Skip to main content

Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation


ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.



adenomatous polyposis coli


basic fibroblast growth factor


cyclin-dependent kinase


casein kinase 1




Dickkopf 1


Dulbecco’s modified Eagle’s medium




epidermal growth factor




glyceraldehyde 3-phosphate dehydrogenase


green fluorescent protein


glycogen synthase kinase 3


Hank’s buffered salt solution


human neural progenitor cell


low-density lipoprotein receptor-related protein 6


microtubuleassociated protein 2


neural progenitor cell


receptor tyrosine kinase-like orphan receptor 2


receptor-like tyrosine kinase


T-cell factor


  1. Lindvall, O., Kokaia, Z. and Martinez-Serrano, A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat. Med. 10Suppl (2004) S42–50.

    PubMed  Article  Google Scholar 

  2. Clelland, C.D., Barker, R.A. and Watts, C. Cell therapy in Huntington disease. Neurosurg. Focus 24 (2008) E9.

    PubMed  Article  Google Scholar 

  3. Locatelli, F., Bersamo, A., Ballabio, E., Lanfranconi, S., Papdimitriou, D., Strazzer, S., Bresolin, N., Comi, G.P. and Corti, S. Stem cell therapy in stroke. Cell. Mol. Life Sci. 66 (2009) 757–772.

    PubMed  Article  CAS  Google Scholar 

  4. Donato, R., Miljan, E.A., Hines, S.J., Aouabdi, S., Pollock, K., Patel, S., Edwards, F.A. and Sinden, J.D. Differential development of neuronal physiological responsiveness in two human neural stem cell lines. BMC Neurosci. 8 (2007) 36.

    PubMed  Article  Google Scholar 

  5. Hoffrogge, R., Mikkat, S., Scharf, C., Beyer, S., Christoph, H., Pahnke, J., Mix, E., Berth, M., Uhrmacher, A., Zubrzycki, I.Z., Miljan, E., Völker, U. and Rolfs, A. 2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM). Proteomics 6 (2006) 1833–1847.

    PubMed  Article  CAS  Google Scholar 

  6. Morgan, P.J., Ortinau, S., Frahm, J., Kruger, N., Rolfs, A. and Frech, M.J. Protection of neurons derived from human neural progenitor cells by veratridine. Neuroreport 20 (2009) 1225–1229.

    PubMed  Article  CAS  Google Scholar 

  7. Logan, C.Y. and Nusse, R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20 (2004) 781–810.

    PubMed  Article  CAS  Google Scholar 

  8. Komiya, Y. and Habas, R. Wnt signal transduction pathways. Organogenesis 4 (2008) 68–75.

    PubMed  Article  Google Scholar 

  9. Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama, T., Masuyama, N. and Gotoh, Y. The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131 (2004) 2791–2801.

    PubMed  Article  CAS  Google Scholar 

  10. Mutoyama, Y., Kondoh, H. and Takada, S. Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochem. Biophys. Res. Commun. 313 (2004) 915–921.

    Article  Google Scholar 

  11. Castelo-Branco, G., Wagner, J., Rodriguez, F.J., Kele, J., Sousa, K., Rawal, N., Pasolli, H.A., Fuchs, E., Kitajewski, J. and Arenas, E. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc. Natl. Acad. Sci. USA 100 (2003) 12747–12752.

    PubMed  Article  CAS  Google Scholar 

  12. Kholodenko B.N. Cell-signaling dynamics in time and space. Nat. Rev. Mol. Cell. Biol. 7 (2006) 165–176.

    PubMed  Article  CAS  Google Scholar 

  13. Nakamura, T., Sano, M., Songyang, Z. and Schneider, M.D. A Wnt- and beta-catenin-dependent pathway for mammalian cardiac myogenesis. Proc. Natl. Acad. Sci. USA 100 (2003) 5834–5839.

    PubMed  Article  CAS  Google Scholar 

  14. Hübner, R., Schmöle, A.C., Liedmann, A., Frech, M.J., Rolfs, A. and Luo, J. Differentiation of human neural progenitor cells regulated by Wnt-3a. Biochem. Biophys. Res. Commun. 400 (2010) 358–362.

    PubMed  Article  Google Scholar 

  15. Schmöle, A.C., Brenführer, A., Karapetyan, G., Jaster, R., Pews_Davtyan, A., Hübner, R., Ortinau, S., Beller, M., Rolfs, A. and Frech, M.J. Novel indolylmaleimide acts as GSK-3β inhibitor in human neural progenitor cells. Bioorg. Med. Chem. 18 (2010) 6785–6795.

