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Multivalent ligands control stem cell behaviour in vitro and in vivo

Nature Nanotechnology volume 8, pages 831–838 (2013)Cite this article

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Abstract

There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions1,2,3,4,5,6,7. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology8,9,10. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering11,12,13,14,15,16,17,18. Cellular mechanisms that orchestrate ligand–receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor–ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.

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Figure 1: Multivalent ephrin-B2 enhances neuronal differentiation of NSCs in vitro.
Figure 2: Multivalent ephrin-B2 enhances receptor clustering.
Figure 3: Multivalent ephrin-B2 enhances downstream signalling.
Figure 4: Multivalent ephrin-B2 enhances in vivo neurogenesis.
Figure 5: Multivalent ephrin-B1 enhances neuronal and midbrain fates in differentiating hESCs.

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Acknowledgements

The authors thank A. Ciesielska (Bankiewicz Lab, UCSF) for help with HPLC analysis of dopamine and J. Martin (RPI) for suggestions regarding the synthesis of multivalent ligands based on monovalent hyaluronic acid scaffolds. This work was supported by the National Institutes of Health (NIH R21 EB007295) and the California Institute for Regenerative Medicine (CIRM) (grant RT2-02022). A.C. and T.V. were partially supported by training grant fellowships from CIRM (T1-00007). D.P.S. was partially supported by a National Science Foundation Graduate Research Fellowship and a training grant fellowship from CIRM (TG2-01164).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, 201 Gilman Hall, University of California, Berkeley, 94720-1462, California, USA

    Anthony Conway, Tandis Vazin & David V. Schaffer

  2. Department of Bioengineering, 306 Stanley Hall MC #1762, University of California, Berkeley, 94720-1762, California, USA

    Tandis Vazin, Dawn P. Spelke, Kevin E. Healy & David V. Schaffer

  3. Department of Material Science and Engineering, 210 Hearst Mining Building, University of California, Berkeley, 94720-1760, California, USA

    Nikhil A. Rode & Kevin E. Healy

  4. Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, 12180, New York, USA

    Ravi S. Kane

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Contributions

A.C. performed all the experiments and analysed all data. A.C. and D.V.S. designed the ephrin-B2 experiments and wrote the manuscript. T.V., A.C. and D.V.S. designed the ephrin-B1 experiments. D.P.S. cloned the EphB4-Dendra2 retroviral vector. N.R. conducted the SEC-MALS experiment. K.E.H. and R.S.K. provided critical feedback on the manuscript.

Corresponding author

Correspondence to David V. Schaffer.

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Competing interests

K.E.H. is an inventor of intellectual property related to HA bioconjugates. T.V. is an inventor of intellectual property related to dopaminergic differentiation of hESCs using SPIE.

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Conway, A., Vazin, T., Spelke, D. et al. Multivalent ligands control stem cell behaviour in vitro and in vivo. Nature Nanotech 8, 831–838 (2013). https://doi.org/10.1038/nnano.2013.205

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