Isolated adult pluripotent stem cells and methods for isolating and cultivating thereof

• US 20050014254A1
• Filed: 04/08/2004
• Published: 01/20/2005
• Est. Priority Date: 06/23/2003
• Status: Active Grant

• Current Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Original Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Inventors: Kruse, Charli
• Granted Patent: US 8,603,809 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

This invention relates to isolated pluripotent adult stem cells which are obtained from exocrine glandular tissue as well as methods of isolating and culturing these pluripotent stem cells.

Claims

1. Isolated pluripotent adult stem cells obtained from exocrine glandular tissue.

2. The pluripotent adult stem cells according to claim 1, wherein the exocrine glandular tissue originates from a vertebrate.

3. The pluripotent adult stem cells according to claim 2, wherein the exocrine glandular tissue originates from a mammal.

4. The pluripotent adult stem cells according to claim 1, wherein the exocrine glandular tissue is derived from a salivary gland, a lacrimal gland, a sudoriferous gland, a sebaceous gland or from gastrointestinal tissue, including the pancreas.

5. The pluripotent adult stem cells according to claim 1, wherein the exocrine glandular tissue is acinar tissue.

6. The pluripotent adult stem cells according to claim 5, wherein the acinar tissue is derived from the pancreas, the parotid gland or the Mandibular gland.

7. The pluripotent adult stem cells according to one of claim 1, being capable of forming organoid bodies.

8. The pluripotent adult stem cells according to claim 1 being capable of differentiating into cell types of all three germ layers in a culture medium that does not contain any additional growth factors or differentiation factors after culturing under spatial conditions which ensure three-dimensional contact of the cells.

9. The pluripotent adult stem cells according to claim 1 wherein even after freezing/cryopreservation, the cells still retain an ability for self-renewal and unlimited division and do not differentiate.

10. A stem cell culture comprising the stem cells according to claims 1 in a culture medium adapted to allow stable maintenance and proliferation of the cells essentially without differentiation.

11. The stem cell culture according to claim 10, wherein the culture medium does not include any feeder cell layer.

12. The stem cell culture according to claim 10, wherein the cells retain an ability for self-renewal and unlimited division for more than 25 passages.

13. The stem cell culture according to claim 12, wherein the cells retain the ability for self-renewal and unlimited division for more than 50 passages.

15. Organoid bodies formed from the stem cells according to claims 1 and obtainable by culturing these stem cells under spatial conditions that ensure three-dimensional contact of the cells.

16. The organoid bodies according to claim 15 obtainable by culturing the stem cells in hanging drops, moving suspension culture or surface culture on surfaces to which the cells substantially do not adhere.

17. The organoid bodies according to claim 15, wherein the cells of these organoid bodies essentially retain a viability and a capacity for differentiation after freezing/cryopreservation.

18. Secondary organoid bodies obtainable from the organoid bodies according to claims 15 by spontaneous growth in surface culture on surfaces without limited surface adhesion.

19. A method of producing adult pluripotent stem cells according to claims 1, wherein exocrine glandular tissue is removed,

the tissue thus removed is divided,

the divided tissue is cultured and

the cells persisting in the culture are cultured.

20. The method according to claim 19, wherein the tissue is divided in such a gentle way that cell structures in resulting tissue fragments are largely preserved and the divided tissue is first cultured under suitable conditions in tissue culture vessels, whereby most of the differentiated cells rapidly die in the course of within days and become detached from the stem cells, whereupon the stem cells adhere on a bottom of a tissue culture vessel, and the remaining tissue and nonadherent differentiated cells are largely separated by a first change of medium and the remaining nonadherent cells are separated by additional changes of medium at intervals of about 2 to 3 days.

21. A method of producing differentiated cells from the stem cells according to claims 1, wherein the undifferentiated stem cells are cultured further under spatial conditions which ensure three-dimensional contact of the cells until organoid bodies are formed, said organoid bodies then being transferred to a suspension culture and cultured further.

