Method for efficient transfer of human blastocyst-derived stem cell (hbs cells) from a feeder-supported to a feeder-free culture system

• US 20070010010A1
• Filed: 05/10/2004
• Published: 01/11/2007
• Est. Priority Date: 05/08/2003
• Status: Active Grant

• Current Assignee: Takara Bio Europe AB
• Original Assignee: Cellartis AB
• Inventors: Eriksson, Peter, Tallheden, Tommi, Enerback, Sven, Karin, Eva
• Granted Patent: US 7,638,328 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0231 Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
• 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: 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 9/04 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 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

A method for the transfer of human blastocyst-derived stem cells (hBS cells) to feeder-free culture system and propagation of the cells in such a feeder-free culture system, the method comprising the following steps of (a) transferring the balstocyst-derived stem cells from feeder to feeder free culture by mechanical treatment, (b) optionally, culturing the blastocyst-derived stem cells under feeder cell free growth conditions in a suitable growth medium and/or on a suitable support substrate, and (c) optionally passaging the blastocyst derived stem cell line every 3-10 days by enzymatic and/or mechanical treatment. The invention also relates to the application of hBS cells cultured under feeder free condition in medicine (e.g., myocardial regeneration) and screening and toxicity tests.

Claims

1. A method for transfer hBS cells into a feeder-free culture system, the method comprising the step of:

a. transferring the hBS cells from feeder to feeder-free culture without enzymatic treatment but solely by mechanical treatment.

2. The method of claim 1 further comprising the step of

b. culturing the blastocyst derived stem cells under feeder cell free growth conditions in a suitable growth medium and/or on a suitable support substrate.

3. The method of claim 1 further comprising the step of:

c. passaging the blastocyst derived stem cell line every 3-10 days by mechanical treatment.

4. The method of claim 1, wherein the mechanical treatment is performed by dissociation or dissection of the hBS in the feeder culture system by means of a suitable cutting tool.

5. The method according to claim 4, wherein the hBS cells to be transferred is a colony of hBS cells, pieces is cut from the centre of the colony and suspended in a suitable medium as cell clusters.

6. The method according to claim 4, wherein the cell clusters are dissociated mechanically one or more times.

7. The method according to claim 6, wherein the mechanical dissociation is performed until the cell clusters have a size that is at least 50% of that of the original colony.

8. The method of claim 7, wherein the size is determined as the diameter of the cluster or colony, respectively.

9. The method according to claim 2, wherein step b) is performed on a support substrate comprising a component, that inhibits differentiation and/or promotes survival and proliferation and/or adhesion of the blastocyst-derived stem cells and/or an extra cellular matrix component.

10. The method according to claim 9, wherein the extra cellular matrix component is Matrigel™

.

11. The method according to claim 2, wherein step b) comprises the use of a growth medium comprising factors that inhibits differentiation and/or promotes survival and proliferation of the hBS cells.

12. A method according to claim 11, wherein the growth medium is a cell-free medium, selected from the group consisting of k-hBS-medium, k-VitroHES™

-medium or VitroHES™

-medium conditioned by previous exposure of feeder cells.

13. A method according to claim 3, wherein step c) comprises dissociation of the hBS cells by enzymatic treatment.

14. A method according to claim 13, wherein the enzyme is a collagenase.

15. A method according to claim 3, wherein step c) comprises dissociation of the hBS cells by mechanical dissection.

16. A method according to claim 3, wherein step c) comprises dissociation of the hBS cell by subjecting the hBS cells to an EDTA solution.

17. A method according to claim 16. wherein the concentration of the EDTA solution is at the most about 100 mM.

18. A method according to claim 1, wherein the bBS cells:

i) exhibit proliferation capacity in an undifferentiated state for more than 12 months when grown under feeder free growth conditions,

ii) exhibit normal euploid chromosomal karyotype,

iii) maintain potential to develop into derivatives of all types of germ layers both in vitro and in vivo,

iv) exhibit at least two of the following markers OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81, and the protein core of a keratin sulfate/chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody GCTM-2,

v) do not exhibit marker SSEA-1 or other differentiation markers,

vi) retain its pluripotency and forms teratomas in vivo when injected into immuno-compromised mice, and/or

vii) are capable of differentiate.

