Generation of anterior foregut endoderm from pluripotent cells

• US 9,719,067 B2
• Filed: 12/04/2015
• Issued: 08/01/2017
• Est. Priority Date: 04/25/2010
• Status: Active Grant

• Current Assignee: Mount Sinai School of Medicine (Sinai Health System)
• Original Assignee: Mount Sinai School of Medicine (Sinai Health System)
• Inventors: Snoeck, Hans-Willem, Green, Michael
• Primary Examiner(s): WILSON, MICHAEL C
• Application Number:
  US14/959,800
• Publication Number:
  US 20160168535A1
• Time in Patent Office: 606 Days
• Field of Search: 435325
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• 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 2501/40 Regulators of development
• 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 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0617 Thyroid and parathyroid glands
• CPC Class Codes: C12N 5/0688 Cells from the lungs or the respiratory tract

Abstract

The invention is directed to in vitro methods of inducing differentiation of anterior foregut endoderm and the enriched populations of anterior foregut endoderm produced by such methods. Such enriched populations are useful for studies of the molecular events that occur during differentiation and for generating cells for cell replacement therapy.

Claims

1. A method of preparing an enriched population of anterior foregut endoderm cells from pluripotent cells, the method comprising:

(i) culturing isolated mammalian pluripotent cells under conditions that induce the pluripotent cells to form definitive endoderm cells; and

(ii) culturing the definitive endoderm cells in the presence of an inhibitor of a bone morphogenetic protein (“

BMP”

) and an inhibitor of a transforming growth factor beta (“

TGF-β

”

) such that definitive endoderm cells form anterior foregut endoderm cells.

2. The method of claim 1, wherein the pluripotent cells are cultured in the presence of basic fibroblast growth factor (“

bFGF”

), bone morphogenetic protein (“

BMP”

) and Activin A to induce said pluripotent cells to form said definitive endoderm cells.

3. The method of claim 1, wherein the definitive endoderm cells are cultured in the absence of Activin A.

4. The method of claim 1, wherein said inhibitor of bone morphogenetic protein (“

BMP”

) is Noggin or Chordin.

5. The method of claim 4, wherein said inhibitor of bone morphogenetic protein (“

BMP”

) is Noggin.

6. The method of claim 1, wherein the inhibitor of transforming growth factor beta (“

TGF-beta”

) signaling is SB-431542.

7. The method of claim 1, wherein the pluripotent cells are cultured for 1-4 days.

8. The method of claim 1, wherein the definitive endoderm cells are cells are cultured for 2-6 days.

9. The method of claim 1, further comprising isolating said anterior foregut endoderm cells.

10. The method of claim 1, wherein the pluripotent cells are ES cells.

11. The method of claim 1, wherein the pluripotent cells are iPS cells.

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

13. The method of claim 1, wherein the definitive endoderm cell is an embryoid body cell.