Technologies, methods, and products of small molecule directed tissue and organ regeneration from human pluripotent stem cells

• US 9,428,731 B2
• Filed: 03/11/2014
• Issued: 08/30/2016
• Est. Priority Date: 12/06/2010
• Status: Active Grant

• Current Assignee: Xcelthera, Inc.
• Original Assignee: San Diego Regenerative Medicine Institute, Xcelthera, Inc.
• Inventors: Parsons, Xuejun H
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US14/204,372
• Publication Number:
  US 20140193380A1
• Time in Patent Office: 903 Days
• US Class Current: 1/1
• CPC Class Codes: 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
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: 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
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2502/02 embryonic cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 : Stem cells

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

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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. The method according to claim 6, 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.

8. The method according to claim 7, 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.

9. The method according to claim 7, 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).

10. The method according to claim 9, 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.

11. The method according to claim 9, wherein said cardiomyocytes comprise beating cardiomyocytes which have spontaneous strong rhythmic contractions.

12. 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.

13. The method according to claim 12, wherein the cells of cardiac lineage comprise human cardiomesodermal cells, human cardiac cells, human cardiac precursors, human cardiovascular cells, and human cardiomyocytes.