# US 10,500,234 B2 · Postpartum cells derived from umbilical cord tissue, and methods of making and using the same

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- Number: US 10,500,234 B2
- Document ID: US10500234B2
- Status: Granted
- Kind: B2
- Application number: 15/661,878
- Filed: 2017-07-27
- Published: 2017-11-16
- Granted: 2019-12-10
- Earliest claimed: 2003-06-27
- Assignees: DePuy Synthes Products, Inc.
- Applicants: DePuy Synthes Products, Inc.
- Inventors: Sanjay Mistry; Anthony J. Kihm; Ian R. Harris; Alexander M. Harmon; Darin J. Messina; Agnieszka Seyda; Chin-Feng Yi; Anna Gosiewska
- Web page: https://patentgrep.com/patent/US10500234B2

## CPC

- A61K 35/51: Umbilical cord; Umbilical cord blood; Umbilical stem cells
- A61K 35/50: Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
- A61K 38/18: Growth factors; Growth regulators
- A61K 38/1808: {Epidermal growth factor [EGF] urogastrone}
- A61K 38/1825: {Fibroblast growth factor [FGF]}
- A61K 38/1833: {Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II}
- A61K 38/1841: {Transforming growth factor [TGF]}
- A61K 38/185: {Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3}
- A61K 38/1858: {Platelet-derived growth factor [PDGF]}
- A61K 38/1866: {Vascular endothelial growth factor [VEGF]}
- A61K 38/1891: {Angiogenesic factors; Angiogenin}
- A61K 38/19: Cytokines; Lymphokines; Interferons
- A61K 38/204: {IL-6}
- A61K 38/2053: {IL-8}
- A61K 38/27: Growth hormone [GH], i.e. somatotropin
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/04: Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
- 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 13/12: of the kidneys
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/04: for non-specific disorders of the connective tissue
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 19/10: for osteoporosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- 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
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 39/06: Free radical scavengers or antioxidants
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/0605: {Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- C12N 2500/32: Amino acids
- C12N 2500/34: Sugars
- C12N 2500/44: Thiols, e.g. mercaptoethanol
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2500/95: Protein-free medium and culture conditions
- C12N 2501/12: Hepatocyte growth factor [HGF]
- C12N 2501/21: Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
- C12N 2501/23: Interleukins [IL]
- C12N 2502/02: embryonic cells
- C12N 2506/02: from embryonic cells
- C12N 2506/03: from non-embryonic pluripotent stem cells
- C12N 2509/00: Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N 2533/50: Proteins
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Cells derived from human umbilical cords are disclosed along with methods for their therapeutic use. Isolation techniques, culture methods and detailed characterization of the cells with respect to their cell surface markers, gene expression, and their secretion of trophic factors are described.

## Claims

### Claim 1

A cell culture comprising a culture medium and an isolated cell derived from human umbilical cord tissue substantially free of blood, wherein said isolated cell is capable of self-renewal and expansion in culture, has the potential to differentiate into cells of other phenotypes, produces granulocyte chemotactic protein-2 (GCP-2), and can double sufficiently to generate yields of greater than about 1×1017 cells in about 60 days in culture when seeded at about 5,000 cells/cm2 in a flask or yields of greater than about 1×1011 cells in about 60 days in culture when seeded at about 1,000 cells/cm2 in a flask.

### Claim 2 (depends on 1)

The cell culture of claim 1, wherein the isolated cell can double sufficiently to generate greater than about 1×1017 cells in about 60 days in culture when seeded at about 5,000 cells/cm2 in a flask.

### Claim 3 (depends on 1)

The cell culture of claim 1, wherein the isolated cell can double sufficiently to generate greater than about 1×1011 cells in about 60 days in culture when seeded at about 1,000 cells/cm2 in a flask.

### Claim 4 (depends on 1)

The cell culture of claim 1, wherein the isolated cell expresses CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A, HLA-B, and HLA-C, does not express CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP and HLA-DQ, and has increased expression, relative to a human fibroblast, mesenchymal stem cell, or iliac crest bone marrow cell, of a gene for interleukin 8, reticulon 1 and chemokine (C-X-C motif) ligand 3.