    PubMed  Article  Google Scholar 

  16. Klipp, E. and Liebermeister, W. Mathematical modeling of intracellular signalling pathways. BMC Neuroscience 7 (2006) S10.

    PubMed  Article  Google Scholar 

  17. Mazemondet, O., John, M., Maus, C., Uhrmacher A. and Rolfs, A. Integrating diverse reaction types into stochastic models — a signaling pathway case study in the imperative pi-Calculus. In: Proceedings of Winter Simulation Conference, 2009, 931–943.

  18. Pfaffl, M.W. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29 (2001) e45.

    PubMed  Article  CAS  Google Scholar 

  19. Ohl, F., Jung, M., Radonic, A., Sachs, M., Loening, S.A. and Jung, K. Identification and validation of suitable endogenous reference genes for gene expression studies of human bladder cancer. J. Urol. 175 (2006) 1915–1920.

    PubMed  Article  CAS  Google Scholar 

  20. Schiling, M., Maiwald, T., Bohl, S., Kollmann, M., Kreuts, C., Timmer, J. and Klinmüller, U. Computational processing and error reduction strategies for standardized quantitative data in biological networks. FEBS J. 272 (2005) 6400–6411.

    Article  Google Scholar 

  21. Angers, S. and Moon, R.T. Proximal events in Wnt signal transduction. Nat. Rev. Mol. Cell Biol. 10 (2009) 468–477.

    PubMed  CAS  Google Scholar 

  22. Stambolic, V., Ruel, L. and Woodgett, J.R. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol. 6 (1996) 1664–1668.

    PubMed  Article  CAS  Google Scholar 

  23. Jho, E.H., Zhang, T., Domon, C., Joo, C.K., Freund, J.N. and Costantini, F. Wnt/beta-catenin/Tcf signalling induces the transcription of Axin2, a negative regulator of the signalling pathway. Mol. Cell Biol. 22 (2002) 1172–1183.

    PubMed  Article  CAS  Google Scholar 

  24. Blagosklonny, M.V. and Pardee, A.B. The restriction point of the cell cycle. Cell Cycle 1 (2002) 103–110.

    PubMed  CAS  Google Scholar 

  25. Li, Y., Wenyan L., Xi, H. and Guojun, B. Modulation of LRP6-mediated Wnt signaling by molecular chaperone Mesd. FEBS Lett. 580 (2006) 5423–5428.

    PubMed  Article  CAS  Google Scholar 

  26. Tamai, K., Zeng, X., Liu, C., Zhang, X., Harada, Y., Chang, Z. and He, X. A mechanism for Wnt coreceptor activation. Mol. Cell 13 (2004) 149–156.

    PubMed  Article  CAS  Google Scholar 

  27. Khan, Z., Vijayakumar, S., De La Torre, T.V., Rotolo, S. and Bafico, A. Analysis of endogenous LRP6 function reveals a novel feedback mechanism by which Wnt negatively regulates its receptor. Mol. Cell Biol. 27 (2007) 7291–7301.

    PubMed  Article  CAS  Google Scholar 

  28. Winer, J., Jung, C.K., Shackel, I. and Williams, P.M. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal. Biochem. 270 (1999) 41–49.

    PubMed  Article  CAS  Google Scholar 

  29. Willems, E., Mateizel, I., Kemp, C., Gauffman, G., Sermon, K. and Leyns, L. Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells. Int. J. Dev. Biol. 50 (2006) 627–635.

    PubMed  Article  CAS  Google Scholar 

  30. Semenov, M.V., Tamai, K., Brott, B.K., Kuehl, M., Sokol, S. and He, X. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr. Biol. 11 (2001) 951–961.

    PubMed  Article  CAS  Google Scholar 

  31. Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, P., Walter, C., Glinka, A. and Niehrs, C. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417(6889) (2004) 664–667.

    Article  Google Scholar 

  32. Semenov, M.V., Zhang, X. and He, X. DKK1 antagonizes Wnt signaling without promotion of LRP6 internalization and degradation. J. Biol. Chem. 283 (2008) 21427–21432.

    PubMed  Article  CAS  Google Scholar 

  33. Yoshikawa, H., Matsubara, K., Zhou, X., Okumara, S., Kubo, T., Murase, Y., Shikauchi, Y., Esteller, M., Herman, J.G., Wei, X. and Harris, C.C. WNT10B functional dualism: beta-catenin/Tcf-dependent growth promotion or independent suppression with deregulated expression in cancer. Mol. Biol. Cell 18 (2007) 4292–4303.

