Isolation, cultivation and uses of stem/progenitor cells

• US 20060078993A1
• Filed: 08/15/2005
• Published: 04/13/2006
• Est. Priority Date: 08/16/2004
• Status: Active Grant

• Current Assignee: CellResearch Corporation Pte Ltd (CellResearch Group of Cos.)
• Original Assignee: CellResearch Corporation Pte Ltd (CellResearch Group of Cos.)
• Inventors: Phan, Toan-Thang, Lim, Ivor Jiun
• Granted Patent: US 10,363,275 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/36 Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells islets of Langerhans A61K35/39
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: A61K 9/0014 Skin, i.e. galenical aspects of topical compositions non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00
• CPC Class Codes: A61K 9/06 Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels composition of ointments, creams or gels A61K47/00
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/16 Emollients or protectives, e.g. against radiation
• CPC Class Codes: A61P 17/18 Antioxidants, e.g. antiradicals preparations for protection against sunlight A61Q17/00
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 5/24 of the sex hormones
• CPC Class Codes: A61P 5/38 of the suprarenal hormones
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C12N 2506/025 : from extra-embryonic cells, e.g. trophoblast, placenta
• CPC Class Codes: C12N 2506/1392 : from mesenchymal stem cells from other natural sources
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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 5/0629 : Keratinocytes; Whole skin
• CPC Class Codes: C12N 5/063 : Kereatinocyte stem cells; Keratinocyte progenitors
• CPC Class Codes: C12N 5/0668 : Mesenchymal stem cells from other natural sources

Abstract

The present invention relates to a method for isolating stem/progenitor cells from the amniotic membrane of umbilical cord, wherein the method comprises separating the amniotic membrane from the other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions allowing cell proliferation, and isolating the stem/progenitor cells from the tissue cultures. The isolated stem cell cells can have embryonic stem cell-like properties and can be used for various therapeutic purposes. In one embodiment, the invention relates to the isolation and cultivation of stem cells such as epithelial and/or mesenchymal stem/progenitor cells under conditions allowing the cells to undergo mitotic expansion. Furthermore, the invention is directed to a method for the differentiation of the isolated stem/progenitor cells into epithelial and/or mesenchymal cells.

Claims

1. A method for isolating stem/progenitor cells from the amniotic membrane of umbilical cord, the method comprising:

(a) separating the amniotic membrane from the other components of the umbilical cord in vitro;

(b) culturing the amniotic membrane tissue obtained in step (a) under conditions allowing cell proliferation; and

(c) isolating the stem/progenitor cells.

2. The method of claim 1, further comprising:

(a″

) separating the cells of the amniotic membrane tissue before cultivation by a method selected from the group consisting of enzymatic separation and direct tissue explant.

3. The method of claim 1, wherein the stem/progenitor cells have embryonic stem cell-like properties.

4. The method of claim 1, wherein the stem/progenitor cells are epithelial and/or mesenchymal stem/progenitor cells.

5. The method of claim 1, further comprising:

(d) culturing the stem/progenitor cells under conditions allowing the cells to undergo clonal expansion.

6. The method of claim 5, further comprising:

(e) culturing the stem/progenitor cells under conditions allowing the differentiation of said cells into epithelial cells and/or mesenchymal cells; and

(f) isolating the differentiated cells.

7. The method of claim 6, wherein the epithelial cells are selected from the group consisting of:

skin epithelial cells, hair follicular cells, cornea epithelial cells, conjunctival epithelial cells, retinal epithelial cells, liver epithelial cells, kidney epithelial cells, pancreatic epithelial cells, oesophageal epithelial cells, small intestinal epithelial cells, large intestinal epithelial cells, lung epithelial cells, airway epithelial cells, bladder epithelial cells and uterine epithelial cells.

8. The method of claim 6, wherein the mesenchymal cells are selected from the group consisting of:

skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, cells derived from endocrine glands, neurectodermal cells, and all varieties and derivatives of neurectodermal cells.

9. The method of claim 1, further comprising

(g) preserving the isolated stem/progenitor cells for further use.

10. The method of claim 9, wherein preserving is carried out by using cryo-preservation.

11. A stem/progenitor cell isolated from the amniotic membrane of umbilical cord by means of the method of claim 1 or a cell differentiated therefrom.

12. A cell bank comprising a stem/progenitor cell isolated by means of the method of claim 1 or a cell differentiated therefrom.

