In vitro production of foregut stem cells
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.
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.