Differentiation and enrichment of islet-like cells from human pluripotent stem cells

• US 8,415,153 B2
• Filed: 05/25/2007
• Issued: 04/09/2013
• Est. Priority Date: 06/19/2006
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Majumdar, Anish Sen, Jiang, JianJie, Au, Melinda
• Primary Examiner(s): Paras, Jr., Peter
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/303,895
• Publication Number:
  US 20100240130A1
• Time in Patent Office: 2,146 Days
• Field of Search: 435/325, 435/366, 435/377
• US Class Current: 435/377
• 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

Methods for differentiating human pluripotent stem cells into islet-like cells are provided. In certain embodiments, the methods utilize sequential culturing of the human pluripotent stem cells with certain factors to produce islet-like cells. In certain embodiments, the population of cells produced by the methods is further enriched for islet-like cells.

Claims

1. A method of enriching the proportion of islet-like cells from a cell population generated by the differentiation of human pluripotent stem cells to islet-like cells, comprising:

a) differentiating human pluripotent stem cells into a cell population comprising islet-like cells by a method comprising,

i. contacting human pluripotent stem cells with a first medium comprising Activin A; and

ii. contacting the cells from step i) with a second medium comprising FGF-2 but lacking Activin A,

thereby generating a cell population comprising islet-like cells, wherein the cell population comprises cell clusters that have formed buds; and

b) separating at least some of the buds from at least some of the clusters in the cell population of step (a) into at least two fractions, wherein the fraction containing separated buds contains a higher proportion of insulin or glucagon-producing islet-like cells than the cell population of step (a) and wherein the buds are separated from the clusters based on the size difference between the buds and the clusters.

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

3. The method of claim 1, wherein the buds are separated from the clusters by use of nylon mesh.

4. The method of claim 3, wherein the nylon mesh is a 200 micron mesh.

5. The method of claim 3, wherein the nylon mesh is a 70 micron mesh.

6. The method of claim 3, wherein the nylon mesh is between 50 and 250 microns.

7. The method of claim 1, wherein at least one of the fractions with a higher proportion of islet-like cells than the cell population of step (a) is a fraction with a smaller particle size than at least one of the other fractions that result from the size separation.

8. The method of claim 7, wherein the at least one fraction with a higher proportion of islet-like cells than the cell population of step (a) has a particle size equal to or less than 200 microns.

9. The method of claim 7 wherein the at least one fraction with a higher proportion of islet-like cells than the cell population of step (a) has a particle size equal to or less than 150 microns.

10. The method of claim 7, wherein the at least one fraction with a higher proportion of islet-like cells than the cell population of step (a) has a particle size equal to or less than 100 microns.

11. The method of claim 1, wherein the cell population of step (a) is fractionated into three fractions by performing two size separation steps, wherein the second separation step is performed on a fraction that results from the first separation step.

12. The method of claim 11, wherein the first separation step is performed using a size separation value equal to or less than 50 microns and the second separation step is performed using a size separation value between 100 microns and 300 microns on the retained fraction from the first separation step;

and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population of step (a).

13. The method of claim 12, wherein the first separation step is performed using a size separation value of 50 microns and the second separation step is performed using a size separation value of 150 microns, and wherein the 50 to 150 micron fraction contains higher proportion of islet-like cells than the cell population of step (a).

14. The method of claim 11, wherein the first separation step is performed using a size separation value equal to or less than 50 microns and the second separation step is performed using a size separation value between 150 microns and 250 microns on the retained fraction from the first separation step;

and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population of step (a).

15. The method of claim 14, wherein the first separation step is performed using a size separation value of 50 microns and the second separation step is performed using a size separation value of 200 microns, and wherein the 50 to 200 micron fraction contains higher proportion of islet-like cells than the cell population of step (a).

16. The method of claim 11, wherein the first separation step is performed using a size separation value equal to or less than 20 microns and the second separation step is performed using a size separation value of 100 microns or greater on the retained fraction from the first separation step;

and wherein the pass-through fraction from the second separation step contains a higher proportion of islet-like cells than the cell population of step (a).

17. The method of claim 16, wherein the first separation step is performed using a size separation value of 20 microns and the second separation step is performed using a size separation value of 200 microns, and wherein the 20 to 200 micron fraction contains higher proportion of islet-like cells than the cell population of step (a).

18. The method of claim 1, wherein the buds are dissociated from the clusters before the separation step b).

19. The method of claim 18, wherein the buds are dissociated from the clusters by repeated pipetting.

20. The method of claim 18, wherein the buds are dissociated from the clusters by enzyme treatment.

21. The method of claim 20, wherein the enzyme is Dispase.

22. The method of claim 20, wherein the enzyme is trypsin.

23. The method of claim 1, wherein the buds are separated from the clusters by manually or robotically picking the buds.

24. The method of claim 1, wherein the buds are separated from at least some of the clusters using flow cytometry.