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Lectins

The selective binding of recombinant BC2LCN (rBC2LCN) to undifferentiated stem cells enables easy identification of undifferentiated cells for the optimisation of culture conditions.

The in vitro culture of induced pluripotent stem (iPS) cells or embryonic stem (ES) cells can be a tricky process.

Exposing cells to the necessary environmental stimuli to maintain them in an undifferentiated state or conversely direct cell differentiation, can mean juggling cocktails of growth factors, cytokines and inhibitory factors.

Working out what is having the desired effect on your culture is not always easy.

Wako’s range of recombinant lectin products can at least help differentiate the undifferentiated.

Easy identification of undifferentiated cells can enable the optimisation of culture conditions to deliver the best results – whether seeking to maintain undifferentiated cultures or to direct cell differentiation.

Stem cell research has a wealth of potential applications, not least the goals of stem cell derived transplant tissues. When proceeding to transplantation models the ability to screen out undifferentiated potentially tumorigenic cells is essential to develop safe methods for regenerative medicine. Wako’s solution to preparing differentiated cultures is rBC2LCN-PE23. This recombinant lectin-toxin fusion protein contains the catalytic domain of Pseudomonas aeruginosa exotoxin A and addition of this fusion protein to culture media selectively eliminates any undifferentiated cell populations contaminating the differentiated cell culture1

Reference
1. Tateno H. Onuma Y, Ito Y, Minoshima F, Saito S, Shimizu M, Aiki Y, Asashima M, Hirabayashi J. 2015.
Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein. Stem Cell Reports. 4(5):811-820

Now to make things even easier pre-labelled probes are available, choose from FITC, 547 or 635 dyes.

  • rBC2LCN-FITC (green fluorescence)                                 λex= 495nm λem=520nm
  • rBC2LCN-547   (yellow fluorescence, similar to Cy3)     λex= 551nm λem=565nm
  • rBC2LCN-635   (red fluorescence, similar to Cy5)           λex= 634nm λem=654nm

These easy to use probes can stain undifferentiated human stem cells just by adding them to culture medium, enabling live imaging with no need to pre-fix cells. The rBC2LCN probes have low cytotoxicity so there is no need to wash them out in order to continue culturing cells. If you do need to remove the stain, Wako’s rBC2LCN Stripping Solution makes this simple too.

There is no need to disperse cells, just incubate in the solution for 30 minutes to remove the rBC2LCN, enabling you to re-stain with another antibody and continue to culture.

  • rBC2LCN Recombinant Lectins

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References

1. Tateno H, Toyota M, Saito S, Onuma Y, Ito Y, Hiemori K, Fukumura M, Matsushima A, Nakanishi M, Ohnuma K, et al. 2011.
Glycome diagnosis of human induced pluripotent stem cells using lectin microarray. J Biol Chem. 286(23):20345-20353.

2. Tateno H, Matsushima A, Hiemori K, Onuma Y, Ito Y, Hasehira K, Nishimura K, Ohtaka M,Takayasu S, Nakanishi M, et al. 2013.
Podocalyxin is a glycoprotein ligand of the human pluripotent stem cell specific probe rBC2LCN. Stem Cells Transl Med. 2(4):265-273.

3. Onuma Y, Tateno H, Hirabayashi J, Ito Y, Asashima M. 2013.
rBC2LCN, a new probe for live cell imaging of human pluripotent stem cells.
Biochem Biophys Res Commun. 431(3):524-529

4. Tateno H, Omnum Y, Ito Y. 2014. Live-cell imaging of human pluripotent stem cells by a novel lectinprobe rBC2LCN.
Methods Mol Biol. 1200:313-318

5. Tateno H. Onuma Y, Ito Y, Minoshima F, Saito S, Shimizu M, Aiki Y, Asashima M, Hirabayashi J. 2015.
Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein. Stem Cell Reports. 4(5):811-820

The selective binding of recombinant BC2LCN (rBC2LCN) to undifferentiated stem cells was discovered by the Centre for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Japan1.

Following microarray profiling of glycan expression in human iPS and ES cells, rBC2LCN was shown to exclusively recognise an intramolecular sugar chain of podocalyxin found on the surface of undifferentiated human iPS/ES cells2.

Wako’s rBC2LCN was developed in collaboration with AIST and has been successfully used in a number of studies to identify undifferentiated cell populations via cell staining (fixed Fig.1 or live imaging Fig.2) and flow cytometry protocols once labelled with FITC or Cy33-4.

Lectins Fig1

Lectins Fig2

Reference
1. Tateno H, Toyota M, Saito S, Onuma Y, Ito Y, Hiemori K, Fukumura M, Matsushima A, Nakanishi M, Ohnuma K, et al. 2011.
    Glycome diagnosis of human induced pluripotent stem cells using lectin microarray. J Biol Chem. 286(23):20345-20353.

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