Cells expressing pluripotency markers and expressing markers characteristic of the definitive endoderm
The present invention is directed to pluripotent cells that can be readily expanded in culture on tissue culture substrate that is not pre-treated with protein or an extracellular matrix, and do not require a feeder cell line. The present invention also provides methods to derive the pluripotent cell line from human embryonic stem cells.
1. A method for deriving a population of cells expressing pluripotency markers and expressing markers characteristic of the definitive endoderm lineage comprising the steps of:
a. Obtaining obtaining a population of cells expressing markers HNF-3β, GATA-4, Mixl1, CXCR4 and SOX-17, characteristic of the definitive endoderm lineage, and
b. Culturing culturing the population of cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix in a medium supplemented with Activin A and wnt-3A a wnt ligand, and IGF-1 or insulin, transferrin and selenium, wherein the cultured cells express the markers of the definitive endoderm lineage and pluripotency markers.
2. The method of claim 1 , wherein the population of cells expressing markers characteristic of the definitive endoderm lineage are cultured in normoxic conditions prior to culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.
3. The method of claim 1 , wherein the population of cells expressing markers characteristic of the definitive endoderm lineage are cultured in hypoxic conditions prior to culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.
4. The method of claim 1 , wherein the hypoxic conditions are an O 2 level from about 1% to about 20%.
5. The method of claim 1 , wherein the hypoxic conditions are an O 2 level from about 2% to about 10%.
6. The method of claim 1 , wherein the hypoxic conditions are an O 2 level of about 3%.
7. The method of claim 1 , wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration from about 2% to about 5%.
8. The method of claim 1 , wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration of about 2%.
9. The method of claim 1 , wherein the population of cells expressing markers characteristic of the definitive endoderm lineage are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration from about 25 ng/ml to about 50 ng/ml.
10. The method of claim 1 , wherein the population of cells expressing markers characteristic of the definitive endoderm lineage are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration of about 50 ng/ml.
11. The method of claim 1 , wherein the population of cells expressing pluripotency markers express at least one of the pluripotency markers selected from the group consisting of ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, and Tral-81.
12. The method of claim 1 , wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing Activin-A at a concentration from about 50 ng/ml to about 100 ng/ml.
13. The method of claim 1 , wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing Activin-A at a concentration of about 100 ng/ml.
14. The method of claim 1 , wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration from about 10 ng/ml to about 20 ng/ml.
15. The method of claim 1 , wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration of about 200 ng/ml.
16. The method of claim 1 , wherein the Wnt ligand is Wnt-3a.