Postpartum cells derived from umbilical cord tissue, and methods of making and using the same

• US 9,717,763 B2
• Filed: 09/05/2013
• Issued: 08/01/2017
• Est. Priority Date: 06/27/2003
• Status: Active Grant

• Current Assignee: Depuy Synthes Products Incorporated (Johnson & Johnson)
• Original Assignee: Depuy Synthes Products Incorporated (Johnson & Johnson)
• Inventors: Mistry, Sanjay, Kihm, Anthony J., Harris, Ian R., Harmon, Alexander M., Messina, Darin J., Seyda, Agnieszka, Yi, Chin-Feng, Gosiewska, Anna
• Primary Examiner(s): Kim, Taeyoon
• Application Number:
  US14/018,842
• Publication Number:
  US 20140045263A1
• Time in Patent Office: 1,426 Days
• CPC Class Codes: 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
• CPC Class Codes: A61K 35/50 Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• CPC Class Codes: A61K 38/1808 Epidermal growth factor [EGF] urogastrone
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/1833 Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
• CPC Class Codes: A61K 38/1841 Transforming growth factor [TGF]
• CPC Class Codes: 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
• CPC Class Codes: A61K 38/1858 Platelet-derived growth factor [PDGF]
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: A61K 38/1891 Angiogenesic factors; Angiogenin
• CPC Class Codes: A61K 38/19 Cytokines; Lymphokines; Interferons
• CPC Class Codes: A61K 38/204 IL-6
• CPC Class Codes: A61K 38/2053 IL-8
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 13/12 : of the kidneys
• CPC Class Codes: A61P 17/02 : for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 : Drugs for skeletal disorders
• CPC Class Codes: A61P 19/04 : for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 : for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 : for osteoporosis
• CPC Class Codes: A61P 21/00 : Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 : Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 : for peripheral neuropathies
• CPC Class Codes: A61P 25/14 : for treating abnormal movements, e.g. chorea, dyskinesia
• 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 27/02 : Ophthalmic agents
• CPC Class Codes: A61P 27/06 : Antiglaucoma agents or miotics
• CPC Class Codes: A61P 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 39/06 : Free radical scavengers or antioxidants
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• 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 2500/32 : Amino acids
• CPC Class Codes: C12N 2500/34 : Sugars
• CPC Class Codes: C12N 2500/44 : Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/90 : Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/95 : Protein-free medium and culture conditions
• CPC Class Codes: C12N 2501/12 : Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/21 : Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
• CPC Class Codes: C12N 2501/23 : Interleukins [IL]
• CPC Class Codes: C12N 2502/02 : embryonic cells
• CPC Class Codes: C12N 2506/02 : from embryonic cells
• CPC Class Codes: C12N 2506/03 : from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2509/00 : Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2533/50 : Proteins
• CPC Class Codes: C12N 5/0605 : Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0606 : Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0607 : Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

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

1. A method of enhancing the yield of cells isolated from human umbilical cord tissue, the method comprising the steps of:

(a) obtaining umbilical cord tissue;

(b) removing substantially all of the blood from the tissue to yield umbilical tissue substantially free of blood;

(c) dissociating said umbilical tissue substantially free of blood by mechanical dissociation;

(d) digesting the dissociated tissue with a mixture of enzymes comprising a metalloprotease, neutral protease and mucolytic enzyme, wherein the mixture of enzymes is a mixture of collagenase, dispase and hyaluronidase;

(e) isolating umbilicus-derived cells from the digested tissue;

(f) resuspending the isolated umbilicus-derived cells in a growth medium; and

(g) culturing the isolated umbilicus-derived cells for about 10 to 100 hours to obtain a homogenous population of isolated umbilicus-derived cells, wherein the isolated umbilicus-derived cells of the homogenous population are capable of self-renewal and expansion in culture, have the potential to differentiate into cells of other phenotypes, 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, and do not produce CD117.

2. The method of claim 1, wherein the removing step comprises removal of free or clotted blood by one or more of washing, suctioning, blotting, centrifugal separation, or enzymatic removal.

3. The method of claim 1, wherein the digestion step comprises incubating the dissociated tissue with the mixture of enzymes at about 37°

C.

4. The method of claim 1, wherein the digestion step comprises incubating the dissociated tissue with the mixture of enzymes for one or more hours.

5. The method of claim 1, wherein the population of isolated cells produces each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C.

6. The method of claim 1, wherein the population of isolated cells does not produce any of CD31, CD34, CD45, CD80, CD86, CD141, CD178, B7-H2 or HLA-DR, DP,DQ, as detected by flow cytometry.

7. The method of claim 1, wherein the population of isolated cells can expand in the presence of oxygen from about 5% to about 20%.

8. The method of claim 1, wherein the population of isolated cells can expand for at least 40 doublings in culture.

9. The method of claim 1, wherein the population of isolated cells can expand to generate at least about 10¹⁷ cells in less than about 65 days in culture when seeded at about 5×

10³ cells/cm² of culture vessel surface.

10. The method of claim 1, wherein the population of isolated cells require L-Valine for growth.

11. The method of claim 1, wherein the population of isolated cells grows in the presence of from about 2% to about 15% Fetal Bovine Serum.

12. The method of claim 1, wherein the population of isolated cells grows in the presence or absence of one or more added growth factors selected from EGF, FGF, PDGF, VEGF, IGF and LIF.

13. The method of claim 1, wherein the population of isolated cells adheres and expands on a coated or uncoated tissue culture vessel.

14. The method of claim 1, further comprising expanding the population of isolated umbilicus-derived cells in culture for about 10 days to confluence.

15. The method of claim 1, wherein the cells are able to differentiate to a hepatocyte phenotype, an adipogenic phenotype, a pancreatic phenotype, a chondrogenic phenotype, and a cardiomyocyte phenotype.

17. The method of claim 1, wherein the isolated umbilicus-derived cells of the homogeneous population secrete MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1, and do not secrete SDF-lalpha, TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF.

16. A method of enhancing the yield of cells isolated from human umbilical cord tissue, the method comprising the steps of:

(a) removing substantially all of the blood from post-partum umbilical cord tissue to yield umbilical tissue substantially free of blood;

(b) dissociating said umbilical tissue substantially free of blood by mechanical dissociation;

(c) isolating umbilicus-derived cells by digesting the dissociated tissue with a mixture of enzymes comprising a metalloprotease, neutral protease and mucolytic enzyme, wherein the mixture of enzymes is a mixture of collagenase, dispase and hyaluronidase;

(d) resuspending the isolated umbilicus-derived cells in a growth medium; and

(e) culturing the isolated umbilicus-derived cells for about 10 to 100 hours to obtain a homogenous population of isolated umbilicus-derived cells,

wherein the isolated umbilicus-derived cells of the homogenous population are capable of self-renewal and expansion in culture, have the potential to differentiate into at least a hepatocyte phenotype, an adipogenic phenotype, a pancreatic phenotype, a chondrogenic phenotype, and a cardiomyocyte phenotype, 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, and do not produce CD 117.

18. The method of claim 16, wherein the isolated umbilicus-derived cells of the homogeneous population secrete MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1b, RANTES, and TIMP1, and do not secrete SDF-lalpha, TGF-beta2, ANG2, PDGFbb, MIP1a, and VEGF.