In vitro production of foregut stem cells

• US 10,000,740 B2
• Filed: 10/06/2014
• Issued: 06/19/2018
• Est. Priority Date: 10/09/2013
• Status: Active Grant

• Current Assignee: Cambridge Enterprise Limited (University of Cambridge)
• Original Assignee: Cambridge Enterprise Limited (University of Cambridge)
• Inventors: Vallier, Ludovic, Hannan, Nicholas
• Primary Examiner(s): Hill, Kevin K
• Application Number:
  US15/028,085
• Publication Number:
  US 20160237401A1
• Time in Patent Office: 1,352 Days
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• 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 2501/12 Hepatocyte growth factor [HGF]
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/91 Heparin
• 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 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors
• CPC Class Codes: C12N 5/0689 : Stem cells; Progenitors
• CPC Class Codes: G01N 33/5023 : on expression patterns

Abstract

This invention relates to the differentiation of pluripotent cells (PSCs) into foregut stem cells (FSCs) using a definitive endoderm induction medium comprising a TGFfi ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor to differentiate the pluripotent cells into definitive endoderm cells and a foregut induction medium comprising a TGFβ ligand to differentiate the definitive endoderm cells into foregut stem cells (FSCs). Methods of differentiation, populations of foregut stem cells, culture media and kits are provided.

Claims

1. A method for producing a population of foregut stem cells (FSCs) comprising:

i) providing a population of definitive endoderm cells (DECs), and

ii) culturing the DECs in a foregut induction medium consisting of a chemically defined nutrient medium supplemented with a TGFβ

ligand to produce a population of foregut stem cells (FSCs).

2. The method according to claim 1 wherein the population of DECs is produced by a method comprising:

i) culturing a population of pluripotent cells (PSCs) in a definitive endoderm (DE) induction medium comprising a TGFβ

ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor, and

ii) allowing the PSCs to differentiate into DECs.

3. The method according to claim 2 wherein the pluripotent cells are human pluripotent cells.

4. The method according to claim 2 wherein the pluripotent cells are IPSCs.

5. The method according to claim 4 wherein the iPSCs are derived from antecedent cells obtained from an individual.

6. The method according to claim 2 wherein the pluripotent cells express one or more of the following pluripotency associated markers:

Oct4, Sox2, Alkaline Phosphatase, POU5f1, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc.

7. The method according to claim 2 wherein the DE induction medium consists of a chemically defined nutrient medium supplemented with a TGFβ

ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor.

8. The method according to claim 2 wherein the TGFβ

ligand is activin and/or the PI3K inhibitor is LY294002.

9. The method according to claim 1 wherein the population of DECs is a homogeneous population or a heterogeneous population.

10. The method according to claim 1 wherein the DECs express one or more of the following endoderm associated markers:

Sox17, foxA2, Lhx1, CXCR4, GATA4, eomesodermin (EOMES), Mix11, HNF-3 beta, Cerberus, OTX4, goosecoid, C-kit, CD99, and Hex.

11. The method according to claim 1 wherein the TGFβ

ligand is activin.

12. The method according to claim 1 wherein the FSCs express one or more of the following markers:

SOX2, HHEX, HOXA3, HNF4α

, SOX17, CXCR4, EpCAM, HNF1β

, GATA4, Cer, and HNF6.