Article

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling

Received:
Accepted:
Published online:

Abstract

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from 0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell–cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

  • Subscribe to Nature Materials for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Stem and progenitor cell–based therapy of the human central nervous system. Nat. Biotechnol. 23, 862–871 (2005).

  2. 2.

    , , & Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell 14, 13–26 (2014).

  3. 3.

    , , , & Preclinical efficacy failure of human CNS-derived stem cells for use in the pathway study of cervical spinal cord injury. Stem Cell Rep. 8, 249–263 (2017).

  4. 4.

    et al. HuCNS-SC human NSCs fail to differentiate, form ectopic clusters, and provide no cognitive benefits in a transgenic model of Alzheimer’s disease. Stem Cell Rep. 8, 235–248 (2017).

  5. 5.

    & Matrix revolutions: a trinity of defined substrates for long-term expansion of human ESCs. Cell Stem Cell 7, 7–8 (2010).

  6. 6.

    , & Natural and synthetic materials for self-renewal, long-term maintenance, and differentiation of induced pluripotent stem cells. Adv. Healthc. Mater. 4, 2342–2359 (2015).

  7. 7.

    , , , & Perturbation of single hematopoietic stem cell fates in artificial niches. Integr. Biol. 1, 59–69 (2009).

  8. 8.

    et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).

  9. 9.

    Scalable culture of human pluripotent stem cells in 3D. Proc. Natl Acad. Sci. USA 110, 20852–20853 (2013).

  10. 10.

    & A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc. Natl Acad. Sci. USA 110, E5039–E5048 (2013).

  11. 11.

    , , & The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials 29, 3946–3952 (2008).

  12. 12.

    et al. Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells. Proc. Natl Acad. Sci. USA 104, 11298–11303 (2007).

  13. 13.

    & Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611 (2008).

  14. 14.

    , & Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues. Annu. Rev. Cell Dev. Biol. 26, 533–556 (2010).

  15. 15.

    , & Growth factors, matrices, and forces combine and control stem cells. Science 324, 1673–1677 (2009).

  16. 16.

    , & Engineering biomaterials for synthetic neural stem cell microenvironments. Chem. Rev. 108, 1787–1796 (2008).

  17. 17.

    , , & Mechanical memory and dosing influence stem cell fate. Nat. Mater. 13, 645–652 (2014).

  18. 18.

    , , , & Dynamic force generation by neural stem cells. Cell. Mol. Bioeng. 2, 464–474 (2009).

  19. 19.

    et al. Mesenchymal stem cells ability to generate traction stress in response to substrate stiffness is modulated by the changing extracellular matrix composition of the heart during development. Biochem. Biophys. Res. Commun. 439, 161–166 (2013).

  20. 20.

    et al. Substrate modulus directs neural stem cell behavior. Biophys. J. 95, 4426–4438 (2008).

  21. 21.

    , , & Rho GTPases mediate the mechanosensitive lineage commitment of neural stem cells. Stem Cells 29, 1886–1897 (2011).

  22. 22.

    et al. The promotion of neuronal maturation on soft substrates. Biomaterials 30, 4567–4572 (2009).

  23. 23.

    & The effect of substrate stiffness on adult neural stem cell behavior. Biomaterials 30, 6867–6878 (2009).

  24. 24.

    et al. The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells. Biomaterials 30, 4695–4699 (2009).

  25. 25.

    & Deconstructing the third dimension—How 3D culture microenvironments alter cellular cues. J. Cell Sci. 125, 3015–3024 (2012).

  26. 26.

    et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15, 326–334 (2016).

  27. 27.

    , , & Poly(ethylene glycol) hydrogel system supports preadipocyte viability, adhesion, and proliferation. Tissue Eng. 11, 1498–1505 (2005).

  28. 28.

    et al. The effect of matrix characteristics on fibroblast proliferation in 3D gels. Biomaterials 31, 8454–8464 (2010).

  29. 29.

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

  30. 30.

    , , , & Biomimetic interfacial interpenetrating polymer networks control neural stem cell behavior. J. Biomed. Mater. Res. A 81A, 240–249 (2007).

  31. 31.

    & Elastin. Adv. Protein Chem. 70, 437–461 (2005).

  32. 32.

    , & Tetrakis(hydroxymethyl) phosphonium chloride as a covalent cross-linking agent for cell encapsulation within protein-based hydrogels. Biomacromolecules 13, 3912–3916 (2012).

  33. 33.

    Stem cells in the central nervous system. Science 276, 66–71 (1997).

  34. 34.

    The culture of neural stem cells. J. Cell. Biochem. 106, 1–6 (2009).

  35. 35.

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

  36. 36.

    & Sliding hydrogels with mobile molecular ligands and crosslinks as 3D stem cell niche. Adv. Mater. 28, 7257–7263 (2016).

  37. 37.

    & Stem cell differentiation: Post-degradation forces kick in. Nat. Mater. 12, 384–386 (2013).

  38. 38.

    & Are in vivo and in situ brain tissues mechanically similar? J. Biomech. 37, 1339–1352 (2004).

  39. 39.

    & Reassessment of brain elasticity for analysis of biomechanisms of hydrocephalus. J. Biomech. 37, 1263–1269 (2004).

  40. 40.

    , , , & The effect of injectable gelatin-hydroxyphenylpropionic acid hydrogel matrices on the proliferation, migration, differentiation and oxidative stress resistance of adult neural stem cells. Biomaterials 33, 3446–3455 (2012).

