Liver organoid, uses thereof and culture method for obtaining them

1. A method for differentiating liver epithelial stem cells, wherein the method comprises culturing the epithelial stem cells in the presence of a culture medium comprising a basal medium for animal or human cells, and further comprising a ligand of an Epidermal Growth Factor (EGF) receptor, a Notch inhibitor, and a Transforming Growth Factor (TGF)-beta inhibitor.

2. The method according to claim 1 , wherein the culture medium further comprises an FGF and/or HGF.

3. The method according to claim 1 , wherein the ligand of an EGF receptor is EGF.

4. The method according to claim 1 , wherein the Notch inhibitor is selected from:

a. a gamma-secretase inhibitor;

b. an inhibitor capable of diminishing ligand mediated activation of Notch; and

c. an inhibitor of A Disintegrin And Metalloproteinase (ADAM) proteases.

5. The method according to claim 4 , wherein the gamma-secretase inhibitor is DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), or LY-411575.

6. The method according to claim 4 , wherein the Notch inhibitor is tert-Butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate (DAPT).

7. The method according to claim 1 , wherein the TGF-beta inhibitor is an inhibitor of Activin Receptor-like Kinase 5 (ALK5), Activin Receptor-like Kinase 4 (ALK4), and/or Activin Receptor-like Kinase 7 (ALK7) signaling.

8. The method according to claim 7 , wherein the TGF-beta inhibitor is selected from A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, SJN-2511.

9. The method according to claim 8 , wherein the TGF-beta inhibitor is A83-01.

10. The method according to claim 1 , wherein the culture medium further comprises dexamethasone.

11. The method according to claim 10 , wherein the dexamethasone is at a concentration of between 10 μM and 1 mM.

12. The method according to claim 1 , wherein: the ligand of an EGF receptor is EGF at a concentration of about 50 ng/ml; the TGF-beta inhibitor is A83-01 at a concentration of about 500 nM; and/or the Notch inhibitor is DAPT at a concentration of about 10 μM.

13. The method according to claim 1 , wherein the basal medium for animal or human cells is selected from Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove's Modified Dulbecco's Media and Minimal Essential Media (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI 1640 Media.

14. The method according to claim 13 , wherein the basal medium for animal or human cells comprises Advanced-DMEM/F12, and optionally further comprises gastrin, N-acetylcystein and/or B27.

15. The method according to claim 1 , wherein the culture medium does not comprise one or more of the components selected from the list: a Wnt agonist, nicotinamide, and a BMP inhibitor.

16. The method according to claim 1 , wherein the method further comprises contacting the cells with an extracellular matrix.

17. The method according to claim 16 , wherein the extracellular matrix is a basement membrane preparations from Engelbreth-Holm-Swarm mouse sarcoma cells, or wherein the extracellular matrix is a synthetic extracellular matrix material.

18. A method according to claim 1 , wherein the liver epithelial stem cells:

a. express Lgr5; and/or

b. do not naturally express one or more of Cd11b, CD13, CD14, Alpha Fetoprotein (AFP), Pdx1, a Cytochrome P450 (CYP) member at a significant level.

19. A method according to claim 1 , wherein the differentiated cells:

a. express one or more of Cd11b, CD13, CD14, AFP, Pdx1, a CYP member at a significant level; and/or

b. express one or more of Tyrosine Aminotransferase (TAT) and/or Albumin; and/or

c. express K19 and/or Fumarylacetoacetate Hydrolase (FAH); and/or

d. do not express Lgr5.