### Claim 5 (depends on 1)

The cell culture of claim 1, wherein the culture medium is a chemically-defined medium or a medium with added mammalian serum.

### Claim 6 (depends on 5)

The cell culture of claim 5, wherein the culture medium comprises L-valine.

### Claim 7 (depends on 1)

The cell culture of claim 1, wherein the isolated cell secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1, and does not secrete SDF-1alpha, TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF.

### Claim 8 (depends on 1)

The cell culture of claim 1, wherein the isolated cell is cryopreserved.

### Claim 9 (depends on 1)

A kit comprising the cell culture of claim 1 and at least one other component selected from the group consisting of a scaffold, a hydrating agent and a cell culture substrate.

### Claim 10

A three-dimensional matrix comprising an isolated cell derived from human umbilical cord tissue substantially free of blood, wherein said isolated cell is capable of self-renewal and expansion in culture, has the potential to differentiate into cells of other phenotypes, produces granulocyte chemotactic protein-2 (GCP-2), and can double sufficiently to generate yields of greater than about 1×1017 cells in about 60 days in culture when seeded at about 5,000 cells/cm2 in a flask or yields of greater than about 1×1011 cells in about 60 days in culture when seeded at about 1,000 cells/cm2 in a flask.

### Claim 11 (depends on 10)

The three-dimensional matrix of claim 10, wherein the isolated cell expresses CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A, HLA-B, and HLA-C, does not express CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP and HLA-DQ, and has increased expression, relative to a human fibroblast, mesenchymal stem cell, or iliac crest bone marrow cell, of a gene for interleukin 8, reticulon 1 and chemokine (C-X-C motif) ligand 3.

### Claim 12 (depends on 10)

The three-dimensional matrix of claim 10, wherein the isolated cell secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1, and does not secrete SDF-1alpha, TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF.

### Claim 13 (depends on 10)

The three-dimensional matrix of claim 10, wherein said matrix comprises a synthetic biocompatible or bioabsorbable polymer.

### Claim 14 (depends on 13)

The three-dimensional matrix of claim 13, wherein said matrix comprises a nonwoven mat comprising a synthetic copolymer of glycolic and lactic acids, a PCL/PGA copolymer, or self-assembling peptides.

### Claim 15

A cryopreserved composition comprising an isolated cell derived from human umbilical cord tissue substantially free of blood, wherein said isolated cell is capable of self-renewal and expansion in culture, has the potential to differentiate into cells of other phenotypes, produces granulocyte chemotactic protein-2 (GCP-2), and can double sufficiently to generate yields of greater than about 1×1017 cells in about 60 days in culture when seeded at about 5,000 cells/cm2 in a flask or yields of greater than about 1×1011 cells in about 60 days in culture when seeded at about 1,000 cells/cm2 in a flask.

### Claim 16 (depends on 15)

The cryopreserved composition of claim 15, wherein the isolated cell can double sufficiently to generate greater than about 1×1017 cells in about 60 days in culture when seeded at about 5,000 cells/cm2 in a flask.

### Claim 17 (depends on 15)

The cryopreserved composition of claim 15, wherein the isolated cell can double sufficiently to generate greater than about 1×1011 cells in about 60 days in culture when seeded at about 1,000 cells/cm2 in a flask.

### Claim 18 (depends on 15)

The cryopreserved composition of claim 15, wherein the isolated cell expresses CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A, HLA-B, and HLA-C, does not express CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP and HLA-DQ, and has increased expression, relative to a human fibroblast, mesenchymal stem cell, or iliac crest bone marrow cell, of a gene for interleukin 8, reticulon 1 and chemokine (C-X-C motif) ligand 3.

### Claim 19 (depends on 15)

The cryopreserved composition of claim 15, wherein the isolated cell secretes MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1, and does not secrete SDF-1alpha, TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF.