# US 11,999,972 B2 · Fibronectin fragment to be used for stem cell production

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- Number: US 11,999,972 B2
- Document ID: US11999972B2
- Status: Granted
- Kind: B2
- Application number: 16/321,112
- Filed: 2017-07-28
- Published: 2021-08-19
- Granted: 2024-06-04
- Earliest claimed: 2016-07-29
- Assignees: Takara Bio Inc.
- Applicants: Takara Bio Inc.
- Inventors: Tomomi Otsuji; Toshikazu Nishie; Risa Kato; Sachiko Okamoto; Tatsuji Enoki; Junichi Mineno
- Web page: https://patentgrep.com/patent/US11999972B2

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K 17/00: Carrier-bound or immobilised peptides (carrier-bound or immobilised enzymes C12N11/00); Preparation thereof
- C12M 25/06: {Plates; Walls; Drawers; Multilayer plates}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0619: {Neurons}
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12N 2510/00: Genetically modified cells
- C12N 2533/52: Fibronectin; Laminin
- 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)
- 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)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

Stem cells can be efficiently proliferated by culturing the stem cells in the presence of a novel recombinant fibronectin fragment.

## Claims

### Claim 1

A recombinant polypeptide comprising in the same molecule:
(a) a polypeptide comprising three repeats selected from the group consisting of human fibronectin III-1 to 7 repeats wherein the III-1 to 7 repeats comprise the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, and 7 respectively, or a variant of the polypeptide comprising three repeats selected from the group consisting of human fibronectin III-1 to 7 repeats wherein the III-1 to 7 repeats comprise an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, and 7 respectively;
(b) a polypeptide comprising human fibronectin III-8 to 10 repeats comprising the amino acid sequences of SEQ ID NO: 8, 9 and 10 respectively, or a variant of the polypeptide comprising human fibronectin III-8 to 10 repeats comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 8, 9 and 10 respectively; and
(c) a polypeptide comprising human fibronectin III-12 to 14 repeats comprising the amino acid sequences of SEQ ID NO: 12, 13 and 14 respectively, or a variant of the polypeptide comprising human fibronectin III-12 to 14 repeats comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12, 13 and 14 respectively;
wherein the recombinant polypeptide retains a function of maintaining an undifferentiated state of stem cells, and
wherein the molecular weight of the recombinant polypeptide is 100 kDa or less.

### Claim 2 (depends on 1)

The recombinant polypeptide according to claim 1, wherein the polypeptide of (a) is a polypeptide comprising human fibronectin III-1 to 3 repeats comprising the amino acid sequences of SEQ ID NO: 1, 2 and 3 respectively or a polypeptide comprising human fibronectin III-4 to 6 repeats comprising the amino acid sequences of SEQ ID NO: 4, 5 and 6 respectively, or the variant of (a) is a variant of the polypeptide comprising human fibronectin III-1 to 3 repeats comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, 2 and 3 respectively or a variant of the polypeptide comprising human fibronectin III-4 to 6 repeats comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4, 5 and 6 respectively.

### Claim 3 (depends on 1)

The recombinant polypeptide according to claim 1, which comprises an amino acid sequence of SEQ ID NO: 19 or 20 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19 or 20, wherein the molecular weight of the recombinant polypeptide is 100 kDa or less.

### Claim 4 (depends on 1)

A solid phase coated with the recombinant polypeptide according to claim 1.

### Claim 5 (depends on 4)

The solid phase according to claim 4, which is a device for cell culture or a carrier for cell culture coated with the recombinant polypeptide.

### Claim 6 (depends on 4)

The solid phase according to claim 4, which is a dish, a plate, a flask, a bag, a bead, a membrane or a slide glass coated with the recombinant polypeptide.