Methods of ganglioside production

Abstract

The invention provides methods for production of gangliosides, e.g., GM1, from cells in culture using, for example, bone marrow cells and neuroblastoma cells. Methods include the treatment of cells with neural induction media and chloroquine or chloroquine alone in the case of, e.g., human bone marrow cells, neuraminidase or glucosamine, to induce the production of gangliosides, e.g., GM1, in the cells. Also provided are methods of long-term high density culturing of cells without passaging to produce gangliosides, e.g., GM1. Methods of quantifying gangliosides, e.g., GM1 in cell culture are also provided.

1 . A method of producing a ganglioside in a cell, comprising:
 (a) treating said cell with chloroquine to accumulate said ganglioside wherein said ganglioside is selected from the group consisting of GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1;   (b) isolating said ganglioside, quantifying said ganglioside, or both, from said chloroquine-treated cell;
 wherein said cell is selected from the group consisting of an immortalized cell, a stromal cell, and a fibroblast; 
 wherein said cell is not a PC 12 cell, an HT22 cell, a brain cell from a sheep afflicted with gangliosidosis, and fibroblast cell from sheep afflicted with gangliosidosis. 
   
     
     
         2 . The method of  claim 1 , wherein said cell is selected from the group consisting of a neuroblastoma cell, a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (HEK), a stromal cell, and a fibroblast cell. 
     
     
         3 . The method of  claim 2 , wherein said cell is a CHO cell, wherein said CHO cell is a CHO-K1 cell. 
     
     
         4 . The method of  claim 2 , wherein said cell is a HEK cell, wherein said HEK cell is a HEK293 cell. 
     
     
         5 . The method of  claim 2 , wherein said cell is a fibroblast cell. 
     
     
         6 . The method of  claim 5 , wherein said fibroblast cell is a dermal fibroblast cell. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 2 , wherein said cell is a neuroblastoma cell. 
     
     
         9 . The method of  claim 8 , wherein said neuroblastoma cell is selected from the group consisting of SHSY-5Y, SHSY-S, and SK-N-AS. 
     
     
         10 . The method of  claim 2 , wherein said cell is a stromal cell. 
     
     
         11 . The method of  claim 10 , wherein said stromal cell is derived from bone marrow. 
     
     
         12 . The method of  claim 11 , wherein said stromal cell is derived from human bone marrow. 
     
     
         13 . The method of  claim 12 , wherein said human bone marrow cell is produced by low density and low oxygen culture methods. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , further comprising treating said cell with neuraminidase. 
     
     
         17 . The method of  claim 1 , further comprising treating said cell with glucosamine. 
     
     
         18 . The method of  claim 1 , further comprising biochemically manipulating said cell to increase ganglioside production. 
     
     
         19 . The method of  claim 1 , wherein said cell produces an amount of 10 to 200 percent or about 10 to 200 percent more ganglioside than a cell that has not been treated with chloroquine. 
     
     
         20 . The method of  claim 19 , wherein said amount is 15 to 125 percent or about 15 to 125 percent. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 21 , wherein said amount is 65 or about 65. 
     
     
         23 - 26 . (canceled) 
     
     
         27 . A method of producing ganglioside, comprising:
 (a) treating human bone marrow cells with chloroquine to accumulate ganglioside, wherein said ganglioside is selected from the group consisting of GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1;   and   (b) isolating said ganglioside, quantifying said ganglioside, or both, from said chloroquine-treated human bone marrow cells.   
     
     
         28 . The method of  claim 27 , wherein said (a) treating said human bone marrow cells with chloroquine comprises contacting said human bone marrow cells with between 5 and 100 micromolar chloroquine. 
     
     
         29 . The method of  claim 27 , wherein said (a) treating said human bone marrow cells with chloroquine comprises contacting said human bone marrow cells with chloroquine for between 2 to 72 hours. 
     
     
         30 - 35 . (canceled)