Methods for producing single pluripotent stem cells and differentiation thereof

• US 10,316,293 B2
• Filed: 06/10/2015
• Issued: 06/11/2019
• Est. Priority Date: 07/01/2007
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Nelson, Shelley
• Primary Examiner(s): Bertoglio, Valarie E
• Application Number:
  US14/735,841
• Publication Number:
  US 20150275179A1
• Time in Patent Office: 1,462 Days
• Field of Search: 435380, 435377
• 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• 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 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0602 Vertebrate 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 5/0608 Germ cells production of embryos, nuclear transfer A01K67/027
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention relates to the field of pluripotent stem cell culture and methods facilitate pluripotent stem cell culture at industrial levels.

Claims

1. A method for differentiating pluripotent stem cells, comprising the steps of:

a) culturing the pluripotent stem cells as clusters in conditioned medium;

b) releasing the pluripotent stem cells as single cells by treatment with TrypLE™

SELECT or TrypLE™

EXPRESS in a concentration of from about 0.5 g/l to about 2.5 g/l for two minutes, wherein from 2.6 to 3.15×

10⁵ single cells are released from the pluripotent stem cell clusters, and wherein there is no gain of chromosome abnormalities after releasing the single cells from the clusters; and

c) differentiating the single pluripotent stem cells plated on a tissue culture substrate into definitive endoderm cells.

2. The method of claim 1, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

SELECT.

3. The method of claim 1, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS.

4. The method of claim 1, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

SELECT or TrypLE™

EXPRESS at a concentration of about 0.5 g/l.

5. The method of claim 1, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

SELECT or TrypLE™

EXPRESS at a concentration of about 2.5 g/l.

6. The method of claim 3, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration from about 1×

to about 0.001×

.

7. The method of claim 3, wherein the pluripotent stem cells are released as single cells by treatment with TrypLE™

EXPRESS at a concentration of 1×

.

8. The method of claim 1, wherein the tissue culture substrate is selected from the group consisting of MATRIGEL™

, fibronectin, laminin, human serum and collagen.

9. The method of claim 8, wherein the tissue culture substrate is MATRIGEL™

.

10. The method of claim 9, wherein the MATRIGEL™

is used at a dilution from about 1;

30 to about 1;

10.

11. The method of claim 10, wherein the MATRIGEL™

is used at a dilution of 1;

10.

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

13. The method of claim 12, wherein the embryonic stem cells are human.

14. The method of claim 1, wherein the single pluripotent stem cells are differentiated into definitive endoderm cells by culturing the cells in a medium comprising activin A.

15. The method of claim 14, wherein the definitive endoderm cells are further differentiated into one or more of pancreatic endoderm cells, pancreatic endocrine cells, pancreatic hormone-secreting cells or β

-cells.

17. The method of claim 1, wherein the step of releasing comprises treatment with TrypLE™

SELECT or TrypLE™

EXPRESS followed by centrifugation.

19. The method of claim 1, wherein the step of releasing results in no loss in pluripotency.

21. The method of claim 1, wherein the step of differentiating the single pluripotent stem cells comprises culturing the cells in a medium supplemented with a Gsk3b inhibitor.

23. The method of claim 1, wherein the method further comprises passaging the single cells prior to differentiation.

16. A method for differentiating pluripotent stem cells, comprising the steps of:

a) culturing the pluripotent stem cells as clusters in MEF conditioned medium;

b) releasing the pluripotent stem cells as single cells by treatment with a recombinant trypsin for at least two minutes, wherein from 2.6 to 3.15×

10⁵ single cells are released from the pluripotent stem cell clusters, and wherein there is no gain of chromosome abnormalities after releasing the single cells from the clusters; and

c) differentiating the single pluripotent stem cells plated on a tissue culture substrate or a MEF feeder layer into definitive endoderm cells.

18. The method of claim 16, wherein the step of releasing comprises treatment with the recombinant trypsin followed by centrifugation.

20. The method of claim 16, wherein the step of releasing results in no loss in pluripotency.

22. The method of claim 16, wherein the step of differentiating the single pluripotent stem cells comprises culturing the cells in a medium supplemented with a Gsk3b inhibitor.

24. The method of claim 16, wherein the method further comprises passaging the single cells prior to differentiation.

25. The method of claim 16, wherein the method comprises differentiating the single pluripotent stem cells plated on a MEF feeder layer into definitive endoderm cells.