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Skeletal muscle stem cells (MuSCs) are essential for efficacious muscle repair, making MuSCs promising therapeutic targets for tissue engineering and regenerative medicine. MuSCs are presented with a diverse and temporally defined set of cues from their microenvironment during regeneration that direct stem cell expansion, differentiation, and return to quiescence. Understanding the complex interplay among these biophysical and biochemical cues is necessary to develop therapies targeting or employing MuSCs. To probe the role of mechanical cues presented by the extracellular matrix, we leverage chemically defined hydrogel substrates with controllable stiffness and adhesive ligand composition to characterize the MuSC response to matrix cues presented during early and late phases of regeneration. We demonstrate that relatively soft hydrogels recapitulating healthy muscle stiffness promote MuSC activation and expansion, while relatively stiff hydrogels impair MuSC proliferation and arrest myogenic progression. These effects are seen on soft and stiff hydrogels presenting laminin-111 and exacerbated on hydrogels presenting RGD adhesive peptides. Soluble factors present in the MuSC niche during different phases of regeneration, prostaglandin E2 and oncostatin M, synergize with matrix-presented cues to enhance stem cell expansion on soft substrates and block myogenic progression on stiff substrates. To determine if temporally varied matrix stiffness reminiscent of the regenerating microenvironment alters MuSC fate, we developed a photoresponsive hydrogel system with accelerated reaction kinetics that can be rapidly softened on demand. MuSCs cultured on these materials revealed that the cellular response to a stiff microenvironment is fixed within the first three days of culture, as subsequent softening back to a healthy stiffness did not rescue MuSC proliferation or myogenic progression. These results highlight the importance of temporally controlled biophysical and biochemical cues in regulating MuSC fate that can be harnessed to improve regenerative medicine approaches to restore skeletal muscle tissue.

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior.

Stem cells are a powerful resource for many applications including regenerative medicine, patient-specific disease modeling, and toxicology screening. However, eliciting the desired behavior from stem cells, such as expansion in a naïve state or differentiation into a particular mature lineage, remains challenging. Drawing inspiration from the native stem cell niche, hydrogel platforms have been developed to regulate stem cell fate by controlling microenvironmental parameters including matrix mechanics, degradability, cell-adhesive ligand presentation, local microstructure, and cell–cell interactions. We survey techniques for modulating hydrogel properties and review the effects of microenvironmental parameters on maintaining stemness and controlling differentiation for a variety of stem cell types. Looking forward, we envision future hydrogel designs spanning a spectrum of complexity, ranging from simple, fully defined materials for industrial expansion of stem cells to complex, biomimetic systems for organotypic cell culture models.

Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.

The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell–matrix and Schwann cell–neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell–matrix and cell–cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell–matrix interactions by presenting integrin-binding RGD (Arg–Gly–Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell–neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2 mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell–neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell–matrix and cell–cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.