Hepatic stellate cell precursors and methods of isolating same

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 Hlx homeobox transcriptional factor.

1 . An isolated hepatic stellate precursor cell which expresses both VCAM antigen and β3-integrin antigen and does not express both MHC class Ia antigen and glial fibrillary acidic protein (GFAP). 
     
     
         2 . The isolated hepatic stellate precursor cell according to  claim 1  in which is a human hepatic stellate precursor cell. 
     
     
         3 . 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 liver tissue; and sequentially, in any order, or substantially simultaneously,
 (i) removing from the single cell suspension those cells that express MHC class Ia antigen and glial fibrillary acidic protein (GFAP); and 
 (ii) 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.   
     
     
         4 . The method of  claim 3  further comprising: sequentially, in any order, or substantially simultaneously (d) isolating those cells that are positive for VCAM. 
     
     
         5 . The method of  claim 3 , wherein step (b) further comprises (iii) isolating those cells that are positive for VCAM. 
     
     
         6 . The method of  claim 3 , wherein step (b) further comprises (iii) removal of those cells expressing CD45. 
     
     
         7 . The method of  claim 3 , wherein step (b) further comprises (iii) isolating those cells that are positive for both VCAM antigen and β3-integrin. 
     
     
         8 . The method of  claim 3 , wherein step (b) further comprises (iii) isolating from the cell suspension those cells that express desmin, nestin, vimentin, smooth muscle alpha-actin or a combination thereof. 
     
     
         9 . The method of  claim 3  in which the isolating and removing steps are carried out using flow cytometry. 
     
     
         10 . The method of  claim 3  in which the hepatic stellate cell progenitors are human hepatic stellate cell progenitors. 
     
     
         11 . A method of clonogenic expansion of stellate precursor cells comprising culturing in serum-free media isolated stellate precursor cells expressing both VCAM antigen and β3-integrin antigen and not expressing both MHC class Ia antigen and glial fibrillary acidic protein (GFAP). 
     
     
         12 . The method of  claim 11  in which the media further comprises a growth factor. 
     
     
         13 . The method of  claim 12  in which the growth factor is insulin, transferrin, LIF or EGF or a combination thereof. 
     
     
         14 . The method of  claim 11  in which the isolated stellate precursor cells are further cultured in the presence of feeder cells. 
     
     
         15 . The method of  claim 14  in which the feeder cells are STO cells.