# US 2014/0193380 A1 · Technologies, Methods, and Products of Small Molecule Directed Tissue and Organ Regeneration from Human Pluripotent Stem Cells

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- Number: US 2014/0193380 A1
- Document ID: US20140193380A1
- Status: Published application
- Kind: A1
- Application number: 14/204,372
- Filed: 2014-03-11
- Published: 2014-07-10
- Earliest claimed: 2011-11-29
- Assignees: San Diego Regenerative Medicine Institute
- Inventors: Xuejun H. Parsons
- Web page: https://patentgrep.com/patent/US20140193380A1

## CPC

- C12N 5/0619: {Neurons}
- 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 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 25/00: Drugs for disorders of the nervous system
- 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
- C12N 5/0618: {Cells of the nervous system}
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- G01N 33/5044: {involving specific cell types}
- G01N 33/5058: {Neurological cells}
- G01N 33/5073: {Stem cells}
- C12N 2500/38: Vitamins
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/33: Insulin
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/40: Regulators of development
- C12N 2502/02: embryonic cells
- C12N 2506/02: from embryonic cells
- C12N 2533/50: Proteins
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)

## Abstract

Pluripotent human embryonic stem cells (hESCs) hold great potential for restoring tissue and organ function, which has been hindered by inefficiency and instability of generating desired cell types through multi-lineage differentiation. This instant invention is based on the discovery that pluripotent hESCs maintained under defined culture conditions can be uniformly converted into a specific lineage by small molecule induction. Retinoic acid induces specification of neuroectoderm direct from the pluripotent state of hESCs and triggers progression to neuronal progenitors and neurons efficiently. Similarly, nicotinamide induces specification of cardiomesoderm direct from the pluripotent state of hESCs and triggers progression to cardiac precursors and cardiomyocytes efficiently. This technology provides a large supply of clinically-suitable human neuronal or cardiac therapeutic products for CNS or myocardium repair. This invention enables well-controlled efficient induction of pluripotent hESCs exclusively to a specific clinically-relevant lineage for tissue and organ engineering and regeneration, cell-based therapy, and drug discovery.

## Claims

### Claim 1

A method of directly differentiating pluripotent human embryonic stem cells (hES cells) into cells of a cardiac lineage, comprising:
(i) providing a culture of hES cells in a defined medium comprising bFGF, insulin, ascorbic acid, and activin-A, wherein said defined medium is free of serum, free of feeder cells, and free of feeder cell conditioned medium; and
(ii) adding nicotinamide (NAM) to the culture, wherein the hES cells are cultured in the presence of NAM for a period of time sufficient to induce the expression of cardiac specific transcription factor (CSX) Nkx2.5 and cause the hES cells to directly differentiate into cells of cardiac lineage.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said defined medium comprises: (a) a basal medium; (b) 20-100 ng/ml bFGF; (c) 10-30 microgram/ml insulin; (d) 40-60 microgram/ml ascorbic acid; and (e) 20-100 ng/ml activin-A.

### Claim 3 (depends on 1)

The method according to claim 1, wherein at least 70% of the hES cells from the culture of step (i) are positive for at least 4 markers selected from the group consisting of alkaline phosphatase, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, acetylated histones, Brg-1, hSNF2H, and microRNA hsa-miR-302.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the hES cells comprise a pluripotent hES cell line which (i) is derived from the inner cell mass or epiblast of a human blastocyst; (ii) is capable of proliferation in culture for over one year in a defined medium comprising bFGF, insulin, ascorbic acid, and activin-A, wherein said defined medium is free of serum, free of feeder cells, and free of feeder cell conditioned medium; (iii) maintains a stable karyotype in which the chromosomes are euploid through prolonged culture; and (iv) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the cells of cardiac lineage are selected from the group consisting of cardiomesodermal cells, cardiac cells, cardiac precursors, cardiovascular cells, and cardiomyocytes.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the NAM is selected from the group consisting of nicotinamide, analogues of nicotinamide, nicotinamide receptors and their ligands, NAD (nicotinamide adenine dinucleotide), and activators and inhibitors of NAD-dependent enzymes.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said hES cells directly differentiate into a population of cells of cardiac lineage from the culture of step (ii) comprising at least 80% of cardiomesodermal cells that are positive for at least 2 markers selected from the group consisting of Nkx2.5, alpha-actinin, and SSEA-1.

