# US 2023/0147883 A1 · Method for prodcing cardiomyocytes from induced pluripotent stem cells in an integrated process

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- Number: US 2023/0147883 A1
- Document ID: US20230147883A1
- Status: Published application
- Kind: A1
- Application number: 17/993,869
- Filed: 2022-11-23
- Published: 2023-05-11
- Earliest claimed: 2021-11-28
- Assignees: Fatemeh Etezadi; Fatemeh Jahanpeyma
- Inventors: Fatemeh Etezadi; Koichi Hasegawa; Fatemeh Jahanpeyma
- Web page: https://patentgrep.com/patent/US20230147883A1

## CPC

- C12N 5/0657: {Cardiomyocytes; Heart cells}
- C12N 2500/92: Medium free of human- or animal-derived components
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/33: Insulin
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/00: Supports or coatings for cell culture, characterised by material
- C12N 2533/30: Synthetic polymers
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N 2535/10: Patterned coating
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for generating a population of cardiomyocytes from induced pluripotent stem cells (iPSCs) in an integrated process. The method may comprise seeding the iPSCs on a modified surface of a modified cell culture substrate, culturing the seeded iPSCs on the modified surface of the modified cell culture substrate in an animal component-free culture medium, and differentiating the cultured iPSCs to the population of cardiomyocytes on the modified surface of the modified cell culture substrate. The modified cell culture substrate may comprise a patterned polydimethylsiloxane (PDMS) substrate, a first coating comprising a plurality of polydopamine molecules, and a second coating comprising a plurality of Laminin 511 E8 Fragment (LME8) molecules.

## Claims

### Claim 1

A method for generating a population of cardiomyocytes from induced pluripotent stem cells (iPSCs) in an integrated process, the method comprising:
seeding the iPSCs on a modified surface of a modified cell culture substrate, the modified cell culture substrate comprising:
a patterned polydimethylsiloxane (PDMS) substrate with a stiffness of 667 kPa, the patterned PDMS substrate comprising an imprinted pattern of human fetus cardiomyocytes on a cell-contacting surface of the patterned PDMS substrate;
a first coating comprising a plurality of polydopamine molecules chemically attached to the cell-contacting surface of the patterned PDMS substrate, wherein the plurality of polydopamine molecules have a coating concentration between 47 μg/cm2 and 53 μg/cm2; and
a second coating covering a top surface of the first coating, the second coating comprising a plurality of Laminin 511 E8 Fragment (LME8) molecules chemically attached to the first coating, wherein the plurality of LME8 molecules have a coating concentration between 0.4 μg/cm2 and 0.6 μg/cm2;
culturing the seeded iPSCs on the modified surface of the modified cell culture substrate in an animal component-free culture medium until reaching a confluency of at least 80%; and
differentiating the cultured iPSCs to the population of cardiomyocytes on the modified surface of the modified cell culture substrate comprising:
inducing differentiation of the cultured iPSCs to a population of mesoderm cells by culturing the cultured iPSCs in a first growth medium comprising a Wnt activator;
inducing differentiation of the population of mesoderm cells to a population of cardiac mesoderm cells by culturing the population of mesoderm cells in a second growth medium comprising a Wnt inhibitor; and
inducing differentiation of the population of cardiac mesoderm cells to the population of cardiomyocytes by culturing the population of cardiac mesoderm cells in a third growth medium comprising Insulin.

### Claim 2 (depends on 1)

The method of claim 1, wherein the animal component-free culture medium comprises a Dulbecco's Modified Eagle Medium F-12 comprising at least one of L-ascorbic acid-2-phosphate magnesium, sodium selenium, fibroblast growth factor 2 (FGF2), Insulin, NaHCO3, transferrin, and transforming growth factor beta 1 (TGFβ1).

### Claim 3 (depends on 1)

The method of claim 1, wherein the animal component-free culture medium comprises an inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) with a concentration between 8 μM and 12 μM.

### Claim 4 (depends on 1)

The method of claim 1, wherein inducing differentiation of the cultured iPSCs to the population of mesoderm cells by culturing the cultured iPSCs in the first growth medium comprising the Wnt activator comprises inducing differentiation of the cultured iPSCs to the population of mesoderm cells by culturing the cultured iPSCs in Roswell Park Memorial Institute Medium (RPMI) comprising CHIR99021 with a concentration between 10 μM and 14 μM for a time duration between 18 and 30 hours.

### Claim 5 (depends on 1)

The method of claim 1, wherein inducing differentiation of the population of mesoderm cells to the population of cardiac mesoderm cells by culturing the population of mesoderm cells in the second growth medium comprising the Wnt inhibitor comprises inducing differentiation of the population of mesoderm cells to the population of cardiac mesoderm cells by culturing the population of mesoderm cells in RPMI comprising XAV939 with a concentration between 1.5 μM and 2.5 μM for a time duration between 48 and 100 hours.

### Claim 6 (depends on 1)

The method of claim 1, wherein inducing differentiation of the population of cardiac mesoderm cells to the population of cardiomyocytes by culturing the population of cardiac mesoderm cells in the third growth medium comprising Insulin comprises inducing differentiation of the population of cardiac mesoderm cells in RPMI comprising Insulin for a time duration between 5 and 15 days.