Enzymatical modification of cell glycosylation using serum albumin and divalent cations

Abstract

The invention is directed to a method and kit to control and modify the status of cells, such as human stem cells, by changing their glycosylation, in particular sialylation and fucosylation, levels in a reaction condition where culture medium reagents, such as divalent cations, are present and cells are kept non-adherent. The invention is further directed to novel stem cells, the glycosylation of which has been specifically altered.

1 .- 41 . (canceled) 
     
     
         42 . A method for in vitro enzymatic modification of the glycosylation of viable human adherent stem cells, the method comprising the step of
 a) culturing human adherent stem cells in non-NeuGc and serum free cell culture medium, wherein said human adherent stem cells are from an aggregating or surface-adherent cell culture,   b) detaching said human adherent stem cells from a cell culture surface, and   c) contacting the human adherent stem cells obtained in step b) in non-NeuGc and serum free culture medium with a glycosyltransferase in conditions that prevent adhesion of the human adherent stem cells, wherein adherence of the human adherent stem cells is inhibited by providing shear force and wherein said culture medium contains at least one divalent cation Mg 2+  or Ca 2+  and wherein Mg 2+  or Ca 2+  ions are used in a concentration of at least 0.05 mM.   
     
     
         43 . The method according to  claim 42 , wherein the glycosyltransferase is a fucosyltransferase or a sialyltransferase. 
     
     
         44 . The method according to  claim 43 , wherein the fucosyltransferase is selected from the group consisting of FTIII, FTIV, FTV, FTVI, FTVII and FTIX. 
     
     
         45 . The method according to  claim 43 , wherein the fucosyltransferase is a human fucosyltransferase. 
     
     
         46 . The method according to  claim 43 , wherein the sialyltransferase is selected from the group consisting of α2,3-sialyltransferase and α2,6-sialyltransferase. 
     
     
         47 . The method according to  claim 43 , wherein the sialyltransferase is a human sialyltransferase. 
     
     
         48 . The method according to  claim 42 , wherein the human adherent stem cells are embryonal stem cells, cord blood stem cells, hematopoietic stem cells, mesenchymal stem cells or human peripheral blood mononuclear cells. 
     
     
         49 . The method according to  claim 42 , wherein the culture medium comprises a non-glycoprotein substantially without acceptor glycans for glycosyltransferase.