22. A method of producing differentiated cells from the stem cells according to claims 1, wherein the undifferentiated stem cells are transferred to a differentiation medium and are cultured further under spatial conditions so as to ensure a three-dimensional contact of the cells until forming primary organoid bodies, the primary organoid bodies are transferred to a surface culture, and secondary organoid bodies having identical properties as the primary organoid bodies are then formed in the surface culture from out-growing individual cells of the primary organoid bodies which then can be cultured further.

23. The method according to claim 21, wherein the culturing is performed under spatial conditions which ensure three-dimensional contact of the cells, culturing in hanging drops, moving suspension culture or surface culture on surfaces to which the cells substantially do not adhere.

24. Differentiated cells obtainable from the stem cells produced by the method according to claims 21.

25. The differentiated cells according to claim 24, being members selected from the group consisting of are bone cells, fibroblasts, muscle cells, endothelial cells, epithelial cells, hematopoietic cells, sensory cells, endocrine and exocrine glandular cells, glial cells, neural cells, oligodendrocytes, blood cells, intestinal cells, heart, lung, liver, kidney and pancreas cells.

26. A differentiation culture comprising the differentiated cells according to claims 24 and/or organoid bodies in a differentiation medium.

27. The differentiation culture according to claim 26, wherein the differentiation medium does not include any additional growth factors or differentiation factors.

28. A method of treating an injury or a disease condition comprising administration of a therapeutically effective amount of isolated pluripotent adult stem cells obtained from exocrine glandular tissue or the differentiated cells according to claims 24 to an individual or extracorporeal contacting of the stem cells or differentiated cells derived therefrom with a body fluid of the individual and then returning the body fluid to the individual.

29. The method according to claim 28, wherein the stem cells are derived from autologous tissue of the individual.

30. The method according to claim 28 wherein the stem cells are introduced into the body of the individual where they differentiate in vivo to take over a function of a missing or damaged organ, tissue type or cell type.

31. The method according to claims 28 wherein the treatment restores or strengthens the individual'"'"'s immune system.

32. The method according to claims 28 wherein the stem cells serve as a vehicle for a therapeutic agent.

33. The method according to claim 32, wherein the therapeutic agent is DNA, RNA, a protein, a peptide or a low-molecular pharmaceutical drug.

34. The method according to claims 28 wherein the method is a gene therapy method.

35. The method according to claims 28 wherein the stem cells are genetically engineered to have certain properties.

36. The method according to claims 28 wherein the stem cells are administered together with a physiologically acceptable matrix or a physiologically acceptable vehicle.

37. The method according to claims 28 wherein the stem cells are administered by local injection, systemic injection, parenteral administration, oral administration or intrauterine injection into an embryo.

38. The method according to claims 28 wherein the disease condition to be treated is a tumor, a liver disease, a connective tissue disease, a cardiovascular disease, a neurodegenerative disease, a metabolic disease, an autoimmune disease, anemia, hemophilia, diabetes, ischemia, an inflammation, an infectious disease, an aging process, a genetic defect or a transplant rejection.

39. A method for developing tissue-like or organ-like multicellular systems in vitro, said method comprising using the stem cells according to claims 1, or stem cell cultures, organoid bodies, or differentiated cells derived therefrom.

40. The method according to claim 39, wherein the multicellular systems comprise several types of cells.

41. A method for reproductive cloning of a nonhuman organism said method comprising using the stem cells according to claims 1 or differentiated cells obtained therefrom.

42. A method for testing chemicals in vitro said method comprising using cells according to claims 1, stem cell cultures, organoid bodies, or differentiated cells derived therefrom.

43. A method for producing substances in vitro said method comprising using the differentiated cells according to claims 24 or a differentiation culture including the differentiated cells.

44. A pharmaceutical composition comprising the stem cells according to claims 1 or differentiated cells obtained therefrom as well as a physiologically acceptable matrix or a physiologically acceptable vehicle.

45. The pharmaceutical composition according to claim 44 also comprising additional excipients or active ingredients.

46. A kit comprising the stem cells according to claims 1, differentiated cells derived therefrom or a pharmaceutical composition including the stem cells.

47. The kit according to claim 46 comprising additional excipients or reagents.

14. A primary stem cell culture obtained from exocrine glandular tissue, wherein a majority of living cells present in the culture are undifferentiated pluripotent adult stem cells.