Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs. We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs. Induced pluripotent stem cells (iPSCs) 1The abbreviations used are:iPSCinduced pluripotent stem cellFucfucoseGald-galactoseGalNAcN-acetyl-d-galactosamineGlcd-glucoseGlcNAcN-acetyl-d-glucosamineHPLChigh performance liquid chromatographyLacNAcN-acetyllactosamineManD-mannoseMALDImatrix-assisted laser desorption-ionizationMSmass spectrometryNeuAcN-acetylneuraminic acidPA-pyridylaminatedSCsomatic cellSiasialic acidTOFtime of flight. 1The abbreviations used are:iPSCinduced pluripotent stem cellFucfucoseGald-galactoseGalNAcN-acetyl-d-galactosamineGlcd-glucoseGlcNAcN-acetyl-d-glucosamineHPLChigh performance liquid chromatographyLacNAcN-acetyllactosamineManD-mannoseMALDImatrix-assisted laser desorption-ionizationMSmass spectrometryNeuAcN-acetylneuraminic acidPA-pyridylaminatedSCsomatic cellSiasialic acidTOFtime of flight. are genetically manufactured pluripotent cells obtained by the transfection of reprogramming factors. Such iPSCs were first reported in 2006 for the mouse (1Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18957) Google Scholar) and in 2007 for humans (2Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15039) Google Scholar, 3Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Induced pluripotent stem cell lines derived from human somatic cells.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8182) Google Scholar). Although iPSCs have already been used in the fields of drug development and disease models (4Hanna J. Wering M. Markoulaki S. Sun C.W. Meissner A. Cassady J.P. Beard C. Brambrink T. Wu L.C. Townes T.M. Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.Science. 2007; 318: 1920-1923Crossref PubMed Scopus (1235) Google Scholar, 5Viczian A.S. Solessio E.C. Lyou Y. Zuber M.E. Generation of functional eyes from pluripotent cells.PLoS Biol. 2009; 7: e1000174Crossref PubMed Scopus (59) Google Scholar, 6Inoue H. Yamanaka S. The use of induced pluripotent stem cells in drug development.Clin. Pharmacol. Ther. 2011; 89: 655-661Crossref PubMed Scopus (181) Google Scholar, 7Rashid S.T. Corbineau S. Hannan N. Marciniak S.J. Miranda E. Alexander G. Huang-Doran I. Griffin J. Ahrlund-Richter L. Skepper J. Semple R. Weber A. Lomas D.A. Vallier L. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells.J. Clin. Invest. 2010; 120: 3127-3136Crossref PubMed Scopus (493) Google Scholar), basic aspects of iPSCs largely remain to be elucidated to provide us with a fuller understanding of their properties and for therapeutic applications to be developed in the field of regenerative medicine. These aspects include the need for a definitive system to be established to evaluate their properties; e.g. pluripotency, differentiation propensity, risk of possible contamination of xenoantigens, and even the potential for tumorigenesis. Cell surface glycans are often referred to as the “cell signature,” which changes dramatically depending on the cell properties and conditions (8Varki A. Lowe J.B. Biological roles on glycans.Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY2009: 75-88Google Scholar) as a result of changes in gene expression, including epigenetic modifications of glycan-related molecules. Glycans, because of their outermost cell-surface locations and structural complexity, are considered to be most advantageous communication molecules, playing roles in various biological phenomena. Indeed, SSEA3/4 and Tra-1–60/81, which have been used to discriminate pluripotency, are cell surface glycan epitopes that respond to some specific antibodies (9Muramatsu T. Muramatsu H. Carbohydrate antigens expressed on stem cells and early embryonic cells.Glycoconj. J. 2004; 21: 41-45Crossref PubMed Scopus (117) Google Scholar, 10Schopperle W.M. DeWolf W.C. The TRA-1–60 and TRA-1–81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma.Stem Cells. 2007; 25: 723-730Crossref PubMed Scopus (110) Google Scholar, 11Natunen S. Satomaa T. Pitkanen V. Salo H. Mikkola M. Natunen J. Otonkoski T. Valmu L. 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M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar), of iPSCs and were and the of the pluripotent state were The glycan profiles of the derived from different were different from one another and from of the iPSCs. the used the to detailed glycan structures or their a on high performance liquid with laser of was for both the definitive of glycan structures and their quantitative which remained in the analysis H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). We structural on N-linked and O-linked glycans derived from the human 201B7 cell (2Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15039) Google Scholar) and human dermal fibroblasts undifferentiated and differentiated cells, respectively. quantitative the glycans were by from similar of cells S. T. A. of to PubMed Scopus Google Scholar, H. T. K. A. T. T. for the of from a by PubMed Scopus Google Scholar, H. Y. N. of and