Method for efficient transfer of human blastocyst-derived stem cells (hBS cells) from a feeder-supported to a feeder-free culture system, long-term propagation of hBS cells under feeder-free conditions and use of cultured hBS cells for applications in myocardial regeneration

• US 7,638,328 B2
• Filed: 05/10/2004
• Issued: 12/29/2009
• Est. Priority Date: 05/08/2003
• Status: Expired due to Fees

• Current Assignee: Takara Bio Europe AB
• Original Assignee: Cellartis AB
• Inventors: Eriksson, Peter, Kilmare, Eva Karin, Tallheden, Tommi, Enerback, Sven
• Primary Examiner(s): Bertoglio; Valarie
• Application Number:
  US10/555,694
• Publication Number:
  US 20070010010A1
• Time in Patent Office: 2,059 Days
• Field of Search: 435/366, 435/325, 435/374, 435/379, 435/378, 435/375, 435/395
• 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 transferring human blastocyst-derived stem cells (hBS cells) into a feeder-free culture system, the method comprising the steps of:

a) mechanically dissociating without an enzymatic treatment hBS cells grown on a feeder cell layer;

b) transferring the mechanically dissociated hBS cells to a feeder cell free culture; and

c) culturing the hBS cells under feeder-free growth conditions in a suitable growth medium and on a suitable support substrate, wherein said suitable growth medium is a cell-free medium conditioned by previous exposure to feeder cells, and said suitable support substrate comprises a cell-free matrix comprising extracellular matrix proteins.

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

d) following step c), passaging the hBS cells every 3-10 days by mechanical and/or enzymatic treatment.

3. The method of claim 1, wherein the mechanical dissociation in step a) is performed by dissection of the hBS cells by means of a suitable cutting tool.

4. The method of claim 3, wherein step a) comprises cutting pieces from the center of a colony of hBS cells grown on a feeder cell layer and step b) comprises transferring the pieces to a feeder cell free culture as cell clusters.

5. The method of claim 4, wherein the cell clusters are dissected mechanically one or more times prior to transferring the cell clusters to the feeder free culture.

6. The method of claim 5, wherein the mechanical dissection is performed until the cell clusters have a size that is at least 50% of that of the original colony.

7. The method of claim 6, wherein the size is determined as the diameter of the cluster or colony, respectively.

8. The method of claim 1, wherein said support substrate further comprises a component that inhibits differentiation and/or promotes survival and proliferation and/or adhesion of the hBS cells of step c).

9. The method of claim 1, wherein said growth medium further comprises factors that inhibit differentiation and/or promote survival and proliferation of the hBS cells of step b).

10. The method of claim 1, wherein the hBS cells of step c):

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

vii) are capable of differentiation.

11. The method of claim 2, wherein the hBS cells of step d) have the ability to differentiate into cells which express at least one cardiomyocyte marker and/or at least one cardiomyocyte-specific gene.

12. The method of claim 11, wherein the at least one cardiomyocyte marker is selected from the group consisting of α

-myosin heavy chain, α

-actin, troponin I, and troponin II; and

wherein the at least one cardiomyocyte-specific gene is selected from the group consisting of GATA4, Mkx2.5, α

-MHC, β

-MHC and ANF.

13. The method of claim 11, wherein the cells which express at least one cardiomyocyte marker and/or at least one cardiomyocyte-specific gene form contracting colonies, which are able to increase or decrease their frequency when a or B agonists or antagonists are administered to the culturing media.

14. The method of claim 11, wherein the amount of cells which express at least one cardiomyocyte marker and/or at least one cardiomyocyte-specific gene is higher than 25% of the total amount of the cells within the culture.

15. The method of claim 2, wherein step d) comprises passaging the hBS cells in feeder-free culture every 3-10 days by mechanical treatment.

16. The method of claim 15, wherein the enzyme is a collagenase.

17. The method of claim 16, wherein the collagenase is collagenase IV.

18. The method of claim 15, wherein step d) comprises dissociation of the hBS cells by subjecting the hBS cells to an EDTA solution.

19. The method of claim 18, wherein the concentration of the EDTA solution is at the most about 100 mM.

20. The method of claim 19, wherein the concentration of the EDTA solution is selected from the following group of concentrations:

from about 0.1 to about 100 mM, from about 0.2 to about 75 mM, from about 0.3 to about 50 mM, from about 0.4 to about 25 mM, about 20 mM or less, about 15 mM or less, about 10 mM or less, about 5 mM or less, about 1 mM or less, and about 0.5 mM or less.