Cardiac ultrastructure inspired matrix induces advanced metabolic and functional maturation of differentiated human cardiomyocytes.

The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30 days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.

A molecular clock controls periodically driven cell migration in confined spaces.

Navigation through a dense, physically confining extracellular matrix is common in invasive cell spread and tissue reorganization but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTPase RhoA, which is dependent on the oscillatory changes in the activity and abundance of the RhoA guanine nucleotide exchange factor, GEF-H1, and triggered by a persistent increase in the intracellular Ca2+ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with a frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments.

Diversity in cancer invasion phenotypes indicates specific stroma regulated programs.

Tumor dissemination into the surrounding stroma is the initial step in a metastatic cascade. Invasion into stroma is a non-autonomous process for cancer, and its progression depends upon the stage of cancer, as well as the cells residing in the stroma. However, a systems framework to understand how stromal fibroblasts resist, collude, or aid cancer invasion has been lacking, limiting our understanding of the role of stromal biology in cancer metastasis. We and others have shown that gene perturbation in stromal fibroblasts can modulate cancer invasion into the stroma, highlighting the active role stroma plays in regulating its own invasion. However, cancer invasion into stroma is a complex higher-order process and consists of various sub-phenotypes that together can result in an invasion. Stromal invasion exhibits a diversity of modalities in vivo. It is not well understood if these diverse modalities are correlated, or they emanate from distinct mechanisms and if stromal biology could regulate these characteristics. These characteristics include the extent of invasion, formation, and persistence of invasive forks by cancer as opposed to a collective frontal invasion, the persistence of invading velocity by leader cells at the tip of invasive forks, etc. We posit that quantifying distinct aspects of collective invasion can provide useful suggestions about the plausible mechanisms regulating these processes, including whether the process is regulated by mechanics or by intercellular communication between stromal cells and cancer. Here, we have identified the sub-characteristics of invasion, which might be indicative of broader mechanisms regulating these processes, developed methods to quantify these metrics, and demonstrated that perturbation of stromal genes can modulate distinct aspects of collective invasion. Our results highlight that the genetic state of stromal fibroblasts can regulate complex phenomena involved in cancer dissemination and suggest that collective cancer invasion into stroma is an outcome of the complex interplay between cancer and stromal fibroblasts.

Regulation of axon growth by myosin II-dependent mechanocatalysis of cofilin activity.

Serotonin (5-HT) is known to increase the rate of growth cone advance via cofilin-dependent increases in retrograde actin network flow and nonmuscle myosin II activity. We report that myosin II activity is regulated by PKC during 5-HT responses and that PKC activity is necessary for increases in traction force normally associated with these growth responses. 5-HT simultaneously induces cofilin-dependent decreases in actin network density and PKC-dependent increases in point contact density. These reciprocal effects facilitate increases in traction force production in domains exhibiting decreased actin network density. Interestingly, when PKC activity was up-regulated, 5-HT treatments resulted in myosin II hyperactivation accompanied by catastrophic cofilin-dependent decreases in actin filament density, sudden decreases in traction force, and neurite retraction. These results reveal a synergistic relationship between cofilin and myosin II that is spatiotemporally regulated in the growth cone via mechanocatalytic effects to modulate neurite growth.

Wound Healing Coordinates Actin Architectures to Regulate Mechanical Work.

How cells with diverse morphologies and cytoskeletal architectures modulate their mechanical behaviors to drive robust collective motion within tissues is poorly understood. During wound repair within epithelial monolayers in vitro, cells coordinate the assembly of branched and bundled actin networks to regulate the total mechanical work produced by collective cell motion. Using traction force microscopy, we show that the balance of actin network architectures optimizes the wound closure rate and the magnitude of the mechanical work. These values are constrained by the effective power exerted by the monolayer, which is conserved and independent of actin architectures. Using a cell-based physical model, we show that the rate at which mechanical work is done by the monolayer is limited by the transformation between actin network architectures and differential regulation of cell-substrate friction. These results and our proposed mechanisms provide a robust physical model for how cells collectively coordinate their non-equilibrium behaviors to dynamically regulate tissue-scale mechanical output.

Cooperation of dual modes of cell motility promotes epithelial stress relaxation to accelerate wound healing.