13. A pharmaceutical composition comprising a member selected from the group consisting of a stem/progenitor cell isolated from the amniotic membrane of umbilical cord by means of the method of claim 1, a cell differentiated therefrom and a cellular extract thereof.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is adapted for systemic or topical application.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition adapted for topical application is selected from the group consisting of an ointment, a cream, and a lotion.

16. A method of treating a subject having a disorder comprising administering to the subject an effective amount of a stem/progenitor cell isolated by means of the method of claim 1.

17. The method of treating of claim 16, wherein the disorder is selected from the group consisting of neoplastic disease, skin disease, visceral endocrine deficiency, and a neural disorder.

18. The method of claim 17, wherein the tissue disorder is a congenital or an acquired tissue deficiency.

19. The method of claim 17, wherein the visceral endocrine deficiency is selected from the group consisting of insulin deficiency, Diabetes mellitus associated with insulin deficiency, testosterone deficiency, anemia, hypoglycemia, hyperglycemia, pancreatic deficiency, adrenal deficiency, and thyroid abnormality.

20. The method of claim 17, wherein the neural disorder is selected from the group consisting of Alzheimer'"'"'s disease, Parkinson'"'"'s disease, Jacob Kreutzfeld'"'"'s disease, Lou Gehrig'"'"'s disease, and Huntington'"'"'s disease.

21. The method of claim 17, wherein the skin disease is accelerated aging or a wound.

22. The method of claim 17, wherein the neoplastic disease is cancer.

23. The method of claim 22, wherein the cancer is a selected from the group consisting of squamous cell carcinoma, breast ductal and lobular carcinoma, hepatocellular carcinoma, nasopharyngeal carcinoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'"'"'s Disease, non-Hodgkin'"'"'s lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemias, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi'"'"'s sarcoma, epidermoid cancer and any combination of such cancers, including disseminated (metastasizing) forms thereof.

24. A method of producing a biological molecule, comprising producing the molecule of interest in a stem/progenitor cell as defined in claim 11.

25. The method of claim 24, wherein the biological molecule is selected from the group consisting of a protein, a peptide, a small organic molecule, an oligosaccharide, a polysaccharide, a proteoglycan and a lipid.

26. A method of cultivation of mammalian cells comprising using a stem/progenitor cell as defined in claim 11 as feeder layer for the cells to be cultivated.

27. A method of cultivating stem/progenitors cells as defined in claim 11, comprising:

Obtaining a tissue explant from the amniotic membrane of umbilical cord;

Cultivating the tissue explant in suitable cultivation media and cultivation conditions over a suitable period of time.

28. The method of claim 27, further comprising exposing the tissue explant to fresh cultivation media and continuing the cultivation under suitable conditions over a suitable period of time.

29. A method of culturing mesenchymal stem cells obtained by the method of claim 1, comprising culturing the cells in serum free medium.

30. The method of claim 24, wherein the biological molecule is endogenously produced in the stem/progenitor cell and the cell is cultivated under conditions suitable for production of the biological molecule.

31. The method of claim 24, wherein the nucleic acid sequence that encodes the biological molecule is introduced into the stem/progenitor cell and the cell is cultivated under conditions suitable for expression of the introduced exogenous nucleic acid sequence.

32. A method of differentiating a stem/progenitor cell isolated by the method as defined in claim 1, comprising culturing the stem/progenitor cells under conditions allowing the cells to undergo clonal expansion.

33. The method of claim 32, further comprising culturing the stem/progenitor cells under conditions allowing the differentiation of said cells into epithelial cells and/or mesenchymal cells.

34. The method of claim 33, wherein the epithelial cells are selected from the group consisting of:

skin epithelial cells, hair follicular cells, cornea epithelial cells, conjunctival epithelial cells, retinal epithelial cells, liver epithelial cells, kidney epithelial cells, pancreatic epithelial cells, oesophageal epithelial cells, small intestinal epithelial cells, large intestinal epithelial cells, lung epithelial cells, airway epithelial cells, bladder epithelial cells and uterine epithelial cells.

35. The method of claim 33, wherein the mesenchymal cells are selected from the group consisting of:

skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, cells derived from endocrine glands, neurectodermal cells, and all varieties and derivatives of neurectodermal cells.

36. A method of storing an umbilical cord obtained from a mammalian donor, comprising cryo-preservation of the umbilical cord or of its separated amniotic membrane.