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

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.

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Figure 1: NPC stemness varies as a function of hydrogel degradability.
Figure 2: Maintenance of NPC stemness in hydrogels requires matrix degradation but does not depend on cytoskeletal tension or engineered matrix-adhesion ligand interactions.
Figure 3: Matrix remodelling is required for NPC proliferation and differentiation capacity.
Figure 4: Matrix remodelling regulates NPC stemness by modulating cadherin-mediated cell–cell contact.
Figure 5: Matrix remodelling modulates β-catenin signalling via cadherin contacts to promote NPC stemness.
Figure 6: NPC stemness maintenance varies with remodelling, but not initial stiffness, in both covalently and physically crosslinked hydrogels.

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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.).

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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.

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Correspondence to Sarah C. Heilshorn.

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Madl, C., LeSavage, B., Dewi, R. et al. Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling. Nature Mater 16, 1233–1242 (2017). https://doi.org/10.1038/nmat5020

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