Stem Cell Aggregate Suspension Compositions and Methods of Differentiation Thereof

• US 20100112691A1
• Filed: 11/04/2008
• Published: 05/06/2010
• Est. Priority Date: 11/04/2008
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Brandon, Eugene, Baetge, E. Edward, Bang, Anne, D'Amour, Kevin A., Yu, Xiaojie, Schulz, Thomas C., Robins, Allan J., Agulnick, Alan D., Kelly, Olivia, Green, Chad
• Granted Patent: US 8,008,075 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• 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 method of generating a hES-derived cell aggregate in suspension from a hES single cell suspension, comprising:

(a) culturing a hES cell in ani adherent growth culture condition which allows for expansion in an undifferentiated state;

(b) disassociating the adherent hES cell culture in (a) into a hES single cell suspension culture; and

(d) contacting the hES single cell suspension culture with a differentiating culture condition which allows for formation of hES-derived cell aggregates in suspension when agitated until such a period of time when the single cell suspension culture forms a hES-derived cell aggregate in suspension,

thereby generating a hES-derived cell aggregate in suspension.

2. The method of claim 1, wherein the hES single cell suspension culture is agitated for at least 30 minutes.

3. The method of claim 1, wherein the hES single cell suspension culture is agitated by orbital rotation of the hES single cell suspension culture at 80 rpm to 160 rpm

4. The method of claim 1, wherein the hES single cell suspension culture is agitated by orbital rotation of the hES single cell suspension culture at 100 rpm to 140 rpm.

5. The method of claim 1, wherein a plurality of hES-derived cell aggregates are produced in suspension and the diameters of the hES-derived cell aggregate in suspension are essentially uniform in size and shape.

6. The method of claim 1, wherein the hES-derived cell aggregate is an endoderm lineage-specific cell aggregate.

7. The method of claim 6, wherein the endoderm lineage-specific cell aggregate is selected from the group consisting of a pancreatic lineage type cell aggregate, a liver lineage type cell aggregate, an epithelial lineage type cell aggregate, a thyroid lineage cell aggregate and a thymus lineage cell aggregate.

8. The method of claim 6, wherein the endoderm lineage-specific cell aggregate is a pancreatic lineage type cell aggregate.

9. The method of claim 1, wherein the hES-derived cell aggregate is a multipotent pancreatic progenitor cell aggregate or an endocrine cell aggregate.

10. The method of claim 1, further comprising subsequent to the step of contacting with the differentiating culture condition, an additional step of selecting or purifying the desired hES-derived cell aggregate by use of one or more selectable markers or agents.

11. The method of claim 10, wherein the selectable marker or agent is selected from the group consisting of CD30, CD49a, CD49e, CD55, CD98, CD99, CD142, CD165, CD200, CD318, CD334 and CD340.

12. The method of claim 10, wherein the selecting or purifying of the desired hES-derived cell aggregates is by use of cell-sorting.

13. The method of claim 1, further comprising subsequent to the step of contacting with the differentiating culture condition, an additional step of enriching the culture with low concentrations of growth factors selected from the group consisting of FGF10, EGF, KGF, noggin and retinoic acid which allows for enrichment of pancreatic endoderm cells.

14. The method of claim 1, further comprising subsequent to the step of contacting with the differentiating culture condition, an additional step of enriching the culture with high concentrations of growth factors selected from the group consisting of FGF10, EGF, KGF, noggin and retinoic acid which allows for enrichment of pancreatic endocrine cells.

15. The method of claim 1, further comprising adding to the hES single cell suspension culture an agent which is capable of promoting cell survival, wherein the agent is selected from the group consisting of Y-27632, fasudil, H-1152P, and a mixture comprising insulin/transferrin/selenium (ITS).

16. The method of claim 1, wherein the adherent culture condition is devoid of substrate or matrix selected from the group consisting of extracellular matrix, extracellular matrix protein and human or mouse feeder layer.

17. The method of claim 1, wherein the adherent growth culture condition is xeno-free, matrix-free and feeder-free.

18. The method of claim 1, wherein the first differentiating culturing condition comprises a TGFbeta family member which activates a TGFbeta family member receptor, wherein the TGFbeta family member is selected from the group consisting of Activin A, Activin B, Nodal, GDF-8 and GDF-11.

19. A method of generating a hES-derived cell aggregate in suspension from a hES-derived single cell suspension, comprising:

(a) culturing a hES cell in an adherent growth culture condition which allows for expansion in an undifferentiated state;

(b) contacting the undifferentiated hES cell with a first differentiating culturing condition suitable for differentiating the hES cell and resulting in an adherent hES-derived cell;

(c) disassociating the adherent hES-derived cell into a single cell suspension culture; and

(d) contacting the single cell suspension culture with a second differentiating culture condition which allows for formation of hES-derived cell aggregates in suspension when agitated until such a period of time when the single cell suspension culture forms a hES-derived cell aggregate in suspension,

thereby generating a hES-derived cell aggregate in suspension.

20. The method of claim 19, wherein the second differentiating culturing condition comprises a non-TGFbeta family member selected from the group consisting of FGF10, EGF, KGF, noggin and retinoic acid.

21. A method for enriching pancreatic progenitor cells comprising using low concentrations of growth factors selected from the group consisting of FGF10, EGF, KGF, noggin and retinoic acid.

22. A method for enriching endocrine progenitor cells comprising using high concentrations of growth factors selected from the group consisting of FGF10, EGF, KGF, noggin and retinoic acid.

23. A method for enriching pancreatic progenitor cells from a hES cell culture comprising increasing the hES cell density culture from which is derived the pancreatic progenitor.

24. A method for enriching endocrine progenitor cells from a hES cell culture comprising decreasing the hES cell density culture from which is derived the pancreatic progenitor.