Collective cell migration in cohesive units is vital for tissue morphogenesis, wound repair, and immune response. While the fundamental driving forces for collective cell motion stem from contractile and protrusive activities of individual cells, it remains unknown how their balance is optimized to maintain tissue cohesiveness and the fluidity for motion. Here we present a cell-based computational model for collective cell migration during wound healing that incorporates mechanochemical coupling of cell motion and adhesion kinetics with stochastic transformation of active motility forces. We show that a balance of protrusive motility and actomyosin contractility is optimized for accelerating the rate of wound repair, which is robust to variations in cell and substrate mechanical properties. This balance underlies rapid collective cell motion during wound healing, resulting from a tradeoff between tension mediated collective cell guidance and active stress relaxation in the tissue.

Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments.

Developmental studies and 3D in vitro model systems show that the production and engagement of extracellular matrix (ECM) often precede stem cell differentiation. Yet, unclear is how the ECM triggers signaling events in sequence to accommodate multistep process characteristic of differentiation. Here, we employ transcriptome profiling and advanced imaging to delineate the specificity of ECM engagement to particular differentiation pathways and to determine whether specificity in this context is a function of long-term ECM remodeling. To this end, human mesenchymal stem cells (hMSCs) were cultured in 3D bioprinted prisms created from ECM proteins and associated controls. We found that exogenous ECM provided in 3D microenvironments at early time points impacts on the composition of microenvironments at later time points and that each evolving 3D microenvironment is uniquely poised to promote stem cell differentiation. Moreover, 2D cultures undergo minimal ECM remodeling and are ill-equipped to stimulate pathways associated with development.

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bidirectional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry.

INTRODUCTION: Migration mis-regulation is a hallmark of cancer, and remains an important problem in cancer biology. We postulate the needs for better in vitro models to understand the details of cell-matrix interactions. Here, we utilized multiphoton excited (MPE) photochemistry to fabricate models to systematically study migration dynamics operative in breast and ovarian cancer. Gradients are a convenient means to modulate concentration and also have been implicated in metastases. METHODS: We specifically pattern sub-micron structured gradients from laminin and fibronectin whose up-regulation is associated with increased metastasis and poor prognosis. We developed a new continuous linear bi-directional gradient design, permitting exploration of the underlying cell-matrix interactions of migration, including speed, directness, and f-actin cytoskeleton alignment as a function of concentration. These new models provide both contact guidance and ECM binding cues, and provide a more relevant environment than possible with existing technologies such as flow chambers or 2D printed surfaces. RESULTS: We found an overall increase in these processes with increasing concentration on both laminin and fibronectin gradients for a series of ovarian and breast cancer lines. Moreover, directness was higher for more metastatic cells, indicating that epithelial or mesenchymal state of the cell type governs the dynamics. However, the specifics of the speed and directedness depend on both the cell type and protein, thus we found that we must consider these processes collectively to obtain a self-consistent picture of the migration. For this purpose, we performed a linear discriminate analysis (LDA) and successfully classified the different cell types on the two protein gradients without molecular biology analysis. CONCLUSIONS: The bi-gradient structures are versatile tools to performing detailed studies of cell migration, specifically haptotxis. We further suggest the can be used in assessing efficacy of drug treatments targeted at specific matrix components.

Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold.

RATIONALE: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. OBJECTIVE: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. METHODS AND RESULTS: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell-derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. CONCLUSIONS: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Ovarian Cancer Cell Adhesion/Migration Dynamics on Micro-Structured Laminin Gradients Fabricated by Multiphoton Excited Photochemistry.

Haptotaxis, i.e., cell migration in response to adhesive gradients, has been previously implicated in cancer metastasis. A better understanding of cell migration dynamics and their regulation could ultimately lead to new drug targets, especially for cancers with poor prognoses, such as ovarian cancer. Haptotaxis has not been well-studied due to the lack of biomimetic, biocompatible models, where, for example, microcontact printing and microfluidics approaches are primarily limited to 2D surfaces and cannot produce the 3D submicron features to which cells respond. Here we used multiphoton excited (MPE) phototochemistry to fabricate nano/microstructured gradients of laminin (LN) as 2.5D models of the ovarian basal lamina to study the haptotaxis dynamics of a series of ovarian cancer cells. Using these models, we found that increased LN concentration increased migration speed and also alignment of the overall cell morphology and their cytoskeleton along the linear axis of the gradients. Both these metrics were enhanced on LN compared to BSA gradients of the same design, demonstrating the importance of both topographic and ECM cues on the adhesion/migration dynamics. Using two different gradient designs, we addressed the question of the roles of local concentration and slope and found that the specific haptotactic response depends on the cell phenotype and not simply the gradient design. Moreover, small changes in concentration strongly affected the migration properties. This work is a necessary step in studying haptotaxis in more complete 3D models of the tumor microenvironment for ovarian and other cancers.

