Differentiation of pluripotent stem cells

• US 9,593,305 B2
• Filed: 03/29/2012
• Issued: 03/14/2017
• Est. Priority Date: 06/30/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Bonnet, Pascal Ghislain Andr, Davis, Janet, Liu, Jiajian, Parmenter, Christine
• Primary Examiner(s): Popa, Ileana
• Application Number:
  US13/434,370
• Publication Number:
  US 20120190111A1
• Time in Patent Office: 1,811 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• 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 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/845 Gamma amino butyric acid [GABA]
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/90 : Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention is directed to methods and compositions to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage comprising culturing the pluripotent stem cells in medium comprising a sufficient amount of GDF-8 to cause the differentiation of the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage.

Claims

1. A method of increasing the yield of cells expressing markers characteristic of the definitive endoderm lineage by differentiating human pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, the method comprising treating the human pluripotent stem cells with a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one, for a period of time sufficient for the human pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage, wherein the method increases the percentage of cells expressing CXCR4.

2. The method of claim 1, wherein the method increases the percentage of cells expressing CXCR4 when compared to cells treated in a medium comprising activin A.

3. The method of claim 1, wherein the medium is further lacking Wnt3A.

4. The method of claim 1, wherein the medium further comprises at least one factor selected from the group consisting of:

EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate.

17. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

5. A method of enhancing the differentiation of pluripotent stem cells comprising culturing the pluripotent stem cells for about one to about three days in a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one to differentiate the pluripotent cells into definitive endoderm cells.

6. The method of claim 5, wherein the method increases the percentage of cells expressing CXCR4 when compared to cells treated in a medium comprising activin A.

7. The method of claim 5, wherein the medium is further lacking Wnt3A.

8. The method of claim 5, wherein the medium further comprises at least one factor selected from the group consisting of:

EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate.

9. The method of claim 5, wherein the medium comprises from about 0.625 μ

M to about 10 μ

M of 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12-]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one.

10. The method of claim 5, wherein the method comprises culturing the pluripotent stem cells for about three days in a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12-]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one.

11. A method of increasing the yield of definitive endoderm cells by differentiating pluripotent stem cells into definitive endoderm cells comprising:

culturing the pluripotent stem cells in a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12-]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one; and

culturing the cells in a medium lacking activin A, and containing GDF-8 to differentiate the pluripotent stem cells into definitive endoderm cells, wherein the method increases the percentage of cells expressing CXCR4.

12. The method of claim 11, wherein the pluripotent stem cells are cultured in the medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one for about one day.

13. The method of claim 11, wherein the cells are cultured in the medium lacking activin A, and containing GDF-8 for about two days.

14. The method of claim 11, wherein the medium lacking activin A further lacks Wnt3A.

15. The method of claim 11, wherein the method increases the percentage of cells expressing CXCR4 when compared to cells treated in a medium comprising activin A.

16. The method of claim 11, wherein the medium further comprises at least one factor selected from the group consisting of:

EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate.

18. A method of differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage comprising treating the pluripotent stem cells with a medium lacking activin A, and containing GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one, for a period of time sufficient for the pluripotent stem cells to differentiate into cells expressing markers characteristic of the definitive endoderm lineage.

19. The method of claim 18, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

20. The method of claim 18, wherein the medium further comprises at least one factor selected from the group consisting of:

EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D, VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium butyrate.

21. The method of claim 18, wherein the cells are cultured in the medium lacking activin A, and containing GDF-8 for about one to seven days.

22. The method of claim 18, wherein the medium is further lacking Wnt3A.