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Successful engineering of functional salivary glands necessitates the creation of cell‐instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels are prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce “hyperGel+” or “hyperGel”, respectively. Differential scanning fluorescence experiments confirm the ability of the sulfated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels are nanoporous, cytocompatible, and cell‐permissive, enabling the development of multicellular hS/PC spheroids in 14 days. The incorporation of sulfated HPG species in the hydrogel enhances cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor Y‐27632 (Y‐27) leads to the development of spheroids with a central lumen, increases the expression of acinar marker aquaporin‐3 at the transcript level (AQP3), and decreases the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF‐β) targets SMAD2/3 is also observed in Y27‐treated cultures, suggesting attenuation of TGF‐β signaling. Thus, hyperGel+ cooperates with the Rho‐associated protein kinase inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.
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Peptide‐modified, hyaluronic acid‐based hydrogels as a 3D culture platform for neural stem/progenitor cell engineering
Abstract Neural stem/progenitor cell (NS/PC)‐based therapies have shown exciting potential for regeneration of the central nervous system (CNS) and NS/PC cultures represent an important resource for disease modeling and drug screening. However, significant challenges limiting clinical translation remain, such as generating large numbers of cells required for model cultures or transplantation, maintaining physiologically representative phenotypesex vivoand directing NS/PC differentiation into specific fates. Here, we report that culture of human NS/PCs in 3D, hyaluronic acid (HA)‐rich biomaterial microenvironments increased differentiation toward oligodendrocytes and neurons over 2D cultures on laminin‐coated glass. Moreover, NS/PCs in 3D culture exhibited a significant reduction in differentiation into reactive astrocytes. Many NS/PC‐derived neurons in 3D, HA‐based hydrogels expressed synaptophysin, indicating synapse formation, and displayed electrophysiological characteristics of immature neurons. While inclusion of integrin‐binding, RGD peptides into hydrogels resulted in a modest increase in numbers of viable NS/PCs, no combination of laminin‐derived, adhesive peptides affected differentiation outcomes. Notably, 3D cultures of differentiating NS/PCs were maintained for at least 70 days in medium with minimal growth factor supplementation. In sum, results demonstrate the use of 3D, HA‐based biomaterials for long‐term expansion and differentiation of NS/PCs toward oligodendroglial and neuronal fates, while inhibiting astroglial fates. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 704–718, 2019.
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- Award ID(s):
- 1653730
- PAR ID:
- 10083646
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Biomedical Materials Research Part A
- Volume:
- 107
- Issue:
- 4
- ISSN:
- 1549-3296
- Format(s):
- Medium: X Size: p. 704-718
- Size(s):
- p. 704-718
- Sponsoring Org:
- National Science Foundation
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