US 2010/0145032 A1Published application

Novel carbohydrate profile compositions from human cells and methods for analysis and modification thereof

AssigneesSuomen Punainen Risti, Veripalelu, Glykos Finland Oy

InventorsJarmo Laine, Tero Satomaa, Jari Natunen, Annamari Heiskanen, Maria Blomqvist, Anne Olonen, Juhani Saarinin, Sari Tiitinen, Ulla Impola, Milla Mikkola, Hanna Salo, Olli Aitio, Leena Valmu, Suvi Natunen, Heidi Anderson, Virve Pitkänen, Jukka Partanen, Taina Jaatinen, Minna Tiitanen, Tia Hirvonen

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Abstract

The invention describes reagents and methods for specific binders to glycan structures of stem cells. Furthermore the invention is directed to screening of additional binding reagents against specific glycan epitopes on the surfaces of the stem cells. The preferred binders of the glycans structures includes proteins such as enzymes, lectins and antibodies.

Claims61

Dependent claims

A method of evaluating the status of a stern cell preparation comprising the step of detecting the presence of an elongated glycan structure or a group, at least two, of glycan structures in said preparation, wherein said glycan structure or a group of glycan structures is according to Formula T1

wherein X is linkage position
R1, R2, and R6 are OH or glycosidically linked monosaccharide residue Sialic acid, preferably Neu5Acα2 or Neu5Gc α2, most preferably Neu5Acα2 or
R3, is OH or glycosidically linked monosaccharide residue Fucα1 (L-fucose) or N-acetyl (N-acetamido, NCOCH3);
R4, is H, OH or glycosidically linked monosaccharide residue Fucα1 (L-fucose),
R5 is OH, when R4 is H, and R5 is H, when R4 is not H;
R7 is N-acetyl or OH
X is natural oligosaccharide backbone structure from the cells, preferably N-glycan, O-glycan or glycolipid structure;
or X is nothing, when n is 0,
Y is linker group preferably oxygen for O-glycans and O-linked terminal oligosaccharides and glycolipids and N for N-glycans or nothing when n is 0;
Z is the carrier structure, preferably natural carrier produced by the cells, such as protein or lipid, which is preferably a ceramide or branched glycan core structure on the carrier or H;
The arch indicates that the linkage from the galactopyranosyl is either to position 3 or to position 4 of the residue on the left and that the R4 structure is in the other position 4 or 3;
n is an integer 0 or 1, and m is an integer from 1 to 1000, preferably 1 to 100, and most preferably 1 to 10 (the number of the glycans on the carrier),
With the provisions that one of R2 and R3 is OH or R3 is N-acetyl,
R6 is OH, when the first residue on left is linked to position 4 of the residue on right:
X is not Galα4Galβ4Glc, (the core structure of SSEA-3 or 4) or R3 is Fucosyl,
for the analysis of the status of stem cells and/or manipulation of the stem cells, and wherein said cell preparation is embryonic type stem cell preparation.
and when the glycan structure is an elongated structure, wherein the binder binds to the structure and additionally to at least one reducing end elongation epitope, preferably monosaccharide epitope, (replacing X and/or Y) according to the Formula E1:
AxHex(NAc)n, wherein A is anomeric structure alfa or beta, X is linkage position 2, 3, or 6;
and
Hex is hexopyranosyl residue Gal, or Man, and n is integer being 0 or 1, with the provisions that
when n is 1 then AxHexNAc is β4GalNAc or β6GalNAc,
when Hex is Man, then AxHex is β2Man, and
when Hex is Gal, then AxHex is β3Gal or β36Gal or α3Gal or α4Gal;
or
the binder epitope binds additionally to reducing end elongation epitope
Ser/Thr linked to reducing end GalNAcα-comprising structures or
βCer linked to Galβ4Glc comprising structures, and the glycan structure is the stem cell population determined from associated or contaminating cell population.

24 more elements

A method for the analysis of the status of the stem cells and/or for manipulation of stem cells comprising a step of detecting an elongated glycan structure or at least two glycan structures from a sample of stem cells, wherein said glycan structure is selected from the group consisting of:

a terminal lactosamine structure
(R1)n1Gal(NAc)n3β3/4(Fucα4/3)n2GlcNAcβR wherein R1 is Fucα2, or SAα3, or SAα6 linked to Galβ4GlcNAc, and
R is the reducing end core structure of N-glycan, O-glycan and/or glycolipid;
a, or structure
(SAα3)n1Galβ3(SAα6)n2GalNAc;
wherein
n1, n2 and n3 are 0 or 1 indicating presence or absence of a structure wherein SA is a sialic acid;
or branched epitope
Galβ3(GlcNAcβ6)GalNAc or
R1Galβ4(R3)GlcNAcβ6(R2Galβ3)GalNAc,
wherein R1 and R2 are independently either nothing or SAα3;
and R3 is independently either nothing or Fucα3 ;
or
Manβ4GlcNAc structure in the core structure of N-linked glycan;
or epitope Galβ4Glc,
or terminal mannose
or terminal SAα3/6Gal, wherein SA is a sialic acid, with the provisions that
i) the stem cells are not cells of a cancer cell line and

15 more elements

Dependencies are inferred from the claim text.

Facts

Application: 12/523,628Filed 18 Jan 2008
Priority: FI 2007503318 Jan 2007
Published: 10 Jun 2010
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

G01N 33/5308
Immunoassay; Biospecific binding assay; Materials therefor: for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
C12N 5/0606
Embryonic cells ; Embryoid bodies: pluripotent embryonic cells, e.g. embryonic stem cells [ES]
C12N 5/0647
Cells from the blood or the immune system: haematopoietic stem cells; Uncommitted or multipotent progenitors
C12N 2500/34
Organic components: sugars