### Claim 8 (depends on 7)

The method according to claim 7, further comprising removing bFGF from the culture once cardiomesodermal cells are obtained, wherein the cardiomesodermal cells further differentiate into a population of cardiac cells comprising at least 90% of cardiac precursor cells that are positive for Nkx2.5.

### Claim 9 (depends on 8)

The method according to claim 8, wherein said cardiac cells are negative for at least 4 markers selected from the group consisting of Oct-4, SSEA-4, Tra-1-60, Tra-1-81, microRNA hsa-miR-302, Sox-2, HNK1, Pdx1, AFP, Pax6, beta-III-tubulin, Map-2, MBP, and GFAP.

### Claim 10 (depends on 8)

The method according to claim 8, wherein said cardiac precursor cells further differentiate into a population of cardiovascular cells comprising at least 50% of cardiomyocytes that are positive for at least 3 markers selected from the group consisting of Nkx2.5, alpha-actinin, cardiac myosin heavy chain (MHC), MEF2c, GATA-4, cardiac troponin I (cTnI), and cardiac troponin T (cTnT).

### Claim 11 (depends on 10)

The method according to claim 10, wherein said cardiovascular cells are positive for at least one marker selected from the group consisting of Nkx2.5, alpha-actinin, cardiac myosin heavy chain (MHC), MEF2c, GATA-4, cardiac troponin I (cTnI), cardiac troponin T (cTnT), VE cadherin, von Willebrand factor, SM22alpha, and h1-calponin.

### Claim 12 (depends on 10)

The method according to claim 10, wherein said cardiomyocytes comprise beating cardiomyocytes which have spontaneous strong rhythmic contractions.

### Claim 13 (depends on 1)

The method according to claim 1, wherein producing the cells of cardiac lineage directly from hES cells comprising steps of:
(i) providing a culture of hES cells in a defined medium comprising bFGF, insulin, ascorbic acid, and activin-A, wherein said defined medium is free of serum, free of feeder cells, and free of feeder cell conditioned medium;
(ii) adding NAM to the culture, wherein the hES cells are cultured in the presence of NAM for a period of time sufficient to cause the hES cells to directly differentiate into cells of cardiac lineage comprising at least 80% of cardiomesodermal cells;
(iii) further differentiating the cardiomesodermal cells into a population of cardiac cells comprising at least 90% of cardiac precursor cells; and
(iv) further differentiating the cardiac precursor cells into a population of cardiovascular cells comprising at least 50% of cardiomyocytes.

### Claim 14 (depends on 13)

The method according to claim 13, wherein the cells of cardiac lineage comprise human cardiomesodermal cells, human cardiac cells, human cardiac precursors, human cardiovascular cells, and human cardiomyocytes.

### Claim 15 (depends on 13)

A method of cardiac cell therapy, comprising (i) producing the cells of cardiac lineage directly from hES cells according to the method of claim 13; and (ii) administering to a patient in need of such cardiac cell therapy product the cells of cardiac lineage.

### Claim 16 (depends on 15)

A method according to claim 15 wherein the patient suffers from a cardiovascular disorder comprising heart disease and failure, myocardial infarction, cardiomyopathy, ischemic heart disease, congestive heart failure, stroke, and coronary artery disease.

### Claim 17 (depends on 13)

A method of cardiac drug discovery, comprising (i) producing the cells of cardiac lineage directly from hES cells according to the method of claim 13; and (ii) a high throughput method to screen a compound for an effect on such cardiac cell product—the cells of cardiac lineage.

### Claim 18 (depends on 13)

A method of cardiac tissue engineering, comprising (i) producing the cells of cardiac lineage directly from hES cells according to the method of claim 13; and (ii) adding such cardiac cell product—the cells of cardiac lineage—to a structural template that comprises extracellular matrix, biomaterial scaffold, whole-heart scaffold, and synthetic or purified matrix proteins.