Method for producing adult liver progenitor cells

Abstract

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 - 31 . (canceled) 
     
     
         32 . A method for evaluating the efficacy, the metabolism, the stability, and/or the toxicity of one or more exogenous components, said method comprising:
 (a) providing:
 (I) a cell, wherein the cell is an adult liver progenitor cell characterized in that said cell is measured positive for:
 (i) at least one hepatic marker selected from albumin, HNF-3B, HNF-4, CYP1 A2, CYP2C9, CYP2E1 and CYP3A4; 
 (ii) at least one mesenchymal marker selected from Vimentin, CD90, CD73, CD44, and CD29; 
 (iii) at least one liver-specific activity selected from urea secretion, bilirubin conjugation, alpha-1 -antitrypsin secretion, and CYP3A4 activity; 
 (iv) Sushi domain containing protein 2 (SUSD2); and 
 (v) Cytokeratin-19 (CK-19); 
 
 (II) an isolated cell population comprising at least 60% or between 60% and 99% or between 70% or 90% of cells of part (I); 
 (III) a biological material isolated from a cell of part (I) or population of part (II), wherein the biological material is a conditioned cell culture media, a protein extract, a membrane vesicle, or any fraction thereof comprising one or more isolated proteins, nucleic acids, metabolites, and/or antigens; or 
 (IV) a composition comprising a cell of part (I), a cell population of part (II), or a biological material of part (III); 
   (b) exposing said cell, said cell population, said composition, or said biological material to one or more exogenous components selected from chemical compounds, proteins, nucleic acids, lipids, sugars, metals, salts, viruses, bacteria, and cells; and   (c) detecting the effects of said one or more exogenous components on said cell, said cell population, said composition, or said biological compound and/or detecting the presence, localization, or modification of said one or more exogenous components following the exposure to said cell, said cell population, said composition, or said biological material.   
     
     
         33 . The method of  claim 32  wherein step (c) of said method comprises detecting the effects on cell morphology, on cell viability, on up- or down-regulation of liver-specific or unspecific proteins, and/or on the degradation, aggregation, activation, or inhibition of proteins within said cell, said cell population, said composition, or said biological material. 
     
     
         34 . The method of  claim 32  wherein step (c) of said method comprises detecting the internalization of said one or more exogenous components into, or the physical association with, said cell, said cell population, said composition, or said biological material. 
     
     
         35 . The method of  claim 32  wherein said cell, said cell population, said composition, or said biological material is provided to an animal in step (a), one or more exogenous components is administered to an animal in step (b), and wherein step (c) comprises detecting the effects of said one or more exogenous components on said cell, said cell population, said composition, said biological material, or on said animal, and/or detecting the presence, localization, or modification of said one or more exogenous components following the exposure to said cell, said cell population, said composition, or said biological material in the animal. 
     
     
         36 . The method  claim 32  wherein said cell, said cell population, said composition, or said biological material is exposed in step (b), simultaneously or sequentially in any order, to (i) one or more exogenous components that have an effect on cell morphology, cell viability, up- or down-regulation of liver-specific or unspecific proteins, and/or that degrade, aggregate, activate, or inhibit proteins within said cell, said cell population, said composition, or said biological material; and (ii) one or more exogenous components intended to block or avoid the effects of the one or more exogenous components of (i) within said cell, said cell population, said composition, or said biological material. 
     
     
         37 . The method of  claim 36  wherein the one or more exogenous components of (i) comprises a infectious, tumorigenic, cytotoxic, or genotoxic agent, and the further exogenous components of (ii) comprises a protein, a nucleic acid, a cell, a virus, or a chemical compound. 
     
     
         38 - 40 . (canceled) 
     
     
         41 . The method of  claim 32  characterized in that the cell of part (a)(I) is measured negative for one or more of CD140b, CD45, CD117, CD31, CD133, and CD326. 
     
     
         42 . The method of  claim 32  wherein the cell of part (a)(I) is further measured positive for Cytokeratin-18 (CK-18) and/or alpha smooth muscle actin (ASMA). 
     
     
         43 . The method of  claim 32  where the cell of part (a)(I) is further measured positive for at least one liver-specific activity selected from sulfotransferase activity, tryptophan-2,3-dioxygenase activity, liver carboxylase activity, ammonia metabolism, and glycogen storage. 
     
     
         44 . The method of  claim 32  wherein the cell of part (a)(I) is further measured positive for at least one coagulation-related secreted protein selected from fibrinogen alpha, fibrinogen beta, fibrinogen gamma, Factor V, Factor VII, Factor VIII, Factor IX, and Factor XIII. 
     
     
         45 . The method of  claim 32  wherein the cell of part (a)(I) is measured:
 (i) 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 
 (ii) negative for CD140b. 
 
     
     
         46 . The method of  claim 32  wherein the cell of part (a)(I) presents cuboidal meso-epithelial morphology. 
     
     
         47 . The method of  claim 32  wherein the cell of part (a)(I) is capable of forming three-dimensional cell clusters in suspension and/or differentiating into cells presenting liver-specific activities. 
     
     
         48 . The method of  claim 32  wherein the population of part (a)(II) comprises adherent cells or form three-dimensional cell clusters in suspension. 
     
     
         49 . The method of  claim 48  wherein the population of part (a)(II) presents inducible Phase I CYP-dependent activity and uptake of at least one of Taurocholate, Estrone-3-sulfate, and 1-methyl-4-phenylpyridinium. 
     
     
         50 . The method of  claim 32  wherein the population of part (a)(II) is differentiated into cells presenting liver-specific activities. 
     
     
         51 . The method of  claim 32  wherein the cell of part (a)(I) or the population of part (a)(II) is modified by means of one or more chemical agents, cell culture medium, growth factors, and/or nucleic acid vectors. 
     
     
         52 . The method of  claim 32  wherein the population of part (a)(I) is obtainable by a method 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.