Undifferentiated stem cell culture systems

• US 20070212777A1
• Filed: 04/02/2007
• Published: 09/13/2007
• Est. Priority Date: 12/29/2004
• Status: Active Grant

• Current Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Original Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Inventors: Reubinoff, Benjamin
• Granted Patent: US 8,597,947 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• 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

Abstract

The present application discloses methods expanding SCs in an undifferentiated state, the methods comprising incubating undifferentiated SCs in suspension within a culture system comprising basic medium and knockout serum replacement (KOSR). The methods may also be applicable for selective spontaneous or directed differentiation of SCs into a selected population of somatic cells from a culture system of SCs in suspension, the method further comprising incubating said undifferentiated SCs in culture system that support respectively, spontaneous or directed differentiation of SCs into the selected population of somatic cells. The present application also discloses a culture system for expansion of stem cells (SCs) comprising a suspension of undifferentiated stem cells within basic medium and knockout serum replacement (KOSR). The methods and culture system of the invention may be used for large scale production of differentiated cells.

Claims

1. A method of expanding SCs in an undifferentiated state, the method comprising incubating undifferentiated SCs in suspension within a culture system comprising basic medium and knockout serum replacement (KOSR).

2. The method of claim 1, wherein said undifferentiated SCs are derived from SC colonies cultivated on a feeder layer or in a feeder free adherent culture system.

3. The method of claim 1, wherein the undifferentiated SCs in said suspension are in form of free floating cells, free floating clusters of cells or free floating aggregates of cells.

4. The method of claim 1, wherein said basic medium is Neurobasal™

.

5. The method of claim 1, wherein said culture system comprises one or more of the following:

a member of FGF family;

a extracellular matrix (ECM) component;

an antibacterial agent;

nonessential amino acids;

a TGFβ

superfamily factor;

a neurotrophin;

nicotinamide (NA);

a bone morphogenic protein (BMP) antagonist;

or

a serum free medium supplement.

6. The method of claim 5, wherein said ECM is selected from fibronectin and gelatin;

said antibacterial agent is selected from penicillin and streptomycin;

said TGFβ

superfamily factor is selected from activin A;

said BMP antagonist is selected from noggin, chordin; and

said serum free medium supplement is selected from Nutridoma-CS or TCH™

.

7. The method of claim 1, for expanding pluripotent hESCs in an undifferentiated state.

8. The method of claim 1, for large-scale expansion and/or long term cultivation of undifferentiated SCs in suspension.

34. The method of any one of claims 1 for large scale expansion and/or long term expansion of pluripotent hESCs in a bioreactor.

9. A method for directing differentiation of SCs into a selected population of somatic cells from a culture system of SCs in suspension, the method comprising:

(a) providing a suspension of undifferentiated SCs comprising a basic medium and knockout serum replacement (KOSR); and

(b) incubating said undifferentiated SCs in culture system that support directed differentiation of SCs into the selected population of somatic cells.

10. The method of claim 9, wherein said SCs are pluripotent hESC.

11. The method of claim 9, wherein the somatic cells are selected from mesodermal, endodermal or ectodermal cells.

12. The method of claim 9, wherein said selected population of somatic cells consists essentially of a single population of differentiated somatic cells.

13. The method of claim 9, wherein said population of somatic cells consists essentially of neural precursor cells.

14. The method of claim 13, wherein the culture system comprises a basic media, FGF2 and/or noggin.

15. The method of claim 12, wherein said selected population of somatic cells consists essentially of dopaminergic neuronal cells.

16. The method of claim 15, comprising directing differentiation of SCs into neural precursor cells and incubating said neural precursor cells in a culture system supporting differentiation of neural precursor cells into dopaminergic neuronal cells.

17. The method of claim 16, wherein said culture system supporting differentiation of neural precursor cells into dopaminergic neuronal cells comprises at least one of sonic hedgehog (SHH), an fibroblast growth factor (FGF), or a member of the Wnt family.

18. The method of claim 9, wherein said SCs are pluripotent hESC.

35. The method of claims 9 for large scale differentiation of pluripotent hESCs in a bioreactor.

19. A method for promoting spontaneous differentiation of SCs into somatic cells, the method comprising:

(a) providing a suspension of undifferentiated SCs in a culture system comprising a basic medium and knockout serum replacement (KOSR);

(b) incubating said undifferentiated SCs in culture system that support spontaneous differentiation of SCs into the somatic cells.

20. The method of claim 19, wherein said somatic cells comprise a mixture or any one of mesodermal cells, ectodermal cells, endodermal cells and combination of same.

21. The method of claim 19, wherein said

22. A culture system for expansion of stem cells (SCs) comprising a suspension of undifferentiated stem cells within basic medium and knockout serum replacement (KOSR).

23. The culture system of claim 22 wherein said suspension of stem cells comprises free floating cells, free floating clusters of cells or free floating aggregates of cells.

24. The culture system of claim 22, wherein said the basic medium is Neurobasal™

.

25. The culture system of claim 22, comprising one or more of the following:

a member of FGF family;

an extracellular matrix (ECM) component;

an antibacterial agent;

non-essential amino acids

a TGFβ

superfamily factor;

a neurotrophin;

nicotinamide (NA);

a bone morphogenic protein (BMP) antagonist;

or

a serum free medium supplement.

26. The culture system of claim 25, wherein said FGF member is FGF-2, ECM is selected from fibronectin, laminin and gelatin;

said antibacterial agent is selected from penicillin and streptomycin;

said TGFβ

superfamily factor is activin A;

said BMP antagonist is selected from noggin, chordin, or gremlin, said neurotrophin is selected from BDNF, NT3, NT4; and

said a serum free medium supplement is selected from Nutridoma-CS or TCH™

.

27. The culture system of claim 22, wherein said SCs are embryonic SCs.

28. The culture system of claim 22, wherein said SCs are pluripotent, human embryonic SCs (hESCs).

29. The culture system of claim 22, being free of laminin.

30. The culture system of claim 22, wherein said SCs are expanded for long term and/or in large scale in an undifferentiated state.

31. Use of KOSR for the preparation of a culture system for the maintenance of SCs in an undifferentiated pluripotent state.

32. Use of claim 31, wherein said SCs are pluripotent hESCs.

33. Use of claim 31, for large scale and/or long term expansion of said SCs in a bioreactor.