Method of profiling a cell population

• US 20100055678A1
• Filed: 06/06/2006
• Published: 03/04/2010
• Est. Priority Date: 06/06/2005
• Status: Active Grant

• Current Assignee: Glykos Finland Oy
• Original Assignee: Glykos Finland Oy, Suomen Punainen Risti Veripalvelu
• Inventors: Blomqvist, Maria, Laine, Jamo, Hemmoranta, Heidi, Satomaa, Tero, Natunen, Jari, Jaatinen, Taina, Partanen, Jukka
• Granted Patent: US 8,440,457 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/5002 Partitioning blood components
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The present invention relates to a method of profiling a cell population comprising a step of detecting the presence or absence of at least two biological markers in said cell population, wherein at least one of said markers is a cell surface marker, which is a sialylated N-glycan marker with structure NeuNAcα3Gal, and at least one of said markers is a mRNA marker related to glycoproteins and/or glycosynthase proteins. The invention also relates to method for purification of cord blood cell population and to a complete cell population from cord blood purified by said method.

Claims

1-72. -72. (canceled)

73. A method for purification of a cell population or a cord blood cell population from a sample containing multiple cell populations, comprising the following subsequent steps of contacting said sample with a specific binding reagent, removing material which is not bound to said specific binding reagent, contacting said sample with an additional amount of the specific binding reagent and removing material which is not bound to said specific binding reagent.

74. The method according to claim 73 comprising the following subsequent steps:

a. contacting said sample with a cell-type-specific binding reagent linked to a polyvalent matrix by mixing said reagent with the sample so that a complex of said binding reagent and a cell population of interest forms;

b. immobilizing the complex obtained from step a);

c. removing the material that is not immobilized;

d. releasing the immobilized complex;

e. contacting the sample obtained from step d) with an additional amount of the specific binding reagent linked to a polyvalent matrix by mixing the additional amount of binding reagent with said sample;

f. immobilizing the complex obtained from step e);

g. removing the material that is not immobilized;

h. releasing the immobilized complex; and

i. recovering a cell population bound to the binding reagent.

75. The method according to claim 74, wherein said polyvalent matrix is selected from the group consisting of:

a solid phase, a solid phase which comprises magnetic material, and a solid phase which comprises a magnetic bead, and said magnetic bead is applied to a magnetic field.

76. The method according to claim 74, wherein the cell population is recovered as a complex of the binding reagent and the cell population, and said cell population is released from the binding reagent.

77. The method according to claim 74, wherein the binding reagent is a monoclonal antibody.

78. The method according to claim 74, wherein the cells in said sample are labelled with a marker and the binding reagent is a labelled antibody applicable for an immunoassay or FACS analysis.

79. The method according to claim 74, wherein the method involves minimum amount of washing and a complete cell population is produced.

80. The method according to claim 74, wherein the steps c) and g) involve up to 1-5 washing steps.

81. The method according to claim 74, wherein the complete cell population is a cell population selected from the group consisting of:

a stem cell population, a natural stem cell population derived from blood, and a stem cell population, which is a cord blood cell population.

82. The method according to claim 81, wherein said cord blood cell population contains CD34 or CD133-antigen.

83. A purified complete cell population obtainable by the method according to claim 73.

84. The cell population according to claim 83 in complex with a specific binding reagent.

85. A complete reproducible cell population purified by a binding reagent from a sample comprising multiple cell populations, wherein sample cell population comprises substantial amount of cells binding weakly to the binding reagent, the amount of weakly binding cells being between 0.1-5% or 5-50% of the cell population.

86. A complete cell population wherein said cell population is from cord blood selected with regard to a single cell surface marker, said cell population being characterized by

a. purity of at least 90%;

b. the presence of at least 50% of available cell surface marker containing cells of cord blood; and

c. the presence of essentially natural ratio of cells expressing low and high levels of the surface marker.

87. The cell population according to claim 86, wherein the cell population is a homogenous cord blood cell population purified with antibody binding marker CD34 and wherein the cell population is a CD34 positive cell population.

88. The cell population according to claim 86, wherein the cell population is a homogenous cord blood cell population purified with antibody binding marker CD133 and the cell population is at least 95% pure.

89. A method of profiling a cell population according to claim 86, wherein the cell population of cord blood is profiled by the method comprising a step of detecting the presence or absence of at least two biological markers in said cell population, wherein at least one of said markers is a cell surface marker, and at least one of said markers is a mRNA marker, with the provision that said cell surface marker is not CD133 or CD34.

90. A method of profiling a cell population of cord blood according to claim 86, preferably a complete CD133 cell population, comprising a step of detecting the presence or absence of at least one mRNA marker listed in Table 5, 6, or 7.

91. The method according to claim 89, wherein said profiling comprises detecting the phenotype(s) of said cell population.

92. A method of profiling a cord blood cell population, preferably according to claim 86, comprising a step of detecting in said cell population the presence or absence of a N-glycan marker having the following core structure:

Manα

3(Manα

6)Manβ

4GlcNAcβ

4(Fucα

6)_0-1GlcNAcβ

Asn

93. The method according to claim 92 comprising a step of detecting the presence or absence of at least two biological markers in said cell population, wherein at least one of said markers is said N-glycan marker, which is a sialylated N-glycan marker with structure NeuNAcα

3Gal, and at least one of said markers is a mRNA marker related to glycoproteins and/or glycosynthase proteins.

