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.

What is claimed is: 
     
         1 . A method of producing a ganglioside in a cell, comprising:
 (a) treating said cell with chloroquine to accumulate said ganglioside;   (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 PC12 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 . The method of  claim 5 , wherein said fibroblast cell is a fibroblast from a human with GM1 gangliosidosis. 
     
     
         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 . The method of  claim 1 , wherein said ganglioside is selected from the group consisting of GM1, GM2, M3, GD1a, GD1b, G D3, and GT1. 
     
     
         15 . The method of  claim 14 , wherein said ganglioside is GM1. 
     
     
         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 . The method of  claim 20 , wherein said amount is selected from the group consisting of 15, 19, 28, 63, 65, 83, 104, and 119 percent or about 15, 19, 28, 63, 65, 83, 104, and 119 percent. 
     
     
         22 . The method of  claim 21 , wherein said amount is 65 or about 65. 
     
     
         23 . A method of producing GM1 ganglioside, comprising:
 (a) isolating bone marrow cells from sheep;   (b) culturing said sheep bone marrow cells in neural-induction media (“NIM”) to produce neuron-like sheep bone marrow cells;   (c) treating said neuron-like sheep bone marrow cells with chloroquine to accumulate GM1; and   (d) isolating GM1, quantifying GM1, or both, from said chloroquine-treated neuron-like sheep bone marrow cells.   
     
     
         24 . The method of  claim 23 , wherein said (c) treating said neuron-like sheep bone marrow cells with chloroquine comprises contacting said neuron-like sheep bone marrow cells with between 5 and 100 micromolar chloroquine. 
     
     
         25 . The method of  claim 23 , wherein said (c) treating said neuron-like sheep bone marrow cells with chloroquine comprises contacting the neuron-like sheep bone marrow cells with chloroquine for between 2 to 72 hours. 
     
     
         26 . The method of  claim 23 , wherein said sheep are afflicted with gangliosidosis. 
     
     
         27 . A method of producing GM1 ganglioside, comprising:
 (a) treating human bone marrow cells with chloroquine to accumulate GM1; and   (b) isolating GM1, quantifying GM1, 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 . The method of  claim 27 , wherein said human is afflicted with GM1 gangliosidosis. 
     
     
         31 . A method of quantifying an amount of GM1 ganglioside in a population of adherent cells, comprising
 (a) contacting the adherent cells with cholera-toxin B conjugated to a dye or an enzyme that generates a colored end-product upon contacting its substrate;   (b) measuring light emitted by or absorbed by the dye or the colored end-product, wherein the light emitted by or absorbed by the dye or colored end product is used to quantitate the amount of GM1 ganglioside in the population of adherent cells.   
     
     
         32 . A ganglioside produced by the method of  claim 1 . 
     
     
         33 . The ganglioside of  claim 32 , wherein said ganglioside is GM1. 
     
     
         34 . A method of treating a disease or disorder comprising administering the ganglioside of  claim 32  to a subject in need thereof, wherein said disease or disorder is treated. 
     
     
         35 . The method of  claim 34 , wherein said disease or disorder is selected from the group consisting of neuronal injury, Parkinson's disease, Alzheimer's disease, stroke, Guillain-Barré syndrome, and cancer.