Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. DeForest, C. A. & Anseth, K. S. Advances in bioactive hydrogels to probe and direct cell fate. Annu. Rev. Chem. Biomol. 3, 421–444 (2012).

    CAS  Google Scholar 

  2. Langer, R. & Tirrell, D. A. Designing materials for biology and medicine. Nature 428, 487–492 (2004).

    CAS  Google Scholar 

  3. Lutolf, M. P., Gilbert, P. M. & Blau, H. M. Designing materials to direct stem-cell fate. Nature 462, 433–441 (2009).

    CAS  Google Scholar 

  4. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Google Scholar 

  5. Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nature Mater. 9, 518–526 (2010).

    CAS  Google Scholar 

  6. Yang, C., Tibbitt, M. W., Basta, L. & Anseth, K. S. Mechanical memory and dosing influence stem cell fate. Nature Mater. 13, 645–652 (2014).

    CAS  Google Scholar 

  7. Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M. & Ingber, D. E. Geometric control of cell life and death. Science 276, 1425–1428 (1997).

    CAS  Google Scholar 

  8. Petersen, O. W., Ronnovjessen, L., Howlett, A. R. & Bissell, M. J. Interaction with basement-membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc. Natl Acad. Sci. USA 89, 9064–9068 (1992).

    CAS  Google Scholar 

  9. Burdick, J. A. & Murphy, W. L. Moving from static to dynamic complexity in hydrogel design. Nature Commun. 3, 1269 (2012).

    Google Scholar 

  10. Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnol. 23, 47–55 (2005).

    CAS  Google Scholar 

  11. Rompolas, P., Mesa, K. R. & Greco, V. Spatial organization within a niche as a determinant of stem-cell fate. Nature 502, 513–518 (2013).

    CAS  Article  Google Scholar 

  12. Katz, J. S. & Burdick, J. A. Light-responsive biomaterials: Development and applications. Macromol. Biosci. 10, 339–348 (2010).

    CAS  Google Scholar 

  13. DeLong, S. A., Moon, J. J. & West, J. L. Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration. Biomaterials 26, 3227–3234 (2005).

    CAS  Google Scholar 

  14. Hahn, M. S., Miller, J. S. & West, J. L. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv. Mater. 18, 2679–2684 (2006).

    CAS  Google Scholar 

  15. DeForest, C. A., Polizzotti, B. D. & Anseth, K. S. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nature Mater. 8, 659–664 (2009).

    CAS  Google Scholar 

  16. DeForest, C. A., Sims, E. A. & Anseth, K. S. Peptide-functionalized click hydrogels with independently tunable mechanics and chemical functionality for 3D cell culture. Chem. Mater. 22, 4783–4790 (2010).

    CAS  Google Scholar 

  17. DeForest, C. A. & Anseth, K. S. Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photocoupling and photodegradation reactions. Nature Chem. 3, 925–931 (2011).

    CAS  Google Scholar 

  18. Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Mater. 3, 249–253 (2004).

    CAS  Google Scholar 

  19. Wylie, R. G. et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nature Mater. 10, 799–806 (2011).

    CAS  Google Scholar 

  20. Adzima, B. J. et al. Spatial and temporal control of the alkyne–azide cycloaddition by photoinitiated Cu(II) reduction. Nature Chem. 3, 258–261 (2011).

    Google Scholar 

  21. Mosiewicz, K. A. et al. In situ cell manipulation through enzymatic hydrogel photopatterning. Nature Mater. 12, 1072–1078 (2013).

    CAS  Article  Google Scholar 

  22. Kloxin, A. M., Kasko, A. M., Salinas, C. N. & Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59–63 (2009).

    CAS  Google Scholar 

  23. Wirkner, M. et al. Triggered cell release from materials using bioadhesive photocleavable linkers. Adv. Mater. 23, 3907–3910 (2011).

    CAS  Google Scholar 

  24. Griffin, D. R. et al. Synthesis of photodegradable macromers for conjugation and release of bioactive molecules. Biomacromolecules 14, 1199–1207 (2013).

    CAS  Google Scholar 

  25. Kloxin, A. M., Tibbitt, M. W. & Anseth, K. S. Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms. Nature Protoc. 5, 1867–1887 (2010).

    CAS  Google Scholar 

  26. Fairbanks, B. D., Singh, S. P., Bowman, C. N. & Anseth, K. S. Photodegradable, photoadaptable hydrogels via radical-mediated disulfide fragmentation reaction. Macromolecules 44, 2444–2450 (2011).

    CAS  Google Scholar 

  27. Griffin, D. R. & Kasko, A. M. Photodegradable macromers and hydrogels for live cell encapsulation and release. J. Am. Chem. Soc. 134, 13103–13107 (2012).

    CAS  Google Scholar 

  28. Azagarsamy, M. A., Alge, D. L., Radhakrishnan, S. J., Tibbitt, M. W. & Anseth, K. S. Photocontrolled nanoparticles for on-demand release of proteins. Biomacromolecules 13, 2219–2224 (2012).

    CAS  Google Scholar 

  29. He, M., Li, J., Tan, S., Wang, R. & Zhang, Y. Photodegradable supramolecular hydrogels with fluorescence turn-on reporter for photomodulation of cellular microenvironments. J. Am. Chem. Soc. 135, 18718–18721 (2013).

    CAS  Google Scholar 

  30. DeForest, C. A. & Anseth, K. S. Photoreversible patterning of biomolecules within click-based hydrogels. Angew. Chem. Int. Ed. 51, 1816–1819 (2011).