94. The method according to claim 92, wherein the cell population is a complete CD133 positive cell population from cord blood.

95. The method according to claim 92, wherein the mRNA marker corresponds to a N-glycoprotein comprising a N-glycosylation site, Asn-X-Ser/Thr/Cys, wherein X is any amino acid except proline.

96. The method according to claim 95, wherein the mRNA marker corresponds to a glycosylated membrane associated protein and it is selected from a group of markers listed in Table 2 or group of N-glycoproteins selected from the group consisting of:

ADAM28, ALCAM, AREG, CRIM1, CRHBP, DSG2, DST, EMP1, FLT3, FSTL1, GPR125, IGFBP7, ITGA9, KIT, MMP28, PILRB, PON2, PTPRD, SCA-1, SV2A, SEPP1, TIE1, TM7SF3, TNFRSF21, LRP6, and VLA4 or mRNA markers related to a potential glycosynthase enzyme;

ST6GALNACIV, CMAH, B3GALT3, GCNT2, and SIAT10.

110. The method according to claim 92 or 97, wherein the cell population is profiled to differentiate cord blood cell populations from other cell populations such as adult peripheral blood or to checked for hematologic malignancy.

97. A method of profiling a cell population of cord blood, preferably CD133-type homogenous cell population, comprising a step of detecting the presence or absence of at least two biological markers in said cell population, wherein at least one of said markers is a cell surface marker, and at least one of said markers is a mRNA marker, with the provision that said cell surface marker is not CD133 or CD34.

98. The method according to claim 97, wherein the cell population is a complete CD133 positive cell population from cord blood or a subpopulation thereof.

99. The method according to claim 97, wherein the mRNA marker is selected from the group consisting of:

glycosylation regulating proteins, glycosyltransferases, nucleotide sugar synthesis associated protein, glycosidases, and glycoproteins.

100. The method according to claim 99, wherein the glycosylation regulating protein is selected from the group consisting of transcription factors, cell cycle related proteins, receptor proteins and growth factors or the cell surface marker is a glycan marker linked to a protein carrier structure and the glycan marker is a N-linked glycan linked to a protein.

101. The method according to claim 100, wherein the glycan marker is a α

3- and/or α

6-sialylated glycan structure.

102. The method according to claim 100, wherein the glycan marker has the structure according to the Formula

SAα

3/6Galβ

3/4GlcNAc

wherein said structure is linked to a N-glycan, and, wherein SA is sialic acid, preferably NeuNAc.

103. The method according to claim 97, wherein said mRNA marker is selected from a group of markers listed in Table 5 or Table 6 or Table 7.

104. The method according to claim 103, wherein the expression of said mRNA marker in CD133 positive cells is altered

105. The method according to claim 103, wherein said mRNA marker is related to a potential membrane protein.

106. The method according to claim 105, wherein said mRNA marker comprises a mRNA sequence translatable as amino acid sequence Asn-X-Ser/Thr/Cys, wherein X can be any amino acid except proline.

107. The method according to claim 104, wherein said mRNA marker is selected form the group consisting of:

markers correlating cord blood cell populations with low association with CD34, or mRNA markers selected from the group consisting of;

ALCAM, SV2A, and DSG2.

108. The method according to claim 97, wherein said mRNA marker is selected from the following group consisting of markers:

mRNA marker relates to a potential glycosylation enzyme and is selected from the group consisting of;

ST6GALNACIV, B3GALT3 GCNT2 and SIAT10;

mRNA marker related to a potential cell cycle and is selected from the TABLE 11, SPINK2, or mRNA marker having an on-off change in expression in early blood cells and is selected from Table 15.

109. The method according to claim 97, wherein said mRNA marker relates to a glycosylated protein and the presence or absence of said mRNA marker is observed indirectly by verifying correlation of said marker with a corresponding protein or carbohydrate level.

111. A method for selecting optimal biomarkers for a stem cell population, preferably for a complete cord blood derived CD133 or CD34 cell population, the method comprising the steps of:

a. producing a pure stem cell population;

b. selecting a marker from markers listed in Tables 1-27 for testing the cell population;

c. producing specific binding molecule recognizing the marker;

d. testing the binding of the binding molecule to the cell population or testing the manipulation of the cell population by the binding molecule;

e. optionally repeating the test with multiple cell populations from different individuals and with a control cell population(s); and

f. selecting the optimal binding protein based on the binding activity and/or specificity and/or reproducibility of the protein.

112. Method for combined mRNA expression analysis and glycome analysis, the method comprising the steps of:

a. producing a pure stem cell population;

b. screening mRNA expression levels of the cell population and screening of N-glycans of the cell population by carbohydrate recognizing screening methods;

c. correlating the glycan expression with mRNA expression; and

d. selecting the structure correlated in the previous step as a marker structure,

wherein the cell population is preferably a complete cord blood derived CD133 or CD34 cell population.

113. Method of assigning glycan structures with specific cell surface marker proteins, the method comprising the steps of:

a. producing a pure stem cell population;

b. isolating glycoprotein(s) from the cell;

c. analyzing glycan structures of the isolated protein and/or the identity of the protein;

wherein the cell population is preferably a complete cord blood derived CD133 or CD34 cell population.