    PubMed  Article  CAS  Google Scholar 

  34. Kirikoshi, H. and Katoh, M. Expression and regulation of WNT10B in human cancer: up-regulation of WNT10B in MCF-7 cells by beta-estradiol and down-regulation of WNT10B in NT2 cells by retinoic acid. Int. J. Mol. Med. 10 (2002) 507–511.

    PubMed  CAS  Google Scholar 

  35. Ishikawa, T., Tamai, Y., Zorn, A.M., Yoshida, H., Seldin, M.F., Nishikawa, S. and Taketo, M.M. Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placenta angiogenesis. Development 128 (2001) 25–33.

    PubMed  CAS  Google Scholar 

  36. Snow, G.E., Kasper, A.C., Busch, A.M., Schwarz, E., Ewings, K.E., Bee, T., Spinella, M.J., Dmitrovsky, E. and Freemantle, S.J. Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting. BMC Cancer 9 (2009) 83.

    Article  Google Scholar 

  37. van Amerongen, R., Mikels, A. and Nusse, R. Alternative Wnt signaling is initiated by distinct receptors. Sci. Signal. 1 (2008) re9.

    PubMed  Article  Google Scholar 

  38. Caricasole, A., Ferraro, T., Iacovelli, L., Barletta, E., Caruso, A., Melchiorri, D., Terstappen G.C. and Nicoletti, F. Functional characterization of WNT7A signaling in PC12 cells: interaction with a FZD5-LRP6 receptor complex and modulation by Dickkopf proteins. J. Biol. Chem. 278 (2003) 37024–37031.

    PubMed  Article  CAS  Google Scholar 

  39. Carmon, K.S. and Loose, D.S. Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells. Mol. Cancer Res. 6 (2008) 1017–1028.

    PubMed  Article  CAS  Google Scholar 

  40. Le Grand, F., Jones, A.E., Seale, V., Scime, A. and Rudnicki, M.A. Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells. Cell Stem Cell 4 (2009) 535–547.

    PubMed  Article  Google Scholar 

  41. Yang, Y., Topol, L., Lee, H. and Wu, J. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development 130 (2003) 1003–1015.

    PubMed  Article  CAS  Google Scholar 

  42. Castelo-Brance, G., Sousa, K.M., Bryja, V., Pinto, L., Wagner, J. and Arenas, E. Ventral midbrain glia express region-specific transcription factors and regulate dopaminergic neurogenesis through Wnt-5a secretion. Mol. Cell Neurosci. 31 (2006) 251–262.

    Article  Google Scholar 

  43. Beagle, B., Mi, K. and Johnson, G.V.W. Phosphorylation of PPP(S/Y)P motif of the free LRP6 intracellular domains is not required to activate the Wnt/beta-catenin pathway and attenuate GSK3beta activity. J. Cell Biochem. 108 (2009) 886–895.

    PubMed  Article  CAS  Google Scholar 

  44. Wu, G., Huang, H., Abreu, J.G. and He, X. Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6. PloS One 4 (2009) e4926.

    PubMed  Article  Google Scholar 

  45. Bryja, V., Schulte, G. and Arenas, E. Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin. Cell Signal. 19 (2007b) 610–616.

    PubMed  Article  CAS  Google Scholar 

  46. Yokoyama, N., Yin, D. and Malbon, C.C. Abundance, complexation, and trafficking of Wnt/beta-catenin signaling elements in response to Wnt3. J. Mol. Signal. 2 (2007) 11.

    PubMed  Article  Google Scholar 

  47. Müller, H.A., Samanta, R. and Wieschaus, E. Wingless signaling in the Drosophila embryo: zygotic requirements and the role of the frizzled genes. Development 126 (1999) 577–586.

    PubMed  Google Scholar 

  48. Cadigan, K.M. and Nusse, R. Wnt signaling: a common theme in animal development. Genes Dev. 11 (1997) 3286–3305.

    PubMed  Article  CAS  Google Scholar 

  49. Sato, A., Kojima, T., Ui-Tei, K., Miyata, Y. and Saigo, K. Frizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants. Development 126 (1999) 4421–4430.

    PubMed  CAS  Google Scholar 

  50. Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van de Wetering, M., Clevers, H., Schlag, P.M., Birchmeier, W. and Behrens, J. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell Biol. 22 (2004) 1184–1193.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Arndt Rolfs or Jiankai Luo.

Additional information

Both authors contributed equally to this work and should be considered co-first authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mazemondet, O., Hubner, R., Frahm, J. et al. Quantitative and kinetic profile of Wnt/β-catenin signaling components during human neural progenitor cell differentiation. Cell Mol Biol Lett 16, 515 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:

Key words

  • Wnt/β-catenin pathway
  • Spatio-temporal dynamics
  • Quantitative kinetics