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Constructing cellular niche properties by localized presentation of Wnt proteins on synthetic surfaces

Nature Protocols volume 12, pages 1498–1512 (2017)Cite this article

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Abstract

Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on Wnt proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize Wnt3a proteins on microbeads or a glass surface. This procedure requires ∼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active Wnt proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for Wnt/β-catenin-dependent transcription. Immobilization efficiency can be measured 12–18 h after seeding the cells and takes 2–4 h. The covalent immobilization of Wnt proteins can also be used for single-cell analysis using Wnt-coated microbeads (12–18 h of live imaging) and to create a Wnt platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for Wnt immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed.

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Figure 1: The chemistry of covalent immobilization of Wnt proteins.
Figure 2: Assessment of biological activity of the Wnt surfaces—quality control assays.
Figure 3: Analysis of the effects of Wnt3a surfaces on the pluripotency of mESCs.

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Acknowledgements

We thank C. Chiappini, C. Garcin and E. Rognoni for comments on the manuscript. This work was supported in part by a Sir Henry Dale Fellowship (102513/Z/13/Z to S.J.H.) and a grant from the UK Regenerative Medicine Platform (MRC Niche Hub Reference MR/K026666/1 to S.J.H.).

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Author notes

  1. Molly Lowndes and Sergi Junyent: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK

    Molly Lowndes, Sergi Junyent & Shukry J Habib

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  1. Molly Lowndes
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Contributions

M.L., S.J. and S.J.H. designed and performed experiments, analyzed the data and wrote the paper.

Corresponding author

Correspondence to Shukry J Habib.

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Competing interests

S.J.H. is named on pending US patent application no. 20140171356 A1 (Chemically immobilized Wnt protein and methods of use).

Integrated supplementary information

Supplementary Figure 1 Flow cytometry gating strategy.

Individual histograms from Figure 3. On the right of each histogram is the gating strategy, showing the subpopulation percentages. The gate for the FSC-A × SSC-A plots was used to determine the cell population from all events. The gate on the FSC-A × Comp-Pacific-Blue-A plots separates the live from the dead cells, which have incorporated the DAPI stain. A chart shows the final number of cells analyzed, along with the statistics for each condition (geometric mean and coefficient of variation).

Supplementary information

Supplementary Figure 1

Flow cytometry gating strategy. Individual histograms from Figure 3. On the right of each histogram is the gating strategy, showing the subpopulation percentages. The gate for the FSC-A × SSC-A plots was used to determine the cell population from all events. The gate on the FSC-A × Comp-Pacific-Blue-A plots separates the live from the dead cells, which have incorporated the DAPI stain. A chart shows the final number of cells analyzed, along with the statistics for each condition (geometric mean and coefficient of variation). (PDF 260 kb)

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Lowndes, M., Junyent, S. & Habib, S. Constructing cellular niche properties by localized presentation of Wnt proteins on synthetic surfaces. Nat Protoc 12, 1498–1512 (2017). https://doi.org/10.1038/nprot.2017.061

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