# US 2013/0157359 A1 · Fgf having enhanced stability

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- Number: US 2013/0157359 A1
- Document ID: US20130157359A1
- Status: Published application
- Kind: A1
- Application number: 13/717,055
- Filed: 2012-12-17
- Published: 2013-06-20
- Earliest claimed: 2011-12-16
- Assignees: Wisconsin Alumni Research Foundation
- Inventors: Guokai Chen; James A. Thomson
- Web page: https://patentgrep.com/patent/US20130157359A1

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 2501/113: Acidic fibroblast growth factor (aFGF, FGF-1)
- 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 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/91: Heparin
- C12N 2506/02: from embryonic cells
- C12N 2506/1307: from adult fibroblasts
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods are provided that exploit thermostable FGF-1 proteins for support of human pluripotent stem cell cultures. Also provided are compositions containing thermostable FGF-1 for culturing of human pluripotent stem cells.

## Claims

### Claim 1

A method for culturing human pluripotent stem cells, comprising the step of culturing a human pluripotent stem cell in a medium comprising at least one thermostable fibroblast growth factor-one (FGF-1) that comprises amino acid substitutions at positions corresponding to positions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2), wherein the medium comprising the thermostable FGF-1 is characterized by an enhanced ability to support pluripotency relative to a medium comprising truncated wild type FGF-1 (SEQ ID NO:2).

### Claim 2 (depends on 1)

The method of claim 1 wherein the amino acid sequence of the thermostable FGF-1 further comprises an amino acid substitution within the heparin binding domain.

### Claim 3 (depends on 2)

The method of claim 2 wherein the position of the amino acid substitution within the heparin binding domain corresponds to position 112 of SEQ ID NO:2.

### Claim 4 (depends on 1)

The method of claim 1, wherein the human pluripotent cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the medium further comprises heparin.

### Claim 6 (depends on 1)

The method of claim 1, wherein the thermostable FGF-1 is provided in the medium at a concentration of less than 40 ng/ml.

### Claim 7 (depends on 1)

The method of claim 1, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:3.

### Claim 8 (depends on 7)

The method of claim 7, wherein the amino acid sequence of the thermostable FGF-1 consists of the amino acid sequence of SEQ ID NO:3.

### Claim 9 (depends on 7)

The method of claim 7, wherein thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:4.

### Claim 10 (depends on 9)

The method of claim 9, wherein the amino acid sequence of the thermostable FGF-1 consists of the amino acid sequence of SEQ ID NO:4.

### Claim 11

A fully-defined medium suitable for culturing human pluripotent cells in an undifferentiated state, the defined medium comprising a thermostable fibroblast growth factor-one (FGF-1) that comprises amino acid substitutions at positions corresponding to positions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2).

### Claim 12 (depends on 11)

The fully-defined medium of claim 11, wherein the amino acid sequence of the thermostable FGF-1 further comprises an amino acid substitution at position 112 of the wild type FGF-1 (SEQ ID NO:2).

### Claim 13 (depends on 11)

The medium of claim 11, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:3.

### Claim 14 (depends on 13)

The medium of claim 13, wherein the amino acid sequence of the thermostable FGF-1 consists of the amino acid sequence of SEQ ID NO:3.

### Claim 14

A composition comprising:
a human pluripotent stem cell and a culture medium comprising a thermostable fibroblast growth factor-one (FGF-1) that comprises amino acid substitutions at positions corresponding to positions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2), and wherein the culture medium comprising the thermostable FGF-2 is suitable for culturing the human pluripotent cell in an undifferentiated state, and is characterized by an enhanced ability to support pluripotency relative to a medium comprising a truncated wild type FGF-1 (SEQ ID NO:2).

### Claim 15 (depends on 13)

The medium of claim 13, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:4.

### Claim 15 (depends on 11)

The medium of claim 11, wherein the thermostable FGF-1 is provided in the medium at a concentration of less than 40 ng/ml.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the amino acid sequence of the thermostable FGF-1 further comprises an amino acid substitution within the heparin binding domain.

### Claim 16 (depends on 15)

The medium of claim 15, wherein the amino acid sequence of the thermostable FGF-1 consists of the amino acid sequence of SEQ ID NO:4.

### Claim 16 (depends on 14)

The composition of claim 14, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:3.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:4.

### Claim 18 (depends on 14)

The composition of claim 14, wherein the thermostable FGF-1 is provided in the medium at a concentration of less than 40 ng/ml.

### Claim 19

A method for differentiating a human pluripotent stem cell into a mesoderm lineage cell, the method comprising the step of:
culturing the human pluripotent stem cell in a medium until the cell expresses mesoderm lineage markers, wherein the medium comprises BMP4 and a thermostable fibroblast growth factor-one (FGF-1) that comprises amino acid substitutions at positions corresponding to positions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2), and wherein the medium comprising the BMP4 and the thermostable FGF-1 is characterized by an enhanced ability to support differentiation of a human pluripotent stem cell into a mesoderm lineage cell relative to a medium comprising BMP4 and a truncated wild type FGF-1 (SEQ ID NO:2).

### Claim 20 (depends on 19)

The method of claim 19, wherein the thermostable FGF-1 further comprises an amino acid substitution at position 112 of the wild type FGF-1 (SEQ ID NO:2).

### Claim 21 (depends on 19)

The method of claim 19, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:3.

### Claim 22 (depends on 21)

The method of claim 21 wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:4.

### Claim 23

A method for reprogramming a human somatic cell into a human pluripotent cell, comprising:
culturing the somatic cell in a medium until the cell expresses markers indicative of a human pluripotent cell, wherein the medium comprises water, salts, amino acids, vitamins, a carbon source, insulin, selenium and at least one thermostable fibroblast growth factor-one (FGF-1) that comprises amino acid substitutions at positions corresponding to positions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2), and wherein the medium comprising the thermostable FGF-1 is characterized by an enhanced ability to support pluripotency relative to a medium comprising a truncated wild type FGF-1 (SEQ ID NO:2).

### Claim 24 (depends on 23)

The method of claim 23, wherein the thermostable FGF-1 further comprises an amino acid substitution at a position corresponding to position 112 of the wild type FGF-1 (SEQ ID NO:2).

### Claim 25 (depends on 23)

The method of claim 23, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:3.

### Claim 26 (depends on 25)

The method of claim 25, wherein the amino acid sequence of the thermostable FGF-1 consists of the amino acid sequence of SEQ ID NO:3.

### Claim 27 (depends on 25)

The method of claim 25, wherein the thermostable FGF-1 comprises the amino acid sequence of SEQ ID NO:4.

### Claim 28 (depends on 27)

The method of claim 27, wherein the amino acid sequence of the thermostable FGF-1 consists of the amino acid sequence of SEQ ID NO:4.

### Claim 29 (depends on 23)

The method of claim 23, wherein the medium is serum-free.

### Claim 30 (depends on 23)

The method of claim 23, wherein the medium further comprises heparin.