Image-inspired 3D multiphoton excited fabrication of extracellular matrix structures by modulated raster scanning.

Multiphoton excited photochemistry is a powerful 3D fabrication tool that produces sub-micron feature sizes. Here we exploit the freeform nature of the process to create models of the extracellular matrix (ECM) of several tissues, where the design blueprint is derived directly from high resolution optical microscopy images (e.g. fluorescence and Second Harmonic Generation). To achieve this goal, we implemented a new form of instrument control, termed modulated raster scanning, where rapid laser shuttering (10 MHz) is used to directly map the greyscale image data to the resulting protein concentration in the fabricated scaffold. Fidelity in terms of area coverage and relative concentration relative to the image data is ~95%. We compare the results to an STL approach, and find the new scheme provides significantly improved performance. We suggest the method will enable a variety of cell-matrix studies in cancer biology and also provide insight into generating scaffolds for tissue engineering.

Spatial and temporal analysis of extracellular matrix proteins in the developing murine heart: a blueprint for regeneration.

The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus, we identified changes in the spatial and temporal localization of the primary ECM of the developing ventricle. This characterization can serve as a blueprint for fabrication techniques, which we illustrate by using multiphoton excitation photochemistry to create a synthetic scaffold based on COLIV organization at P2. Similarly, fabricated scaffolds generated using ECM components, could be utilized for ventricular repair.

Cell Adhesion on Micro-Structured Fibronectin Gradients Fabricated by Multiphoton Excited Photochemistry.

Concentration gradients of ECM proteins play active roles in many areas of cell biology including wound healing and metastasis. They may also form the basis of tissue engineering scaffolds, as these can direct cell adhesion and migration and promote new matrix synthesis. To better understand cell-matrix interactions on attractive gradients, we have used multiphoton excited (MPE) photochemistry to fabricate covalently linked micro-structured gradients from fibronectin (FN). The gradient design is comprised of a parallel series of individual linear gradients with overall dimensions of approximately 800 × 800 µm, where a linear dynamic range of nearly 10-fold in concentration was achieved. The adhesion dynamics of 3T3 fibroblasts were investigated, where the cell morphology and actin cytoskeleton became increasingly elongated and aligned with the direction of the gradient at increasing protein concentration. Moreover, the cell morphologies are distinct when adhered to regions of differing FN concentration but with similar topography. These results show that the fabrication approach allows investigating the roles of contact guidance and ECM cues on the cell-matrix interactions. We suggest this design overcomes some of the limitations with other fabrication methods, especially in terms of 3D patterning capabilities, and will serve as a new tool to study cell-matrix interactions.

Structural changes in mixed Col I/Col V collagen gels probed by SHG microscopy: implications for probing stromal alterations in human breast cancer.

Second Harmonic Generation (SHG) microscopy has been previously used to describe the morphology of collagen in the extracellular matrix (ECM) in different stages of invasion in breast cancer. Here this concept is extended by using SHG to provide quantitative discrimination of self-assembled collagen gels, consisting of mixtures of type I (Col I) and type V (Col V) isoforms which serve as models of changes in the ECM during invasion in vivo. To investigate if SHG is sensitive to changes due to Col V incorporation into Col I fibrils, gels were prepared with 0-20% Col V with the balance consisting of Col I. Using the metrics of SHG intensity, fiber length, emission directionality, and depth-dependent intensities, we found similar responses for gels comprised of 100% Col I, and 95% Col I/5% Col V, where these metrics were all significantly different from those of the 80% Col I/20% Col V gels. Specifically, the gels of lower Col V content produce brighter SHG, are characterized by longer fibers, and have a higher forward/backward emission ratio. These attributes are all consistent with more highly organized collagen fibrils/fibers and are in agreement with previous TEM characterization as well as predictions based on phase matching considerations. These results suggest that SHG can be developed to discriminate Col I/Col V composition in tissues to characterize and follow breast cancer invasion.