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A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells.

Tateno H, Onuma Y, Ito Y, Hiemori K, Aiki Y, Shimizu M, Higuchi K, Fukuda M, Warashina M, Honda S, Asashima M, Hirabayashi J - Sci Rep (2014)

Bottom Line: We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN.The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells.The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.

ABSTRACT
While human pluripotent stem cells are attractive sources for cell-replacement therapies, a major concern remains regarding their tumorigenic potential. Thus, safety assessment of human pluripotent stem cell-based products in terms of tumorigenicity is critical. Previously we have identified a pluripotent stem cell-specific lectin probe rBC2LCN recognizing hyperglycosylated podocalyxin as a cell surface ligand. Here we demonstrate that hyperglycosylated podocalyxin is secreted from human pluripotent stem cells into cell culture supernatants. We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN. The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells. This work provides a proof of concept for the noninvasive and quantitative detection of tumorigenic human pluripotent stem cells using cell culture supernatants. The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

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Schematic representation of the principle of the GlycoStem test.Hyperglycosylated podocalyxin, a type1 transmembrane protein, carries a hiPSC/hESC marker (H type3, Fucα1-2Galβ1-3GalNAc) recognized by the hiPSC/hESC-specific lectin probe rBC2LCN (discriminator). Podocalyxin (soluble form) is secreted into cell culture supernatants, and is captured by rBC2LCN immobilized on a microtiter plate. The rBC2LCN-captured podocalyxin is detected with HRP-labeled rABA (signal enhancer) recognizing mucin-type O-glycans heavily displayed on podocalyxin. The number of hPSCs is quantified with the GlycoStem test using cell culture supernatants.
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f1: Schematic representation of the principle of the GlycoStem test.Hyperglycosylated podocalyxin, a type1 transmembrane protein, carries a hiPSC/hESC marker (H type3, Fucα1-2Galβ1-3GalNAc) recognized by the hiPSC/hESC-specific lectin probe rBC2LCN (discriminator). Podocalyxin (soluble form) is secreted into cell culture supernatants, and is captured by rBC2LCN immobilized on a microtiter plate. The rBC2LCN-captured podocalyxin is detected with HRP-labeled rABA (signal enhancer) recognizing mucin-type O-glycans heavily displayed on podocalyxin. The number of hPSCs is quantified with the GlycoStem test using cell culture supernatants.

Mentions: We then attempted to establish a practical system to measure rBC2LCN-positive media. For this purpose, it seemed reasonable to adopt a sandwich assay system to enhance the significant but relatively weak signals. Assuming hyperglylcosyated podocalyxin as a major target molecule10, selection of an overlay probe which works best as a “signal enhancer” is critical, (Fig. 1). Cell culture supernatants of TIG3 hiPSCs (TIG/MKOS #19) or control media were incubated with rBC2LCN immobilized on a glass slide. After washing, Cy3-labeled overlay-probe candidates were incubated at 20°C for 3 h, and their binding was analyzed by an evanescence-field fluorescence scanner12. We first tried goat anti-podocalyxin polyclonal antibody (pAb) as an overlay probe toward a conventional antibody-lectin sandwich assay system1314, but without success, although the antibody could be used for western blotting and immunoprecipitation of podocalyxin10. We then challenged a novel system of “lectin-lectin” sandwich assay, which utilizes two glycan-specific probes (Fig. 1). As a result of screening 44 recombinant lectins to enhance the rBC2LCN signals, 17 were shown to give higher signals to TIG3 hiPSCs (TIG/MKOS #19) supernatants than control cell culture media (Fig. S2). Four recombinant lectins, Sclerotium rolfsii lectin (rSRL), Coprinopsis cinerea lectin 2 (rCGL2), Agaricus bisporus lectin (rABA), and Xerocomus chrysenteron (rXCL) exhibited strong enough signals (>10,000) to cell culture supernatants of TIG3 hiPSCs (TIG/MKOS #19), while giving only little or no signal to control media (<2,500). This result demonstrates that the four lectins could serve as strong signal enhancers. For the subsequent studies, rABA was used as an overlay molecule, which gave the best S/N ratio in the ELISA-type assay described below.


A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells.

