HEPATIC STELLATE CELL PRECURSORS AND METHODS OF ISOLATING SAME

• US 20080009057A1
• Filed: 05/24/2007
• Published: 01/10/2008
• Est. Priority Date: 05/26/2006
• Status: Active Grant

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Kubota, Hiroshi, Reid, Lola
• Granted Patent: US 7,824,911 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/00 Specific components of cell culture medium
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/00 Active agents used in cell culture processes, e.g. differentation
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The present invention relates to precursor cells to hepatic stellate cells, compositions comprising same and methods of isolating same. The surface antigenic profile of the precursors is MHC class Ia negative, ICAM-1⁺, VCAM-1⁺, β3-integrin⁺. In addition to expression of these surface markers, the cells also express the intracellular markers desmin, vimentin, smooth muscle α-actin, nestin, hepatocyte growth factor, stromal derived factor-1α and H1x homeobox transcriptional factor.

Claims

1. A method of obtaining a population of cells enriched in hepatic stellate cell progenitors comprising:

(a) providing a single cell suspension of cells from mammalian tissue; and

sequentially, in any order, or substantially simultaneously,

(b) removing from the single cell suspension those cells that express MHC class Ia antigen; and

(c) isolating from the cell suspension those cells that are positive for Vitamin A fluorescence,

to obtain a population of cells enriched in hepatic stellate cell progenitors.

2. The method of claim 1 in which the mammalian tissue is liver, pancreas, gut, lung, or bone marrow cells.

3. The method of claim 1 further comprising:

sequentially, in any order, or substantially simultaneously (d) isolating those cells that are positive for VCAM.

4. The method of claim 1 further comprising removal of those cells expressing CD45.

5. The method of claim 1 and further comprising:

sequentially, in any order, or substantially simultaneously (d) isolating those cells that are positive for both VCAM antigen and β

3-integrin.

6. The method of claim 1 further comprising:

sequentially, in any order, or substantially simultaneously, (e) isolating from the cell suspension those cells that express desmin, nestin, vementin, smooth muscle alpha-actin or a combination thereof.

7. The method of claim 1 in which the isolating and removing steps are carried out using flow cytometry.

8. The method of claim 2 in which the mammalian tissue is liver tissue.

9. The method of claim 1 in which the hepatic stellate progenitor cells are human hepatic stellate cell progenitors.

10. A method of obtaining a population of cells enriched in isolated hepatic stellate cell progenitors comprising:

(a) obtaining a cell suspension of hepatic cells; and

(b) sequentially, in any order, or substantially simultaneously, (i) isolating from the single cell suspension of liver cells those cells that are positive for ICAM antigen (ii) removing those cells that are positive for MHC class I antigen, and (iii) isolating those cells that are positive for Vitamin A fluorescence as measured in a flow cytometer, to obtain a population of cells enriched in progenitors.

11. The method of claim 10 further comprising:

sequentially, in any order, or substantially simultaneously, (c) isolating from the cell suspension those cells that express desmin, nestin, vimentin, smooth muscle alpha actin or a combination thereof.

12. The method of claim 10 in which the isolating and removing steps are carried our in a flow cytometer.

13. The method of claim 10 in which the removal of those cells that express MHC class I antigen is carried out with an antibody.

14. The method of claim 10 in which the hepatic stellate progenitor cells are human hepatic stellate cell progenitors.

15. An isolated hepatic stellate precursor cell which expresses both VCAM antigen and β

3-integrin antigen.

16. A method of clonogenic expansion of stellate precursor cells comprising culturing isolated stellate precursor cells expressing both VCAM antigen and β

3-integrin antigen in serum-free media.

17. The method of claim 16 in which the media further comprises a growth factor.

18. The method of claim 17 in which the growth factor is insulin, transferrin, LIF or EGF or a combination thereof.

19. The method of claim 16 in which the isolated stellate precursor cells are further cultured in the presence of feeder cells.

20. The method of claim 19 in which the feeder cells are STO cells.