HEPATOCYTE PRECURSOR CELL LINES

• US 20100086936A1
• Filed: 11/16/2009
• Published: 04/08/2010
• Est. Priority Date: 02/15/2002
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Sherley, James L., Lee, Hsuan-Shu, Crane, Gracy G.
• Granted Patent: US 8,404,481 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

The present invention is directed to methods for readily generating hepatocyte precursor cell lines that retain hepatocyte-specific functions after extensive in vitro culturing. The methods comprise isolating and culturing hepatocyte precursor cell lines under permissive culture conditions that suppress asymmetric cell kinetics and allow exponential growth of the precursor cells, followed by transferring the hepatocyte precursor cell lines to non-permissive culture conditions that allow expression of asymmetric cell kinetics and induce expression of hepatocyte-specific characteristics.

Claims

1. (canceled)

18. A method for identifying molecular probes specific for hepatic precursor cells, comprising culturing and expanding said single hepatic precursor cells using the method of claim 1, and using said population of expanded hepatic precursor cells for comparison to a second population of non-stem cells to identify differences in gene and/or protein expression between the two said populations.

2. (canceled)

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17. (canceled)

19. A method of culturing and expanding hepatic precursor cells in vitro, comprising culturing hepatic precursor cells isolated from a mammal in a culture medium which permits cell growth under conditions and for a time sufficient to permit cell growth, wherein the expression of a protein downstream of the guanine nucleotide biosynthesis pathway in said hepatic precursor cells is modulated by an agent present in the culture medium or by a genetic manipulation to said hepatic precursor cells such that asymmetric cell kinetics are suppressed.

20. The method of claim 19, wherein the modulation is increased expression of the protein.

21. The method of claim 19, wherein the modulation is decreased expression of the protein.

22. A method of culturing and expanding hepatic precursor cells such that the hepatic precursor cells express hepatic cell markers comprising:

a) isolating hepatic precursor cells from a mammal;

b) culturing said hepatic precursor cells in culture medium comprising at least 50 μ

M of a guanine nucleotide precursor, or an analogue or derivative thereof, wherein said guanine nucleotide precursor suppresses asymmetric cell kinetics thereby allowing exponential growth of said hepatic precursor cells;

c) passaging said cultured hepatic precursors in the culture medium of step (b) to allow expansion of said hepatic precursor cells; and

d) contacting said expanded hepatic precursors of step (c) with a culture medium that does not suppress asymmetric cell kinetics (or that allows expression of asymmetric kinetics), such that the expression of a hepatic cell marker is induced, wherein the hepatic cell marker is selected from the group consisting of;

H4 antigen, α

1-antitrypsin (AAT), cytokeratin 7 (CK7), cytokeratin 8 (CK8), α

-fetoprotein (AFP), albumin, cytochrome p450 1A1, hepatocyte nuclear transcription factor (HNF-3), CAAT enhancer-binding protein (CEBP)-alpha and CAAT enhancer-binding protein (CEBP)-beta.