Method for evaluating cell populations

摘要

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.

主权项

1. An in vitro method of altering fucosylation of a human mesenchymal stem cell surface, wherein sialyl-Lewis x structures are produced to on protein linked O-glycans and N-glycans on said mesenchymal stem cell surface and wherein a sialyl-structure of said sialyl Lewis x structures is α3-linked Neu5Ac (N-acetylneuraminic acid), comprising the steps of:
a) culturing mesenchymal stem cells in non-NeuGc (N-glycolylneuraminic acid) and serum free culture medium; b) subculturing said mesenchymal stem cells of step a) at least once in non-NeuGc and serum free culture medium so that said sialyl Lewis x structures on the said mesenchymal stem cell surface protein linked O-glycans consist essentially of only α3-sialylated Lewis x structures, wherein said α3-sialylated Lewis x structures are α3-sialylated N-acetyllactosamines; c) α3-fucosylating said mesenchymal stem cells of step b) with guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) and recombinant α1,3-fucosyltransferase enzyme, wherein α3-fucosylation results in formation of α3-fucosylated sialyl-Lewis x structures on said mesenchymal stem cell surface, wherein said α3-fucosylated sialyl-Lewis x structures are α3-fucosylated N-acetyllactosamines; d) assaying levels of α3-linked Neu5Ac structures, and optionally α6-linked sialic acid structures, on said mesenchymal stem cell surface prior to and/or after step c), wherein said assaying is performed by mass spectrometry or a glycan binding molecule, wherein said glycan binding molecule comprises a protein, an enzyme, a lectin or an antibody that recognizes an α3-sialylated glycan structure; and e) contacting said α3-sialylated and α3-fucosylated sialyl-Lewis x structures on said mesenchymal stem cell surface with selectin for selectin directed targeting of said mesenchymal stem cells.