Stem cell aggregate suspension compositions and methods of differentiation thereof

• US 8,445,273 B2
• Filed: 08/29/2011
• Issued: 05/21/2013
• Est. Priority Date: 11/04/2008
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Green, Chad, Yu, Xiaojie, Bang, Anne, Brandon, Eugene, Kelly, Olivia, Schulz, Thomas C, Robins, Allan J, Agulnick, Alan D, Baetge, E Edward, D'Amour, Kevin A
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/220,590
• Publication Number:
  US 20120045830A1
• Time in Patent Office: 631 Days
• Field of Search: 435/363
• US Class Current: 435/363
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• 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 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/195 Heregulin, neu differentiation factor
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention relates to methods for production of undifferentiated or differentiated embryonic stem cell aggregate suspension cultures from undifferentiated or differentiated embryonic stem cell single cell suspensions and methods of differentiation thereof.

Claims

1. A bioreactor comprising primate pluripotent stem cell-derived cell aggregates in suspension, wherein the primate pluripotent stem cell-derived cell aggregates in suspension are generated by a method comprising:

a. providing a culture of undifferentiated adherent primate pluripotent stem cells;

b. disassociating the adherent primate pluripotent stem cell culture of step (a) into a primate pluripotent stem single cell suspension;

c. seeding the primate pluripotent stem cell single cell suspension in a defined culture medium that supports undifferentiated growth of the primate pluripotent stem cells, thereby forming a primate pluripotent stem cell single cell suspension culture;

d. agitating the primate pluripotent stem single cell suspension culture at low shear, wherein the primate pluripotent stem single cells in the suspension form substantially uniform primate pluripotent stem cell aggregates; and

e. contacting the primate pluripotent stem cell aggregates in suspension with a differentiating culture condition wherein primate pluripotent stem cell aggregates in suspension form primate pluripotent stem derived cell aggregates in suspension, thereby generating primate pluripotent stem derived cell aggregates in suspension.

2. The bioreactor of claim 1, wherein the differentiating culture condition comprises at least one component selected from the group consisting of members of the fibroblast growth factor family, epidermal growth factor family, keratinocyte growth factor family, growth and differentiation factor (GDF) family antagonists, retinoic acid, apoptotic inhibitors, TTNPB, Rho-kinase inhibitors, TGFβ

superfamily growth factors inhibitors, TGFβ

superfamily growth factors, WNT factor family, hedgehog factor family inhibitors, insulin like growth factor family inhibitors and notch inhibitors.

3. The bioreactor of claim 1, wherein the primate pluripotent stem cell aggregates are differentiated without passaging.

4. The bioreactor of claim 1, wherein the primate pluripotent stem cell aggregates are differentiated in a feeder-, matrix- and adherent growth culture -free environment.

5. The bioreactor of claim 1, wherein agitating the primate pluripotent stem single cell suspension culture consists of rotational culture.

6. The bioreactor of claim 5, wherein the primate pluripotent stem cell aggregates are rotated at about 60 rpm to 160 rpm.

7. The bioreactor of claim 1, wherein the adherent primate pluripotent stem cells are human embryonic stem cells.

8. The bioreactor of claim 1, wherein the bioreactor is a large scale culture.

9. The bioreactor of claim 1, wherein the bioreactor comprises a plurality of primate pluripotent stem cell-derived cell aggregates that are essentially uniform in size, shape or both size and shape.

10. The bioreactor of claim 1, wherein the primate pluripotent stem cell-derived cell aggregates are suspended in a physiologically acceptable culture medium.

11. The bioreactor of claim 10, wherein the culture medium is substantially free of insulin, IGF-1 or both insulin and IGF-1.

12. The bioreactor of claim 1, wherein the primate pluripotent stem cell-derived cell aggregates express at least one marker selected from the group consisting of brachyury, SOX17, FOXA2, HNF1 beta, PDX1, NKX6.1, NKX2.2, INS, GCG, SST, SOX7,and PTF1A.

13. The bioreactor of claim 1, wherein the primate pluripotent stem cell-derived cell aggregates are cells selected from the group consisting of endoderm, ectoderm and mesoderm.

14. The bioreactor of claim 1, wherein the primate pluripotent stem cell-derived cell aggregates are cell aggregates selected from the group consisting of definitive endoderm cell aggregates, foregut endoderm cell aggregates or PDX1+endoderm cell aggregates.

15. The bioreactor of claim 1, wherein the bioreactor is closed loop.

16. The bioreactor of claim 1, further comprising a means for controlling at least one environmental condition comprising ph, gases or temperature.

17. The bioreactor of claim 1, wherein the primate pluripotent stem cell-derived cell aggregates are batch fed.

18. The bioreactor of claim 1, wherein the bioreactor comprises a vessel with a fluid nutrient medium in which the primate pluripotent stem cell-derived cell aggregates are suspended.