to the of cell surface by Biol. Full Text PDF PubMed Google Scholar, Y. Y. J. N. K. T. and of and from PubMed Scopus Google Scholar) with their S. T. Y. of by of the with a PubMed Scopus Google Scholar, S. liquid of PubMed Scopus Google Scholar), which the derived glycans were by and structures were and by with and the first structural the of a “glycome shift” upon induction of pluripotency. iPSCs, 201B7 cells, the first iPSCs established from human dermal fibroblasts by the Yamanaka were used (2Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15039) Google Scholar). The cells were in with and human basic on mouse fibroblast cells. a we human dermal fibroblasts which were in fibroblast with fibroblast human and both differentiated fibroblasts and undifferentiated 201B7 cells, cultures of the were prepared for quantitative structural was obtained from was from and was from The was from The the and the were from and respectively. and were from and were from α-1–2 and α-2–3 from and expressed in E. was from was from was from and were of while the were of glycans were from and by the established for S. T. A. of to PubMed Scopus Google Scholar, H. T. K. A. T. T. for the of from a by PubMed Scopus Google Scholar, H. Y. N. of and to the of cell surface by Biol. Full Text PDF PubMed Google Scholar, Y. Y. J. N. K. 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N. of Glycobiology. Cold Spring Harbor Laboratory, NY2009: Scholar), these be to of the expression of which to as the first in the of or possible to that for structural the expression of the gene was in iPSCs to as in a analysis H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). the it well in iPSCs, while the of type the we could not a of the which a of the specific to and but not to it not a in the in The of type N-linked glycans were increased in iPSCs and in a expression of which to considered of gene expression a in which a in iPSCs H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). the that a of glycans with are in iPSCs. the to because the of glycans that of fully but glycans these results to the that structures of type glycans of iPSCs are to of in to the of type glycan N-linked glycans of the N-linked glycans in iPSCs. the linkage was of the type and in to N-linked glycans of the N-linked but were of the α-2–3 in N-linked glycans of iPSCs was dramatically changed from the α-2–3 to the was by of iPSCs, including are in with their to reported and H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google of glycan and of glycan to and glycosyltransferase of structural to for the of or glycan from H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google of which are for the glycan structures from H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). in a a of structural to I. for the of or glycan from H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). of which are for the glycan structures from H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). to the no α-2–3 Sia be in iPSCs with in Sia we fractions Sia fractions and were with different of C. and S. α-2–3 and the were by and fractions to N-linked and were with α-2–3 of the glycans derived from the fractions were that of α-2–3 and respectively. the of iPSCs was to α-2–3 the to and glycans α-2–3 it evident that of of a of glycans with a of α-2–3 The to α-2–3 in of analysis a in upon induction of pluripotency H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis the expression of 1 and in of these observations unambiguously that the linkage type of in changed to the type upon analysis of fractions suggested that and fractions to O-linked glycans and the in the linkage type of in O-linked glycans iPSCs and was for that of the of O-linked glycans was in iPSCs with that of of α-1–2 fucose were in both N- and O-linked glycans from iPSCs, while were detected in well with the results obtained by and with the expression of the for α-1–2 and H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. M. of human induced pluripotent stem cells using Biol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar) α-1–2 N-linked glycans and were in a glycan as to glycan in in glycan was with α-1–2 and with a the for the with of and was as α-1–2 and glycan a glycan as was as α-1–2 and and glycan no α-1–2 N-linked glycan was detected in O-linked glycans were detected in and fractions the in to a in and their to that of with α-1–2 was to the with structures of and were considered to be and we that α-1–2 fucose a to iPSCs. the a in from to was The of iPSCs the of the type 1 LacNAc the a N-linked glycan of the N-linked was derived from of which the with The of a was by O-linked was to and the most was The of type 1 LacNAc in was from a quantitative because such glycans were not detected in while the of glycan was as high as of the with the for the increased expression of which the involved in the of type 1 was in human iPSCs and to H. M. S. Y. Y. K. M. A. M. K. H. A. K. J. 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