19. A method according to claim 3, wherein passaged hBS cells have the ability of further differentiating into cardio-like cells relating to cardiac tissue.

20. A method to claim 19. wherein the passaged hBS cells have the ability of differentiating into cardio-like cells relating to cardiac tissue, which display the expression of cardiomyocyte markers, including at least one of α

-myosin heavy chain α

-actin, troponin land troponin II or at least one of the cardiomyocyte specific genes, including GATA4, Mkx2.5, α

-MHC, β

-MHC or ANF.

21. A method according to claim 19, wherein the cardio-like cells are cardiomyocyte-like cells characterized by their organization into contracting colonies, which are able to increase or decrease their frequency when α

or β

agonists or antagonists are administered to the culturing media.

22. A method according to claim 19, wherein the amount of cardio-like cells which are derived from the pluripotent human BS cell line is higher than 25% of the total amount of the cells within the culture.

23. The method according to claim 1, further comprising the step of:

c. passaging the blastocyst derived stem cell line every 3-10 days by enzymatic and/or mechanical treatment.

24. A medicine comprising an effective concentration of the hBS cells obtained by a method of claim 1.

25. A medicament for transplantation of hBS cells into a mammal for the prevention or treatment of a disease comprising an effective concentration of cells obtained by the method of claim 1.

26. A medicament for the prevention or treatment of cardio-related diseases comprising an effective concentration of cells obtained by the method of claim 1.

27. A medicament for transplantation of human blastocyst-derived stem cells into the myocardium or the circulation of a mammal for the prevention or treatment of a cardio-related disease comprising an effective concentration of cells obtained by the method of claim 1.

28. A medicament according to claim 25 wherein the disease is selected from the group consisting of late myocardial infarction, chronic ischemic cardiomyopathy, idiopathic dilated cardiomyopathy, secondary cardiomyopathies such as toxic, diabetic, pregnancy, amyloidosis, sarcoidosis, Fabry and haemochromatosis, end stage hypertrophic cardiomyopathy with LV dysfunction or heart failure, restrictive cardiomyopathy, end-stage hypertensive heart disease, acute myocardial infarction, angina pectoris, fulminant myocarditis, AV-block III, specific forms of congenital heart diseases in children and adults, such as noncompaction LV, atrial and ventricular septal defects tetralogi Fallot and other similar conditions, reconstruction of the valves and heart failure secondary to valvular disease.

29. A medicament according to claim 25 comprising undifferentiated hBS cells or differentiated hBS cells dispersed in a pharmaceutically acceptable medium.

30. A medicament according to claim 29 wherein the medium is an aqueous medium.

31. A medicament according to claim 29 comprising one or more additives selected from the group consisting of pH adjusting agents, stabilizers, preservatives, osmotic pressure adjusting agents, and physiologically acceptable salts.

32. A medicament according to claim 25 comprising one or more agents selected from the group consisting of therapeutically active substances, prophylactically active substances, engraftment improving agents, viability improving agents, differentiation improving agent and immunosuppressive agents.

33. A method for treating a cardio-related disease in a mammal comprising transplanting hBS cells obtained by the method of claim 1 into cardio-like cells in an effective amount to the mammal in need thereof.

34. A kit comprising at least two of the following components in separate compartments, an agent that improves the engraftment and viability of hBS cells, the hBS cells obtainable by the method of claim 1, one or more agents that improve differentiation of hBS cell and one or more pharmaceutical and/or immunosuppressive agents.

35. A kit according to claim 34, further comprising a second cell-type that improves engraftment and survival of the hBS cells.

36. A kit according to claim 34 further comprising undissoclated or dissociated differentiated hBS-cell colonies.

37. A method for the production of monoclonal antibodies comprising utilizing the hBS cells obtained by the method of claim 1.

38. A method for in vitro toxicity testing comprising utilizing the hBS cells obtained by the method of claim 1.

39. A method for in vitro screening of potential drug substances comprising utilizing the hBS cells obtained by the method of claim 1.

40. A method for identification of potential drug substances comprising utilizing the hBS cells obtained by the method of claim 1.