Protein delivery is often used in tissue engineering applications to control differentiation processes, but is limited by protein instability and cost. An alternative approach is to control the cellular microenvironment through biomaterial-mediated sequestration of cell-secreted proteins important to differentiation. Thus, we utilized heparin-based microparticles to modulate cellular differentiation via protein sequestration in an in vitro model system of endochondral ossification. Heparin and poly(ethylene-glycol) (PEG; a low-binding material control)-based microparticles were incorporated into ATDC5 cell spheroids or incubated with ATDC5 cells in transwell culture. Reduced differentiation was observed in the heparin microparticle group as compared to PEG and no microparticle-containing groups. To determine if observed changes were due to sequestration of cell-secreted protein, the proteins sequestered by heparin microparticles were analyzed using SDS-PAGE and mass spectrometry. It was found that heparin microparticles bound insulin-like growth factor binding proteins (IGFBP)-3 and 5. When incubated with a small-molecule inhibitor of IGFBPs, NBI 31772, a similar delay in differentiation of ATDC5 cells was observed. These results indicate that heparin microparticles modulated chondrocytic differentiation in this system via sequestration of cell-secreted protein, a technique that could be beneficial in the future as a means to control cellular differentiation processes. Statement of Significance: In this work, we present a proof-of-principle set of experiments in which heparin-based microparticles are shown to modulate cellular differentiation through binding of cell-secreted protein. Unlike existing systems that rely on expensive protein with limited half-lives to elicit changes in cellular behavior, this technique focuses on temporal modulation of cell-generated proteins. This technique also provides a biomaterials-based method that can be used to further identify sequestered proteins of interest. Thus, this work indicates that glycosaminoglycan-based biomaterial approaches could be used as substitutes or additions to traditional methods for modulating and identifying the cell-secreted proteins involved in directing cellular behavior.
The initiation of heterogeneity within a population of phenotypically identical progenitors is a critical event for the onset of morphogenesis and differentiation patterning. Gap junction communication within multicellular systems produces complex networks of intercellular connectivity that result in heterogeneous distributions of intracellular signaling molecules. In this study, we investigate emergent systems-level behavior of the intercellular network within embryonic stem cell (ESC) populations and corresponding spatial organization during early neural differentiation. An agent-based model incorporates experimentally-determined parameters to yield complex transport networks for delivery of pro-differentiation cues between neighboring cells, reproducing the morphogenic trajectories during retinoic acid-accelerated mouse ESC differentiation. Furthermore, the model correctly predicts the delayed differentiation and preserved spatial features of the morphogenic trajectory that occurs in response to intercellular perturbation. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells.
Interrogating fundamental cell biology principles that govern tissue morphogenesis is critical to better understanding of developmental biology and engineering novel multicellular systems. Recently, functional micro-tissues derived from pluripotent embryonic stem cell (ESC) aggregates have provided novel platforms for experimental investigation; however elucidating the factors directing emergent spatial phenotypic patterns remains a significant challenge. Computational modelling techniques offer a unique complementary approach to probe mechanisms regulating morphogenic processes and provide a wealth of spatio-temporal data, but quantitative analysis of simulations and comparison to experimental data is extremely difficult. Quantitative descriptions of spatial phenomena across multiple systems and scales would enable unprecedented comparisons of computational simulations with experimental systems, thereby leveraging the inherent power of computational methods to interrogate the mechanisms governing emergent properties of multicellular biology. To address these challenges, we developed a portable pattern recognition pipeline consisting of: the conversion of cellular images into networks, extraction of novel features via network analysis, and generation of morphogenic trajectories. This novel methodology enabled the quantitative description of morphogenic pattern trajectories that could be compared across diverse systems: computational modelling of multicellular structures, differentiation of stem cell aggregates, and gastrulation of cichlid fish. Moreover, this method identified novel spatio-temporal features associated with different stages of embryo gastrulation, and elucidated a complex paracrine mechanism capable of explaining spatiotemporal pattern kinetic differences in ESC aggregates of different sizes.
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Kristin M. French;
Joshua Maxwell;
Srishti Bhutani;
Shohini Ghosh-Choudhary;
Marcos J. Fierro;
Todd D. Johnson;
Karen L. Christman;
W Taylor;
Michael Davis
Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.
The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra = 0.2 μm), sand-blasted and acid-etched SLA (Ra = 3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.
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Donald R Griffin;
Jacob Borrajo;
Allyson Soon;
Giovanny F. Acosta-Velez;
Victor Oshita;
Nicole Darling;
Julia Mack;
Thomas Barker;
M. Luisa Iruela-Arispe;
Tatiana Segura
The ability to design artificial extracellular matrices as cell-instructive scaffolds has opened the door to technologies capable of studying the fate of cells in vitro and to guiding tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of biochemical cues to guide cell phenotype and multicellular organization. The extracellular matrix (ECM) is composed of a heterogeneous mixture of proteins that present a variety of spatially discrete signals to residing cell populations. In contrast, most engineered ECMs do not mimic this heterogeneity. In recent years, photo-deprotection has been used to spatially immobilize signals. However, this approach has been limited mostly to small peptides. Here we combine photo-deprotection with enzymatic reaction to achieve spatially controlled immobilization of active bioactive signals that range from small molecules to large proteins. A peptide substrate for transglutaminase factor XIII (FXIIIa) was caged with a photo-deprotectable group, which was then immobilized to the bulk of a cell-compatible hydrogel. With focused light, the substrate can be deprotected and used to immobilize patterned bioactive signals. This approach offers an innovative strategy to immobilize delicate bioactive signals, such as growth factors, without loss of activity and enables in situ cell manipulation of encapsulated cells. Signal patterns: A peptide substrate has been caged with a photo-deprotectable group, and then immobilized to the bulk of a cell-compatible hydrogel. With focused light, the substrate can be deprotected and used to immobilize patterned bioactive signals without loss of activity and thereby enable in situ cell manipulation of encapsulated cells.