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The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein-based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self-assembly or crosslinking, engineered protein-based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain-length, folded structure, and cell-interaction sites. In this review, the modular design of engineered protein-based hydrogels from both a molecular- and network-level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli-responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein-based hydrogels as biomimetic cellular scaffolds is concluded., (© 2024 Wiley‐VCH GmbH.)

Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self-healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC-crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, hydrogels comprised of two recombinant biopolymers: 1) a hyaluronic acid (HA) modified with benzaldehyde and 2) an elastin-like protein (ELP) modified with hydrazine (ELP-HYD), are formulated. Several hydrogel families are synthesized with distinct HA MW and DoF while keeping the ELP-HYD component constant. The resulting hydrogels have a range of stiffnesses, G' ≈ 10-1000 Pa, and extrudability, which is attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations require lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibit more rapid self-healing. Gel extrusion through a cannula (2 m length, 0.25 mm diameter) demonstrates the potential for minimally invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC-crosslinked hydrogels and aims to guide future design of injectable hydrogels., (© 2023 Wiley-VCH GmbH.)

Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli through cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), thereby reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report the insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of Sortase A, a mammalian-inert enzyme. Notably, Sortase A exposure preserves stem cell surface markers, which are an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to produce artificial stem cell niches with tunable biophysical properties, intrinsic cell-interaction motifs, and orthogonal addition of bioactive crosslinks. STATEMENT OF SIGNIFICANCE: We describe a maleimide-functionalized gelatin hydrogel that can be crosslinked via a thiol-maleimide mediated click reaction to form a stable hydrogel without the production of reactive oxygen species typical in light-based crosslinking. The mechanical properties can be tuned to match the in vivo bone marrow microenvironment for hematopoietic stem cell culture. Additionally, we report inclusion of a peptide crosslinker that can be cleaved via the proteolytic action of Sortase A and show that Sortase A exposure does not degrade sensitive surface marker expression patterns. Together, this approach reduces stem cell exposure to reactive oxygen species during hydrogel gelation and enables post-culture quantitative assessment of stem cell phenotype., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Biomaterials that replicate patterns of microenvironmental signals from the stem cell niche offer the potential to refine platforms to regulate stem cell behavior. While significant emphasis has been placed on understanding the effects of biophysical and biochemical cues on stem cell fate, vascular‐derived or angiocrine cues offer an important alternative signaling axis for biomaterial‐based stem cell platforms. Elucidating dose‐dependent relationships between angiocrine cues and stem cell fate are largely intractable in animal models and 2D cell cultures. In this study, microfluidic mixing devices are leveraged to generate 3D hydrogels containing lateral gradients in vascular density alongside murine hematopoietic stem cells (HSCs). Regional differences in vascular density can be generated via embossed gradients in cell, matrix, or growth factor density. HSCs co‐cultured alongside vascular gradients reveal spatial patterns of HSC phenotype in response to angiocrine signals. Notably, decreased Akt signaling in high vessel density regions led to increased expansion of lineage‐positive hematopoietic cells. This approach offers a combinatorial tool to rapidly screen a continuum of microenvironments with varying vascular, biophysical, and biochemical cues to reveal the influence of local angiocrine signals on HSC fate. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct coculture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial least squares regression (PLSR), along with an iterative filter method, identified TGFβ-1, MMP-3, c-RP and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52 ± 3.65-fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplifies hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route toward identifying feeder-free culture platforms for HSC expansion. Insight Hematopoietic stem cells within an artificial niche receive maintenance cues in the form of soluble factors from hematopoietic and mesenchymal progeny. Applying a proteomic regression analysis, we identify a reduced set of soluble factors correlated to maintenance of a hematopoietic phenotype during culture in a biomaterial model of the bone marrow niche. We identify a minimum factor cocktail that promotes hematopoietic maintenance potential in a gelatin-based culture, regardless of the presence of mesenchymal feeder cells. By combining empirical and computational methods, we report an experimentally feasible number of factors from a large dataset, enabling exogenous integration of soluble factors into an engineered hematopoietic stem cell for enhanced maintenance potential of a quiescent stem cell population. [ABSTRACT FROM AUTHOR]