Tateno H, Onuma Y, Ito Y, Hiemori K, Aiki Y, Shimizu M, Higuchi K, Fukuda M, Warashina M, Honda S, Asashima M, Hirabayashi J - Sci Rep (2014)

Schematic representation of the principle of the GlycoStem test.Hyperglycosylated podocalyxin, a type1 transmembrane protein, carries a hiPSC/hESC marker (H type3, Fucα1-2Galβ1-3GalNAc) recognized by the hiPSC/hESC-specific lectin probe rBC2LCN (discriminator). Podocalyxin (soluble form) is secreted into cell culture supernatants, and is captured by rBC2LCN immobilized on a microtiter plate. The rBC2LCN-captured podocalyxin is detected with HRP-labeled rABA (signal enhancer) recognizing mucin-type O-glycans heavily displayed on podocalyxin. The number of hPSCs is quantified with the GlycoStem test using cell culture supernatants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921628&req=5

f1: Schematic representation of the principle of the GlycoStem test.Hyperglycosylated podocalyxin, a type1 transmembrane protein, carries a hiPSC/hESC marker (H type3, Fucα1-2Galβ1-3GalNAc) recognized by the hiPSC/hESC-specific lectin probe rBC2LCN (discriminator). Podocalyxin (soluble form) is secreted into cell culture supernatants, and is captured by rBC2LCN immobilized on a microtiter plate. The rBC2LCN-captured podocalyxin is detected with HRP-labeled rABA (signal enhancer) recognizing mucin-type O-glycans heavily displayed on podocalyxin. The number of hPSCs is quantified with the GlycoStem test using cell culture supernatants.
Mentions: We then attempted to establish a practical system to measure rBC2LCN-positive media. For this purpose, it seemed reasonable to adopt a sandwich assay system to enhance the significant but relatively weak signals. Assuming hyperglylcosyated podocalyxin as a major target molecule10, selection of an overlay probe which works best as a “signal enhancer” is critical, (Fig. 1). Cell culture supernatants of TIG3 hiPSCs (TIG/MKOS #19) or control media were incubated with rBC2LCN immobilized on a glass slide. After washing, Cy3-labeled overlay-probe candidates were incubated at 20°C for 3 h, and their binding was analyzed by an evanescence-field fluorescence scanner12. We first tried goat anti-podocalyxin polyclonal antibody (pAb) as an overlay probe toward a conventional antibody-lectin sandwich assay system1314, but without success, although the antibody could be used for western blotting and immunoprecipitation of podocalyxin10. We then challenged a novel system of “lectin-lectin” sandwich assay, which utilizes two glycan-specific probes (Fig. 1). As a result of screening 44 recombinant lectins to enhance the rBC2LCN signals, 17 were shown to give higher signals to TIG3 hiPSCs (TIG/MKOS #19) supernatants than control cell culture media (Fig. S2). Four recombinant lectins, Sclerotium rolfsii lectin (rSRL), Coprinopsis cinerea lectin 2 (rCGL2), Agaricus bisporus lectin (rABA), and Xerocomus chrysenteron (rXCL) exhibited strong enough signals (>10,000) to cell culture supernatants of TIG3 hiPSCs (TIG/MKOS #19), while giving only little or no signal to control media (<2,500). This result demonstrates that the four lectins could serve as strong signal enhancers. For the subsequent studies, rABA was used as an overlay molecule, which gave the best S/N ratio in the ELISA-type assay described below.

Bottom Line: We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN.The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells.The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.

ABSTRACT
While human pluripotent stem cells are attractive sources for cell-replacement therapies, a major concern remains regarding their tumorigenic potential. Thus, safety assessment of human pluripotent stem cell-based products in terms of tumorigenicity is critical. Previously we have identified a pluripotent stem cell-specific lectin probe rBC2LCN recognizing hyperglycosylated podocalyxin as a cell surface ligand. Here we demonstrate that hyperglycosylated podocalyxin is secreted from human pluripotent stem cells into cell culture supernatants. We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN. The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells. This work provides a proof of concept for the noninvasive and quantitative detection of tumorigenic human pluripotent stem cells using cell culture supernatants. The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

Show MeSH
Related in: MedlinePlus