Generation of thymic epithelial progenitor cells in vitro

• US 9,850,465 B2
• Filed: 02/26/2014
• Issued: 12/26/2017
• Est. Priority Date: 02/27/2013
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Parent, Audrey, Hebrok, Matthias, Anderson, Mark Stuart
• Primary Examiner(s): DAVIS, RUTH A
• Application Number:
  US14/770,625
• Publication Number:
  US 20160010055A1
• Time in Patent Office: 1,399 Days
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/385 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 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/065 Thymocytes

Abstract

Methods for generating thymic epithelial progenitor (TEP) cells from pluripotent stem (PS) cells in vitro are provided. Compositions and systems of cell populations of TEP cells as well as cells formed during different stages of differentiation of PS cells into TEP cells are also disclosed. The methods, isolated in vitro cell populations, compositions, and systems disclosed provide functional TEP cells that mature into thymic epithelial cells in vivo.

Claims

1. A method for generating thymic epithelial progenitor (TEP) cells, the method comprising:

culturing definitive endodermal (DE) cells, obtained from pluripotent stem cells, in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforming growth factor-β

(TGF-β

) signaling to produce anterior foregut endodermal (AFE) cells;

culturing the AFE cells in a medium comprising an activator of retinoic acid receptor, an activator of bone morphogenetic protein (BMP) signaling, and an inhibitor of transforming growth factor-β

(TGF-β

) signaling to produce ventral pharyngeal endodermal (VPE) cells; and

culturing the VPE cells in a medium comprising an activator of retinoic acid receptor and an activator of bone morphogenetic protein (BMP) signaling to produce the TEP cells.

2. The method of claim 1, wherein the DE cells are obtained from pluripotent stem cells by culturing pluripotent stem cells in a medium comprising a growth factor selected from the group consisting of Nodal, Activin A, and Activin B.

3. The method of claim 2, wherein the medium for culturing the AFE cells further comprises a Wnt family member, a fibroblast growth factor (FGF), and an inhibitor of hedgehog signaling.

4. The method of claim 3, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

5. The method of claim 3, wherein the TEP cells express FOXN1.

6. The method of claim 2, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

7. The method of claim 6, wherein the TEP cells express FOXN1.

8. The method of claim 2, wherein the TEP cells express FOXN1.

9. The method of claim 1, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cell, embryonic germ cells, and induced pluripotent stem cell.

10. The method of claim 9, wherein the pluripotent stem cells are primate pluripotent stem cells (pPS) cells.

11. The method of claim 10, wherein the pPS cells are human pluripotent stem (hPS) cells.

12. The method of claim 11, wherein the hPS cells are human embryonic stem (hES) cells.

13. The method of claim 11, wherein the hPS cells are induced pluripotent stem (iPS) cells.

14. The method of claim 1, wherein the medium for culturing the AFE cells further comprises a Wnt family member, a fibroblast growth factor (FGF), and an inhibitor of hedgehog signaling.

15. The method of claim 14, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

16. The method of claim 14, wherein the TEP cells express FOXN1.

17. The method of claim 1, wherein the medium for culturing the VPE cells further comprises a Wnt family member, a fibroblast growth factor, and an inhibitor of hedgehog signaling.

18. The method of claim 17, wherein the TEP cells express FOXN1.

19. The method of claim 1, wherein the TEP cells express FOXN1.