Method for producing adult liver progenitor cells
Novel adult liver progenitor cells (called H2Stem Cells) have been have been characterized on the basis of a series of biological activities and markers. Methods for producing H2Stem Cells allow providing such cells in the form of adherent cells and three-dimensional cell clusters in suspension that can be differentiated into cells having strong liver-specific activities and/or that can be used for treating liver diseases or for evaluating the efficacy, the metabolism, and/or toxicity of a compound.
1. An isolated, human adult liver progenitor cell characterized in that said cell is measured positive for expression of:
(a) at least one hepatic marker selected from the group consisting of albumin, HNF-3B, HNF-4, CYP1A2, CYP2C9, CYP2E1 and CYP3A4;
(b) at least one mesenchymal marker selected from the group consisting of Vimentin, CD90, CD73, CD44, and CD29;
(c) at least one liver-specific activity selected from the group consisting of urea secretion, bilirubin conjugation, alpha-1-antitrypsin secretion, and CYP3A4 activity;
(d) Sushi domain containing protein 2 (SUSD2); and
(e) Cytokeratin-19 (CK-19);
wherein said cell is also optionally measured
(f) negative for one or more markers selected from the group consisting of CD140b, CD45, CD117, CD31, CD133, and CD326;
(g) positive for Cytokeratin-18 (CK-18) and/or alpha smooth muscle actin (ASMA); and/or
(h) positive for at least one liver-specific activity selected from the group consisting of sulfotransferase activity, tryptophan-2,3-dioxygenase activity, liver carboxylase activity, ammonia metabolism, and glycogen storage;
wherein said cell has a cuboidal meso-epithelial morphology.
2. The cell of claim 1 wherein said cell is measured:
(a) positive for albumin, Vimentin, CD90, CD73, urea secretion, bilirubin conjugation, alpha-1-antitrypsin secretion, CYP3A4 activity, Sushi domain containing protein 2, Cytokeratin-19, and liver carboxylase activity; and
(b) negative for CD140b.
3. The cell of claim 1 wherein said cell is capable of forming three-dimensional cell clusters in suspension and/or differentiating into cells presenting liver-specific activities.
4. An isolated cell population comprising at least 60%, or between 60% and 99%, or between 70% and 90%, of cells of claim 1 .
5. The cell population of claim 4 wherein the population comprises adherent cells or forms three-dimensional cell clusters in suspension, and optionally presents inducible Phase I CYP-dependent activity and uptake of at least one compound selected from the group consisting of Taurocholate, Estrone-3-sulfate, and 1-methyl-4-phenylpyridinium.
6. The cell of claim 1 wherein said cell or a population thereof is modified by means of one or more chemical agents, cell culture medium, growth factors, and/or nucleic acid vectors.
7. A method for obtaining adult liver progenitor cells of human origin as set forth in claim 1 , comprising:
(a) disassociating adult liver or a part thereof to form a population of primary liver cells;
(b) generating a preparation of the population of primary liver cells of (a);
(c) culturing cells comprised in the preparation of (b) onto a support that allows adherence and growth of cells thereto and the emergence of a population of cells having cuboidal meso-epithelial morphology;
(d) passaging the cells of (c) at least once; and
(e) isolating a population of cells obtained after the passaging of step (d) that maintain a cuboidal meso-epithelial morphology, that are positive for at least one hepatic marker and at least one mesenchymal marker, and that have at least one liver-specific activity,
wherein
(f) the cells of step (c) and/or the cell population of step (e) are measured positive for cytokeratin 19, albumin, alpha-1-antitrypsin secretion, Sushi domain containing protein 2, and CYP3A4.
8. The method of claim 7 wherein the cells of step (c) and/or the cell population of step (e) are further measured positive for Cytokeratin-18 (CK-18) and/or alpha smooth muscle actin (ASMA) and are measured negative for CD140b.
9. The method of claim 7 wherein the cell population of step (e) is maintained in cell culture conditions that allow formation of three-dimensional cell clusters in suspension.
10. A composition comprising the cell of claim 1 or a population thereof.
11. A method for treating a liver disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the cells of claim 1 , or a population or composition thereof, to the subject.
12. The method of claim 11 wherein the liver disease is an inborn error of liver metabolism, an inherited Blood Coagulation Disorder, progressive familial intrahepatic cholestasis type 1/2/3, alpha 1-Antitrypsin Deficiency, defect of liver cell transporters, Porphyria, fatty liver or fibrotic liver disease, primary biliary cirrhosis, sclerosing cholangitis, liver degenerative disease, or acute or chronic liver failure.
13. A kit comprising a cell of claim 1 or a population or composition thereof.
14. The kit of claim 13 wherein said kit further comprises one or more vials containing said cell, said cell population, or said composition, and one or more of the following elements: devices, disposable materials, solutions, chemical products, biological products, and/or instructions for using elements of said kit.