages are generated by:
(a) contacting and incubating said stem cells with a serum-free culture media comprising VEGF, BMP4 and a GSK3 inhibitor under normoxic conditions such that said stem cells differentiate into cells of the mesoderm lineage;
(b) contacting and incubating said cells of the mesoderm lineage under hypoxic conditions with a culture media comprising SCF, FGF-2, IL-3, IL-6, VEGF and DKK1 such that the cells of the mesoderm lineage differentiate into cells of the hematopoietic cell lineage;
(c) culturing said cells of the hematopoietic cell lineage with a culture media comprising SCF, FGF-2, IL-3 and IL-6 under normoxic conditions such that the cells of the hematopoietic cell lineage mature;
(d) contacting and incubating said mature cells of the hematopoietic cell lineage with a culture media comprising M-CSF under normoxic conditions such that said hematopoietic cells differentiate into primitive macrophages that express CD163, CD11b, HLA-DR, IBA-1, CX3CR1 and CD14;
(ii) generating neurons from said human stem cells, wherein the neurons are generated by:
(e) contacting and incubating said stem cells with neural progenitor cell (NPC) medium for up to 7 days such that said stem cells differentiate to neural precursor cells;
(f) contacting and incubating the neural precursor cells from operation (e) with a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor;
(g) contacting and incubating the cells from operation (f) with NPC media comprising epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF);
(h) contacting and incubating the cells from operation (g) with neuronal differentiation media such that the cells from operation (g) generate neurons; and
(iii) contacting and incubating the primitive macrophages from (i) with the neurons from (ii) for up to 21 days such that the neurons from (ii) generate the cells expressing the microglial marker.