Visar Ajeti

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.

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.

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.

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bi-directional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry

Migration mis-regulation is a hallmark of cancer, and remains an important problem in cancer biology. We postulate the need 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. 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. 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. 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.

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.

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.

Abstract TMEM-015: QUANTITATIVE ASSESSMENT OF THE ROLE OF COLLAGEN ALTERATIONS IN OVARIAN CANCER

A profound remodeling of the extracellular matrix (ECM) occurs in human ovarian cancer but it unknown how this affects tumor growth, where this understanding could lead to better diagnostics and therapeutic approaches. To this end, we utilized collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe structural differences in the extracellular matrix of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous (LGS and HGS) tumors. The SHG and scattering metrics are sensitive to the organization of collagen on the sub-micron size. We found these sub-resolution determinations are consistent with the dualistic classification of type I and II serous tumors. However, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. Moreover, our analyses are further consistent with LGS and benign tumors having similar etiology. Further, the SHG metrics and optical scattering measurements were then used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (~90%) between the tissue types, and this delineation is superior to current clinical performance. To also quantify these alterations we implemented a new form of 3D texture analysis to classify the collagen morphologies in these tissues. We developed a tailored set of 3D filters which extract textural features in each tissue class and we achieved 83-91% accuracies for the six classes. This classification based on ECM structural changes will complement conventional classification based on genetic profiles and can serve as an additional biomarker. We further investigate the role of these ECM alterations by using multiphoton excited (MPE) polymerization to fabricate biomimetic models to investigate operative cell-matrix interactions in invasion/metastasis. This process is akin to 3D printing except is performed at much higher resolution and with the proteins that comprise the native ECM. We specifically use this technique to create collagen scaffolds with complex, 3D submicron morphology as ovarian stromal models. The scaffold designs are derived directly from “blueprints” based on the SHG images of normal, high risk, benign tumors, and malignant ovarian tissues. The models are seeded with different cancer cell lines and this allows decoupling of the roles of cell characteristics (metastatic potential) and ECM structure and composition (normal vs cancer) on adhesion/migration dynamics. We found the malignant stroma structure promoted enhanced migration persistence and cell proliferation and also cytoskeletal alignment. Moreover, the method allows varying fiber properties such as fiber diameters and characteristic frequency as well as overall alignment. While alignment has been well studied, we found that the migration dynamics are highly dependent upon the morphological properties of the fibers themselves. These models cannot be synthesized by other conventional fabrication methods and we suggest the MPE image-based fabrication method will enable a variety of studies in cancer biology. This work is currently by a new grant from NCI R01 CA206561-01. Citation Format: Visar Ajeti, Manish Patankar, Kevin Eliceiri, and Paul J. Campagnola. QUANTITATIVE ASSESSMENT OF THE ROLE OF COLLAGEN ALTERATIONS IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-015.

Modeling extracellular matrix (ECM) alterations in ovarian cancer by multiphoton excited fabrication of stromal models(Conference Presentation)

A profound remodeling of the extracellular matrix (ECM) occurs in human ovarian cancer but it unknown how this affects tumor growth, where this understanding could lead to better diagnostics and therapeutic approaches. We investigate the role of these ECM alterations by using multiphoton excited (MPE) polymerization to fabricate biomimetic models to investigate operative cell-matrix interactions in invasion/metastasis. First, we create nano/microstructured gradients mimicking the basal lamina to study adhesion/migration dynamics of ovarian cancer cells of differing metastatic potential. We find a strong haptotactic response that depends on both contact guidance and ECM binding cues. While we found enhanced migration for more invasive cells, the specifics of alignment and directed migration also depend on cell polarity. We further use MPE fabrication to create collagen scaffolds with complex, 3D submicron morphology. The stromal scaffold designs are derived directly from “blueprints” based on SHG images of normal, high risk, and malignant ovarian tissues. The models are seeded with different cancer cell lines and this allows decoupling of the roles of cell characteristics (metastatic potential) and ECM structure and composition (normal vs cancer) on adhesion/migration dynamics. We found the malignant stroma structure promotes enhanced migration and proliferation and also cytoskeletal alignment. Creating synthetic models based on fibers patterns further allows decoupling the topographic roles of the fibers themselves vs their alignment within the tissue. These models cannot be synthesized by other conventional fabrication methods and we suggest the MPE image-based fabrication method will enable a variety of studies in cancer biology.