Compositions and methods useful for culturing differentiable cells

• US 8,658,352 B2
• Filed: 03/12/2013
• Issued: 02/25/2014
• Est. Priority Date: 02/23/2006
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Robins, Allan J., Schulz, Thomas C.
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/795,628
• Publication Number:
  US 20130309708A1
• Time in Patent Office: 350 Days
• Field of Search: None
• US Class Current: 435/4
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/195 Heregulin, neu differentiation factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• 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: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention relates to cell culture methods and compositions that are essentially serum-free and comprise a basal salt nutrient solution and an ErbB3 ligand.

Claims

1. A method of identifying a compound that modulates proliferation and/or differentiation of pluripotent stem cells, the method comprising:

culturing the pluripotent stem cells in a cell medium comprising a basal salt nutrient solution, an ErbB3 ligand, and a candidate compound, wherein the cell medium is essentially free of serum, wherein the pluripotent stem cells can give rise to all three germ layers, and wherein the ErbB3 ligand can bind to an ErbB3 receptor, which in turn dimerizes with an ErbB2 receptor to form an ErbB2/ErbB3 heterodimer, thereby activating the tyrosine kinase activity of the ErbB2 receptor in the ErbB2/ErbB3 heterodimer;

determining whether an increase or decrease in proliferation and/or differentiation occurs in the pluripotent stem cells cultured with the compound relative to a control, wherein the increase or decrease indicates that the compound modulates proliferation and/or differentiation of the pluripotent stem cells.

2. The method of claim 1, wherein the culturing comprises culturing the pluripotent stem cells on a solid surface in an adherent culture.

3. The method of claim 1, wherein the culturing comprises culturing the pluripotent stem cells in a suspension culture.

4. The method of claim 1, wherein the cell medium is free of exogenous insulin and insulin substitutes.

5. The method of claim 1, wherein the cell medium further comprises insulin-like growth factors or a functional fragment thereof.

6. The method of claim 1, wherein the ErbB3 ligand in the cell medium is selected from the group consisting of Neuregulin-β

, Heregulin-β

(HRG-β

), Heregulin-α

(HRG-α

), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β

(NRG2-β

), Epiregulin, Biregulin and functional fragments thereof.

7. The method of claim 6, wherein the ErbB3 ligand is HRG-β

or a functional fragment thereof.

8. The method of claim 7, wherein the cell medium further comprises transforming growth factor beta (TGF-β

), a TGF-β

family member or a functional fragment thereof.

9. The method of claim 8, wherein the TGF-β

family member is selected from the group consisting of Nodal, Activin A, Activin B, bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-4 (BMP4), and functional fragments thereof.

10. The method of claim 9, wherein said TGF-β

family member is Activin A.

11. The method of claim 10, wherein the cell medium further comprises insulin-like growth factors or a functional fragment thereof.

12. The method of claim 11, wherein the cell medium is free of exogenous fibroblast growth factor.

13. The method of claim 11, wherein the cell medium further comprises at least one fibroblast growth factor (FGF) selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 and variants and functional fragments thereof.

14. The method of claim 13, wherein the at least one FGF is FGF-7, FGF-10 and FGF-22.

15. The method of claim 14, wherein the cell medium comprises a serum albumin (SA).

16. The method of claim 15, wherein the SA is bovine SA (BSA) or human SA (HSA).

17. The method of claim 15, wherein the concentration of the SA is more than about 0.2%, volume to volume (v/v).

18. The method of claim 15, wherein the concentration of SA is less than about 5% v/v.

19. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells.

20. The method of claim 1, wherein the pluripotent stem cells are selected from the group consisting of ES cells, EPL cells, ICM/epiblast cells, EG cells, and pluripotent cells derived by dedifferentiation or nuclear transfer.