    Google Scholar 

  31. Lutolf, M. P. Materials science: Cell environments programmed with light. Nature 482, 477–478 (2012).

    CAS  Google Scholar 

  32. Alge, D. L. & Anseth, K. S. Bioactive hydrogels: Lighting the way. Nature Mater. 12, 950–952 (2013).

    CAS  Google Scholar 

  33. Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    CAS  Google Scholar 

  34. Lutolf, M. P. et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics. Proc. Natl Acad. Sci. USA 100, 5413–5418 (2003).

    CAS  Google Scholar 

  35. Patterson, J. & Hubbell, J. A. Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2. Biomaterials 31, 7836–7845 (2010).

    CAS  Google Scholar 

  36. Codelli, J. A., Baskin, J. M., Agard, N. J. & Berozzi, C. R. Second-generation difluorinated cyclooctynes for copper-free click chemistry. J. Am. Chem. Soc. 130, 11486–11493 (2008).

    CAS  Google Scholar 

  37. Sims, E. A., DeForest, C. A. & Anseth, K. S. A mild, large-scale synthesis of 1,3-cyclooctanedione: Expanding access to difluorinated cyclooctyne for copper-free click chemistry. Tetrahedron Lett. 52, 1871–1873 (2011).

    CAS  Google Scholar 

  38. Ning, X. H., Guo, J., Wolfert, M. A. & Boons, G. J. Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions. Angew. Chem. Int. Ed. 47, 2253–2255 (2008).

    CAS  Google Scholar 

  39. Debets, M. F. et al. Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition. Chem. Commun. 46, 97–99 (2010).

    CAS  Google Scholar 

  40. Tibbitt, M. W., Kloxin, A. M., Sawicki, L. A. & Anseth, K. S. Mechanical properties and degradation of chain and step-polymerized photodegradable hydrogels. Macromolecules 46, 2785–2792 (2013).

    CAS  Google Scholar 

  41. Dendane, N. et al. Efficient surface patterning of oligonucleotides inside a glass capillary through oxime bond formation. Bioconjug. Chem. 18, 671–676 (2007).

    CAS  Google Scholar 

  42. Park, S. & Yousaf, M. N. An interfacial oxime reaction to immobilize ligands and cells in patterns and gradients to photoactive surfaces. Langmuir 24, 6201–6207 (2008).

    CAS  Google Scholar 

  43. Woll, D. et al. Intramolecular sensitization of photocleavage of the photolabile 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group: Photoproducts and photokinetics of the release of nucleosides. Chem. Eur. J. 14, 6490–6497 (2008).

    Google Scholar 

  44. Odian, G. G. Principles of Polymerization 4th edn (Wiley-Interscience, 2004).

    Google Scholar 

  45. Jevševar, S., Kunstelj, M. & Porekar, V. G. PEGylation of therapeutic proteins. Biotechnol. J. 5, 113–128 (2010).

    Google Scholar 

  46. Dirksen, A. & Dawson, P. E. Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjug. Chem. 19, 2543–2548 (2008).

    CAS  Google Scholar 

  47. Blanden, A. R., Mukherjee, K., Dilek, O., Loew, M. & Bane, S. L. 4-aminophenylalanine as a biocompatible nucleophilic catalyst for hydrazone ligations at low temperature and neutral pH. Bioconjug. Chem. 22, 1954–1961 (2011).

    CAS  Google Scholar 

  48. Zustiak, S. P. & Leach, J. B. Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels. Biotechnol. Bioeng. 108, 197–206 (2011).

    CAS  Google Scholar 

  49. Phillies, G. D. Diffusion of bovine serum albumin in a neutral polymer solution. Biopolymers 24, 379–386 (1985).

    CAS  Google Scholar 

  50. Kopan, R. & Ilagan, M. X. G. The canonical Notch signaling pathway: Unfolding the activation mechanism. Cell 137, 216–233 (2009).

    CAS  Google Scholar 

  51. Beckstead, B. L., Santosa, D. M. & Giachelli, C. M. Mimicking cell–cell interactions at the biomaterial-cell interface for control of stem cell differentiation. J. Biomed. Mater. Res. A 79A, 94–103 (2006).

    CAS  Google Scholar 

  52. Varnum-Finney, B. et al. Immobilization of Notch ligand, Delta-1, is required for induction of Notch signaling. J. Cell Sci. 113, 4313–4318 (2000).

    CAS  Google Scholar 

  53. Khetan, S. et al. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nature Mater. 12, 458–465 (2013).

    CAS  Google Scholar 

  54. Hayman, E. G., Pierschbacher, M. D., Ohgren, Y. & Ruoslahti, E. Serum spreading factor (vitronectin) is present at the cell surface and in tissues. Proc. Natl Acad. Sci. USA 80, 4003–4007 (1983).

    CAS  Google Scholar 

  55. Lin, C. C., Metters, A. T. & Anseth, K. S. Functional PEG-peptide hydrogels to modulate local inflammation induced by the pro-inflammatory cytokine TNFα. Biomaterials 30, 4907–4914 (2009).

    CAS  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 9484 kb)

Supplementary Information

Supplementary Movie 1 (WMV 7072 kb)

Supplementary Information

Supplementary Movie 2 (WMV 6900 kb)

Supplementary Information

Supplementary Movie 3 (WMV 4594 kb)

Supplementary Information

Supplementary Movie 4 (WMV 8097 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmat4219

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing