Unknown,Transcriptomics,Genomics,Proteomics

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Effect of rBC2LCN on human embryonic stem cells

ABSTRACT: We show a recombinant lectin probe, rBC2LCN, is a new tool for fluorescence-based imaging and flow cytometry analysis of human pluripotent stem cells, as an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized using fluoresceinated rBC2LCN. Fluoresceinated rBC2LCN also separated live pluripotent stem cells from mixed cell population by flow cytometry. Induced gene expression in human ES cells was measured at 3 days treatment of rBC2LCN lectin. WA01 (H1) cells were treated with 1, 10 and 100 ug/ml of rBC2LCN or 1 of FITC-rBC2LCN for three days. Two independent experiments were performed at each treatment experiment. Samples of human ES cells (KhES-1 and 3), human iPS cells (201B7 and 253G1) and HDF in each conventional culture condition were used for outgroup examples.

ORGANISM(S): Homo sapiens

SUBMITTER: Yuzuru Ito

PROVIDER: E-GEOD-42976 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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rBC2LCN, a new probe for live cell imaging of human pluripotent stem cells.

Onuma Yasuko Y Tateno Hiroaki H Hirabayashi Jun J Ito Yuzuru Y Asashima Makoto M

Biochemical and biophysical research communications 20130112 3

Cell surface biomarkers have been applied to discriminate pluripotent human embryonic stem cells and induced pluripotent stem cells from differentiated cells. Here, we demonstrate that a recombinant lectin probe, rBC2LCN, a new tool for fluorescence-based imaging and flow cytometry analysis of pluripotent stem cells, is an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized in cultu ...[more]

PMID: 23321312

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Project description:In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type1 transmembrane protein, is the predominant glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds predominantly to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched Oglycan comprising an H type3 structure as the most probable rBC2LCN glycan ligand (Kd, 4.0 x 10-5 M) prepared from human 201B7 iPS cells, suggesting that H type3 is a novel potential pluripotency marker. We conclude that podocalyxin is the predominant glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. Gene expressions in human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs). Human iPS cells (MRC5-iPS, AM-iPS, UtE-iPS, PAE-iPS) were induced from four different somatic cells by infection of the retroviral vectors pMXs encoding OCT3/4, SOX2, KLF4 and c-MYC, simultaneously. Human iPS cells (TIG/MKOS#19, #56, #106) were generated through reprogramming by Sendai virus infection-mediated expression of OCT4, SOX2, KLF4, and c-MYC.

Dataset Information

Effect of rBC2LCN on human embryonic stem cells

Publications

rBC2LCN, a new probe for live cell imaging of human pluripotent stem cells.

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