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A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels

Nature Materials volume 14, pages 523–531 (2015)Cite this article

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Abstract

Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection–oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.

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Figure 1: Hydrogel components and reactions used for photoreversible protein patterning.
Figure 2: Patterning of proteins into SPAAC-based gels by means of photomediated oxime ligation.
Figure 3: Photoremoval of pre-patterned proteins from SPAAC-based gels through o-nitrobenzyl ester linker photocleavage.
Figure 4: Interconnected dual-protein patterns generated through protein photorelease and oxime ligation.
Figure 5: Proteins remain bioactive on photoreversible patterning of gels.
Figure 6: Spatial and temporal control of hMSC differentiation by photoreversible patterning of vitronectin.

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Acknowledgements

The authors thank P. Rapp for discussions on FRAP and FEM analysis, as well as for his constructive comments on the manuscript; M. Shahgholi and N. Torian for assistance with HRMS; J. Heath, J. Pfeilsticker and R. Henning for advice on peptide work and for use of their peptide synthesizer and HPLC; D. Koos and the Caltech Biological Imaging Center for use of confocal microscopes; and K. Beres and C. Murry for assistance with all Notch-related studies. This work was supported by the National Science Foundation Grant NSF-DMR 1206121 and a University of Washington Faculty Startup Grant (C.A.D.).

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  1. Division of Chemistry and Chemical Engineering California Institute of Technology, 1200 East California Boulevard Pasadena, California 91125, USA,

    Cole A. DeForest & David A. Tirrell

  2. Department of Chemical Engineering, University of Washington, 4000 15th Avenue NE Seattle, Washington 98195, USA,

    Cole A. DeForest

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  1. Cole A. DeForest
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C.A.D. and D.A.T. designed the experiments. C.A.D. conducted the experiments. C.A.D. and D.A.T. interpreted the data and composed the manuscript.

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Correspondence to Cole A. DeForest or David A. Tirrell.

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DeForest, C., Tirrell, D. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels. Nature Mater 14, 523–531 (2015). https://doi.org/10.1038/nmat4219

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