Differentiation of pluripotent stem cells

• US 10,233,421 B2
• Filed: 03/13/2017
• Issued: 03/19/2019
• Est. Priority Date: 06/30/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Davis, Janet, Liu, Jiajian, Parmenter, Christine, Bonnet, Pascal Ghislain Andre
• Primary Examiner(s): Popa, Ileana
• Application Number:
  US15/457,893
• Publication Number:
  US 20170183625A1
• Time in Patent Office: 736 Days
• Field of Search: None
• 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 to differentiate pluripotent stem cells into definitive endoderm cells comprising culturing the pluripotent stem cells with a medium supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜

3,7˜

.1˜

9,13˜

.1˜

14,18˜

] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one and one or more of EGF and FGF4.

2. The method of claim 1, wherein the medium is supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜

3,7˜

.1˜

9,13˜

.1˜

14,18˜

] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one and EGF.

3. The method of claim 1, wherein the medium is supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜

3,7˜

.1˜

9,13˜

.1-14,18˜

] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one and FGF-4.

4. The method of claim 1, wherein the medium is supplemented with activin A, Wnt3a, 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜

3,7˜

.1˜

9,13˜

.1˜

14,18˜

] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one EGF and FGF-4.

5. The method of claim 4, wherein the medium is further supplemented with PDGF-AB.

6. The method of claim 5 wherein the medium is further supplemented with VEGF.

7. The method of claim 4, wherein the medium is further supplemented with GDF-8 and muscimol.

8. The method of claim 4, wherein the medium is further supplemented with PDFG-A, VEGF, PDGF, muscimol and GDF-8.

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

10. The method of claim 9, wherein the human pluripotent stem cells are human embryonic stem cells.

11. A method to differentiate pluripotent stem cells into definitive endoderm cells comprising culturing the pluripotent stem cells with a medium supplemented with activin A and 5-chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1˜

3,7˜

.1˜

9,13˜

.1˜

14,18˜

] tritriaconta-3(33),4,6,9(32),10,12,14(31),15,17-nonaen-23-one.

12. The method of claim 11, wherein the medium is further supplemented with Wnt3a.

13. The method of claim 11, wherein the pluripotent stem cells are human pluripotent stem cells.

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