Hydrogel physical properties are tuned by altering synthesis conditions such as initial polymer concentration and polymer–cross-linker stoichiometric ratios. Traditionally, differences in hydrogel synthesis schemes, such as end-linked poly(ethylene glycol) diacrylate hydrogels and cross-linked poly(vinyl alcohol) hydrogels, limit structural comparison between hydrogels. In this study, we use generalized synthesis variables for hydrogels that emphasize how changes in formulation affect the resulting network structure. We identify two independent linear correlations between these synthesis variables and swelling behavior. Analysis through recently updated swollen polymer network models suggests that synthesis-swelling correlations can be used to make a priori predictions of the stiffness and solute diffusivity characteristics of synthetic hydrogels. The same experiments and analyses performed on methacrylamide-modified gelatin hydrogels demonstrate that complex biopolymer structures disrupt the linear synthesis-swelling correlations. These studies provide insight into the control of hydrogel physical properties through structural design and can be used to implement and optimize biomedically relevant hydrogels.

Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues, bound and diffusible biomolecules, and heterotypic cell-cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platforms that promote ex vivo HSC expansion without exhaustion. A library of methacrylamide-functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from murine bone marrow MSCs on the expansion and quiescence of primary murine HSCs. The use of a degradable GelMA hydrogel enables MSC-mediated remodeling, yielding dynamic shifts in the matrix environment over time. An initially low-diffusivity hydrogel combined with a 1:1 HSPC:MSC co-culture was observed to facilitate maintenance of a hematopoietic stem and progenitor cell population over 7-days. Excitingly, this platform promoted retention of a quiescent HSC population compared to HSC monocultures. Our studies reveal MSC-density This article is protected by copyright. All rights reserved. dependent upregulation of MMP-9 and changes in hydrogel mechanical properties (ΔE = 2.61±0.72) suggesting MSC-mediated matrix remodeling may contribute to a dynamic culture environment. Together, we report a three-dimensional hydrogel for ex vivo HSC culture and reveal HSC expansion and quiescence is sensitive to hydrogel properties, MSC co-culture, and MSC-mediated hydrogel remodeling.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix (ECM); however, the role that altered cell-ECM signaling may play in driving PDAC phenotype has historically been difficult to dissect. Here, we design an engineered matrix that recapitulates key hallmarks of the tumor ECM and show that patient-derived PDAC organoids develop gemcitabine chemoresistance when cultured within high stiffness matrices mechanically matched to in vivo tumors. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, stiffness-induced chemoresistance occurs due to the development of a plastic cancer stem cell phenotype – mediated by hyaluronan mechanosignaling – with increased expression of drug efflux transporters. Moreover, PDAC chemoresistance is reversible following transfer from high to low stiffness matrices, suggesting that mechanotherapeutics targeting the fibrotic ECM may sensitize chemoresistant tumors. Overall, we demonstrate the power of engineered matrices and patient-derived organoids to elucidate how ECM properties influence human disease pathophysiology.

No abstract

We identify a combination of an initially low-diffusivity (autocrine dominated) hydrogel and a 1:1 HSCP:MSC seeding ratio as conducive to enhanced HSC population maintenance. Further, gene expression and serial mechanical testing data suggests that MSC-mediated matrix remodeling is significant for the long-term HSC culture, reducing HSC autocrine feedback and potentially enhancing MSC-mediated paracrine signaling over 7-day culture in vitro. This work demonstrates the design of an HSC culture system that couples initial hydrogel properties, MSC co-culture, and concepts of dynamic reciprocity mediated by MSC remodeling to achieve enhanced HSC maintenance.

Hematopoietic stem cells are the progenitors of the blood and immune system and represent the most widely used regenerative therapy. However, their rarity and limited donor base necessitate the design of ex vivo systems that support HSC expansion without the loss of long-term stem cell activity. This review describes recent advances in biomaterials systems to replicate features of the hematopoietic niche. Inspired by the native bone marrow, these instructive biomaterials provide stimuli and cues from cocultured niche-associated cells to support HSC encapsulation and expansion. Engineered systems increasingly enable study of the dynamic nature of the matrix and biomolecular environment as well as the role of cell-cell signaling (e.g., autocrine feedback vs paracrine signaling between dissimilar cells). The inherent coupling of material properties, biotransport of cell-secreted factors, and cell-mediated remodeling motivate dynamic biomaterial systems as well as characterization and modeling tools capable of evaluating a temporally evolving tissue microenvironment. Recent advances in HSC identification and tracking, model-based experimental design, and single-cell culture platforms facilitate the study of the effect of constellations of matrix, cell, and soluble factor signals on HSC fate. While inspired by the HSC niche, these tools are amenable to the broader stem cell engineering community.

Abstract 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.

Biomaterial microarrays are being developed to facilitate identifying the extrinsic cues that elicit stem cell fate decisions to self-renew, differentiate and remain quiescent. Raman microspectroscopy, often combined with multivariate analysis...