  41. 41.

    et al. Cortical neural precursors inhibit their own differentiation via N-cadherin maintenance of β-catenin signaling. Dev. Cell 18, 472–479 (2010).

  42. 42.

    et al. E-cadherin regulates neural stem cell self-renewal. J. Neurosci. 29, 3885–3896 (2009).

  43. 43.

    & Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297, 365–369 (2002).

  44. 44.

    & Independent tuning of multiple biomaterial properties using protein engineering. Soft Matter 5, 114–124 (2009).

  45. 45.

    , , , & Hybrid elastin-like polypeptide–polyethylene glycol (ELP-PEG) hydrogels with improved transparency and independent control of matrix mechanics and cell ligand density. Biomacromolecules 15, 3421–3428 (2014).

  46. 46.

    , & Coherent anti-Stokes Raman scattering microscopy of cellular lipid storage. IEEE J. Sel. Top. Quantum Electron. 16, 506–515 (2010).

  47. 47.

    , & Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density. Biomater. Sci. 1, 1082–1090 (2013).

  48. 48.

    , , & A method improving the accuracy of fluorescence recovery after photobleaching analysis. Biophys. J. 95, 5334–5348 (2008).

  49. 49.

    , , , & Enriched monolayer precursor cell cultures from micro-dissected adult mouse dentate gyrus yield functional granule cell-like neurons. PLoS ONE 2, e388 (2007).

  50. 50.

    , & Bio-orthogonally crosslinked, engineered protein hydrogels with tunable mechanics and biochemistry for cell encapsulation. Adv. Funct. Mater. 26, 3612–3620 (2016).

  51. 51.

    & Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc. Natl Acad. Sci. USA 108, 20404–20409 (2011).

  52. 52.

    & A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl Acad. Sci. USA 105, 2415–2420 (2008).

  53. 53.

    , , , & Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids. Biomater. Sci. 3, 1376–1385 (2015).

  54. 54.

    et al. Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research. Cell Rep. 10, 1681–1691 (2015).

  55. 55.

    et al. Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity. J. Biol. Chem. 274, 3531–3540 (1999).

  56. 56.

    , , , & Fluorescent substrates useful as high throughput screening tools for ADAM9. Comb. Chem. High Throughput Screen. 13, 358–365 (2010).

  57. 57.

    , , , & Cell-responsive synthetic hydrogels. Adv. Mater. 15, 888–892 (2003).

  58. 58.

    , & Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20, 45–53 (1999).

  59. 59.

    , , , & Versatile click alginate hydrogels crosslinked via tetrazine-norbornene chemistry. Biomaterials 50, 30–37 (2015).

  60. 60.

    , & Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth. Acta Biomater. 11, 48–57 (2015).

Download references

Acknowledgements

The authors thank T. Palmer and H. Babu (Stanford Neurosurgery) for providing the murine NPCs, A. Proctor (Stanford Chemical Engineering) for assistance with ELP expression and purification, K. Dubbin (Stanford Materials Science & Engineering) for assistance with FRAP, and C. Kuo (Stanford Medicine) for providing the TOP-FLASH plasmid. Sorting of the lentivirally transduced NPCs for the ADAM9-knockdown experiments was performed with the assistance of C. Crumpton and B. Gomez on an instrument in the Stanford Shared FACS Facility obtained using NIH S10 shared instrument grant S10RR025518-01. C.M.M. acknowledges support from an NIH NRSA pre-doctoral fellowship (F31 EB020502) and the Siebel Scholars Program. This work was supported by funding from the National Institutes of Health (S.C.H.: U19 AI116484 and R21 EB018407), National Science Foundation (S.C.H.: DMR 1508006), California Institute for Regenerative Medicine (S.C.H.: RT3-07948), and Trygger Foundation (A.E.).

Author information

Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, California 94305, USA

    • Christopher M. Madl
    • , Bauer L. LeSavage
    •  & Margarita Khariton
  2. Department of Materials Science & Engineering, Stanford University, Stanford, California 94305, USA

    • Ruby E. Dewi
    • , Cong B. Dinh
    • , Kyle J. Lampe
    • , Annika Enejder
    •  & Sarah C. Heilshorn
  3. Department of Mechanical Engineering, Stanford University, Stanford, California 94305, USA

    • Ryan S. Stowers
    •  & Ovijit Chaudhuri
  4. Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, USA

    • Kyle J. Lampe
  5. Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE-412 96, Sweden

    • Duong Nguyen
    •  & Annika Enejder

Authors

  1. Search for Christopher M. Madl in:

  2. Search for Bauer L. LeSavage in:

  3. Search for Ruby E. Dewi in:

  4. Search for Cong B. Dinh in:

  5. Search for Ryan S. Stowers in:

  6. Search for Margarita Khariton in:

  7. Search for Kyle J. Lampe in:

  8. Search for Duong Nguyen in:

  9. Search for Ovijit Chaudhuri in:

  10. Search for Annika Enejder in:

  11. Search for Sarah C. Heilshorn in:

Contributions

Experiments were designed by C.M.M. and S.C.H., and carried out by C.M.M., B.L.L., R.E.D., C.B.D., R.S.S., M.K., K.J.L. and D.N. CARS experiments were performed with D.N. and A.E. Alginate hydrogel experiments were performed with R.S.S. and O.C. The manuscript was written by C.M.M. and S.C.H. The principal investigator is S.C.H.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sarah C. Heilshorn.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information