Undifferentiated stem cell culture systems

• US 8,597,947 B2
• Filed: 04/02/2007
• Issued: 12/03/2013
• 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
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US11/730,560
• Publication Number:
  US 20070212777A1
• Time in Patent Office: 2,437 Days
• Field of Search: None
• US Class Current: 435/384
• 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 human pluripotent stem cells (SCs), the method comprising

incubating human pluripotent SCs in suspension within a culture system comprising basic medium, a knockout serum replacement (KOSR) and a TGFβ

superfamily factor,

wherein the human pluripotent SCs do not undergo differentiation during the expansion.

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

3. The method of claim 1, wherein the suspension of human pluripotent SCs comprises free floating cells, free floating clusters of cells or free floating aggregates of cells.

4. The method of claim 1, wherein the basic medium has the following composition:

5. The method of claim 1, wherein the culture system further comprises one or more element selected from the group consisting of a member of the FGF family, an extracellular matrix (ECM) component, an antibacterial agent, a non-essential amino acid, a neurotrophin, nicotinamide (NA), a bone morphogenic protein (BMP) antagonist and a serum free medium supplement.

6. The method of claim 5, wherein the ECM is selected from the group consisting of fibronectin, laminin and gelatin;

the antibacterial agent is selected from the group consisting of penicillin and streptomycin;

the TGFβ

superfamily factor is activin A;

the BMP antagonist is selected from the group consisting of noggin, chordin and gremlin;

the neurotrophin is selected from the group consisting of BDNF, NT3 and NT4; and

the serum free medium supplement is Nutridoma-CS.

7. The method of claim 1, wherein the human pluripotent SCs comprise human embryonic stem cells (hESCs).

8. The method of claim 1, wherein the expanding is effected in a bioreactor.

17. The method of claim 1, wherein the TGFβ

superfamily factor is activin A.

18. The method of claim 1, wherein the culture system further comprises a member of the FGF family.

19. The method of claim 18, wherein the member of the FGF family factor is FGF-2.

9. A culture system comprising a suspension of human pluripotent stem cells (SCs) in a culture medium comprising a basic medium, knockout serum replacement (KOSR), and a TGFβ

superfamily factor.

10. The culture system of claim 9 wherein the suspension of human pluripotent stem cells comprises free floating cells, free floating clusters of cells or free floating aggregates of cells.

11. The culture system of claim 9, wherein the basic medium has the following composition:

12. The culture system of claim 9, further comprising one or more element selected from the group consisting of a member of FGF family, an extracellular matrix (ECM) component, an antibacterial agent, a non-essential amino acid, a neurotrophin, nicotinamide (NA), a bone morphogenic protein (BMP) antagonist and a serum free medium supplement.

13. The culture system of claim 12, wherein the FGF member is FGF-2, the ECM is selected from the group consisting of fibronectin, laminin and gelatin;

the antibacterial agent is selected from the group consisting of penicillin and streptomycin;

the TGFβ

superfamily factor is activin A;

the BMP antagonist is selected from the group consisting of noggin, chordin and gremlin;

the neurotrophin is selected from the group consisting of BDNF, NT3, and NT4; and

the serum free medium supplement is Nutridoma-CS.

14. The culture system of claim 9, wherein the human pluripotent SCs are pluripotent, human embryonic SCs (hESCs).

15. The culture system of claim 9, being free of laminin.

16. The culture system of claim 9, wherein the human pluripotent SCs are cultured in a bioreactor.

20. A culture system comprising a suspension of human pluripotent stem cells (SCs) within in a culture medium comprising a basic medium, knockout serum replacement (KOSR), a TGFβ

superfamily member, and one or more element selected from the group consisting of a member of the FGF family, an extracellular matrix (ECM) component, an antibacterial agent, a non-essential amino acid, a neurotrophin, nicotinamide (NA), a bone morphogenic protein (BMP) antagonist and a serum free medium supplement.