Method for evaluating cell populations

Abstract

The invention describes specific sialylated structures present on human stem cells and cell populations derived thereof. The invention is especially directed to methods to control the status of stem cells by observing changes in sialylation of the cells; and control of potential contaminations of biological materials; and reagents and methods used in connection with the cells in order to avoid alterations of the cell glycosylation by contaminating materials. The invention is further directed to novel stem cells, the glycosylation of which has been specifically altered.

1 . A method of altering the fucosylation of human hematopoietic stem cells, wherein sialyl-Lewis x structures are produced to protein-linked glycans on the cell surface, the method comprising the step of incubating the cells with GDP-Fuc and α1,3-fucosyltransferase enzyme. 
     
     
         2 . The method of  claim 1  comprising an additional step of α3-sialylating the cells. 
     
     
         3 . The method of  claim 2 , wherein the α3-sialylation of the cells is performed either by i) culturing the cells in conditions wherein said cells express increased amounts of α3-linked sialic acid compared to fresh isolated cells that have not been cultivated in a cell culture, or ii) incubating the cells with CMP-sialic acid and α2,3-sialyltransferase enzyme. 
     
     
         4 . The method of  claim 1 , wherein said α1,3-fucosyltransferase enzyme reacts with α3-sialylated N-acetyllactosamines in the protein-linked glycans according to the Formula (III):
   (SAα3) s1 Galβ4GNβ2Mα3[(Saα3) s2 Galβ4GNβ2Mα6]Mβ4GNβ4(Fucα6) r3 GN  (III)
 
 with 0, 1 or 2 additional α3-sialylated N-acetyllactosamine branches according to the Formula (IIIb):
   (SAα3) s3 Galβ4GNP  (IIIb)
 
 
 wherein r3, s1, s2 and s3 are either 0 or 1, 
 with the proviso that at least one of s1, s2 and s3 is 1, 
 GN is GlcNAc, M is Man, SA is sialic acid, and ( ) indicates branching, 
 Mα6 residue and/or Mα3 residue can be further substituted with one or two β6- or β4-finked additional branches according to the formula (IIIb), Mβ4 can be further substituted by GNβ4, and 
 the reducing end GN of the N-glycan is linked to a protein on cell surface by N-glycosidic linkage to Asn-residue of N-glycosylation site. 
 
     
     
         5 . The method of  claim 4 , wherein at least two of s1, s2 and/or s3 are 1, or s1=s2=s3=1. 
     
     
         6 . The method of  claim 4 , wherein the Mα6 residue and/or the Mα3 residue are further substituted with one or two β6- or β4-linked additional branches according to the Formula (IIIb). 
     
     
         7 . The method of  claim 4 , wherein the α3-sialylated N-acetyllactosamines are further substituted by α3-linked fucose residues during the fucosylation and form at least one, at least two, at least three or at least four sialyl-Lewis x structures in the protein-linked glycans according to the Formula (III). 
     
     
         8 . The method of  claim 1 , wherein the produced protein-linked glycans on the cell surface comprise N-glycans with at least two, at least three or at least four sialyl-Lewis x structures. 
     
     
         9 . The method of  claim 1 , wherein the protein-linked glycans are modified with structure types of fucosylated glycans selected from the group consisting of terminal Lewis x and sialyl-Lewis x epitopes in protein-linked N-glycans and/or in O-glycans. 
     
     
         10 . The method of  claim 1 , wherein the structure types of fucosylated glycans selected from the group consisting of Lewis x and/or sialyl-Lewis x, are analyzed. 
     
     
         11 . The method of  claim 1 , wherein the method produces a cell population prepared or derived from human hematopoietic stem cells, wherein the cells in said cell population comprise in vitro enzymatically produced sialyl-Lewis x structures in the protein-linked glycans on the cell surface. 
     
     
         12 . The method of  claim 11 , wherein the cell population comprises in vitro increased α3-sialylation by Neu5Ac in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of sialylated N-glycans. 
     
     
         13 . The method of  claim 11 , wherein the cell population comprises increased amount of α3-linked fucose in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12% of sialylated N-glycans. 
     
     
         14 . The method of  claim 1 , wherein the α1,3-fucosyltransferase enzyme is a recombinant human α1,3-fucosyltransferase VI and/or glycan controlled enzyme lacking immunologically active and/or harmful glycan structures. 
     
     
         15 . The method of  claim 3 , wherein the α2,3-sialyltransferase enzyme is a recombinant human α2,3-sialyltransferase and/or glycan controlled enzyme lacking immunologically active and/or harmful glycan structures. 
     
     
         16 . The method of  claim 1 , wherein the enzymes are washed or removed from the cells after the reaction. 
     
     
         17 . The method of  claim 1 , wherein the enzymes are tagged and the tagged enzymes are bound to a matrix and removed from the cells after the reaction by separating the matrix from the cells. 
     
     
         18 . The method of  claim 11 , wherein the enzymes are removed from the cells after the reaction, after which the cells contain reduced amount of the α1,3-fucosyltransferase and/or α2,3-sialyltransferase enzymes compared to cells from which the enzymes are not removed. 
     
     
         19 . The method of  claim 1 , wherein the human hematopoietic stem cells are cord blood cells, peripheral blood mononuclear cells, or bone marrow cells. 
     
     
         20 . The method of  claim 19 , wherein the cord blood cells are human cord blood mononuclear cells, CD133+ cells, Lin− (lineage negative) cells, or CD34+ cells. 
     
     
         21 . The method of  claim 20 , wherein said human cord blood mononuclear cells are produced by density gradient centrifugation. 
     
     
         22 . The method of  claim 1 , wherein the sialyl-Lewis x structures on the cell surfaces are used for targeting of the cells, optionally, for selectin directed targeting of the cells. 
     
     
         23 . The method of  claim 1 , wherein the cells are depleted of divalent cations prior to the fucosylation reaction. 
     
     
         24 . The method of  claim 2 , wherein the step of α3-sialylation is performed prior to step of α3-fucosylation, or the steps of α3-sialylation and α3-fucosylation are performed simultaneously.