Single cell decisions in endothelial population in the context of inflammatory angiogenesis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 161-171).Normalizing angiogenesis is a promising strategy for treatments of cancer and several disorders plagued by misregulated blood supplies. To address the daunting complexity of angiogenesis arising from multiple phenotypic behaviors governed by multiple stimuli, computational approaches have been developed to predict sprouting angiogenic outcomes. In recent years, the agent based model, in which individual cells are modeled as autonomous decision making entities, has become an important tool for simulating complex phenomena including angiogenesis. The reliability of these models depends on model validation by quantitative experimental characterization of the cellular (agent) behaviors which so far has been lacking. To this end, I develop an experimental and computational method to semi-automatically estimate parameters describing the single-cell decision in the agent based model based on flow cytometry aggregate headcount data and single cell microscopy which yields full panel single cell trajectories of individual endothelial cells. Applying thees method to the single cell decision data, I propose two conceptual models to account for the different state transition patterns and how they are modulated in the presence of opposing inflammatory cytokines. The observed unique state transition patterns in the angiogenic endothelial cell population are consistent with one of these descriptions, the diverse population model (DPM). The DPM interpretation offers an alternative view from the traditional paradigm of cell population heterogeneity. This understanding is important in designing appropriate therapeutic agents that take effect at the cellular level to meet a tissue level therapeutic goal.by Tharathorn Rimchala.Ph.D

Endothelial cell phenotypic behaviors cluster into dynamic state transition programs modulated by angiogenic and angiostatic cytokines

Angiogenesis requires coordinated dynamic regulation of multiple phenotypic behaviors of endothelial cells in response to environmental cues. Multi-scale computational models of angiogenesis can be useful for analyzing effects of cell behaviors on the tissue level outcome, but these models require more intensive experimental studies dedicated to determining the required quantitative “rules” for cell-level phenotypic responses across a landscape of pro- and anti-angiogenic stimuli in order to ascertain how changes in these single cell responses lead to emerging multi-cellular behavior such as sprout formation. Here we employ single-cell microscopy to ascertain phenotypic behaviors of more than 800 human microvascular endothelial cells under various combinational angiogenic (VEGF) and angiostatic (PF4) cytokine treatments, analyzing their dynamic behavioral transitions among sessile, migratory, proliferative, and apoptotic states. We find that an endothelial cell population clusters into an identifiable set of a few distinct phenotypic state transition patterns (clusters) that is consistent across all cytokine conditions. Varying the cytokine conditions, such as VEGF and PF4 combinations here, modulates the proportion of the population following a particular pattern (referred to as phenotypic cluster weights) without altering the transition dynamics within the patterns. We then map the phenotypic cluster weights to quantified population level sprout densities using a multi-variate regression approach, and identify linear combinations of the phenotypic cluster weights that associate with greater or lesser sprout density across the various treatment conditions. VEGF-dominant cytokine combinations yielding high sprout densities are characterized by high proliferative and low apoptotic cluster weights, whereas PF4-dominant conditions yielding low sprout densities are characterized by low proliferative and high apoptotic cluster weights. Migratory cluster weights show only mild association with sprout density outcomes under the VEGF/PF4 conditions and the sprout formation characteristics explored here.National Science Foundation (U.S.) (NSF grant EFRI-0735007)National Institutes of Health (U.S.) (NIH grant R01-GM081336)National Institutes of Health (U.S.) (NIH grant R01-EB010246

Engineering of In Vitro 3D Capillary Beds by Self-Directed Angiogenic Sprouting

In recent years, microfluidic systems have been used to study fundamental aspects of angiogenesis through the patterning of single-layered, linear or geometric vascular channels. In vivo, however, capillaries exist in complex, three-dimensional (3D) networks, and angiogenic sprouting occurs with a degree of unpredictability in all x,y,z planes. The ability to generate capillary beds in vitro that can support thick, biological tissues remains a key challenge to the regeneration of vital organs. Here, we report the engineering of 3D capillary beds in an in vitro microfluidic platform that is comprised of a biocompatible collagen I gel supported by a mechanical framework of alginate beads. The engineered vessels have patent lumens, form robust ~1.5 mm capillary networks across the devices, and support the perfusion of 1 µm fluorescent beads through them. In addition, the alginate beads offer a modular method to encapsulate and co-culture cells that either promote angiogenesis or require perfusion for cell viability in engineered tissue constructs. This laboratory-constructed vascular supply may be clinically significant for the engineering of capillary beds and higher order biological tissues in a scalable and modular manner.Singapore-MIT Alliance for Research and Technolog

A Microtiter Assay for Quantifying Protein-Protein Interactions Associated with Cell-Cell Adhesion

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein β-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of β-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z′ factor of 0.74. The quantitative nature of the E-cadherin:β-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:β-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and β-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: β-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression

A platform for rapid prototyping of synthetic gene networks in mammalian cells.

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines

A platform for rapid prototyping of synthetic gene networks in mammalian cells

International audienceMammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines

Confocal microscopy reconstruction of the 3D capillary bed.

<p>(<b>a</b>) Reconstructed <i>z</i>-stack at 40× magnification on Day 7 of hMVEC seeding. (<b>b</b>) Cross-sectional profile of the <i>z</i>-stack shows lumens on both the <i>x</i>,<i>y</i>-axis planes (VE-cadherin, green; actin, red; cell nuclei, blue). Dotted lines indicate where the PDMS micropillars are located. Scale bars, 50 µm (<b>a, b</b>).</p

Alginate-collagen hydrogels as a blueprint for 3D capillary bed design.

<p>(<b>a</b>) Phase contrast image of the alginate-collagen gel region. The alginate beads form 40% of the gel volume. (<b>b</b>) Fluorescent image of the alginate-collagen gel region using PLL–FITC coated alginate beads. Scale bars, 250 µm (<b>a, b</b>).</p

Anastamoses within the 3D capillary bed.

<p>(<b>a</b>) Fluorescent images of hMVECs undergoing angiogenic sprouting at Day 1–6 after seeding. Arrows indicate the points of contact where vessels anastamose and form lumens. (<b>b</b>) Phase contrast images of 3D microvascular networks in alginate-collagen gels at 4<b>×</b> magnification and (<b>c</b>) 20<b>×</b> magnification. (<b>d</b>) High resolution confocal images taken at Day 6 show specific sites of anastomoses, and dotted boxes correspond to images at (<b>e</b>) higher magnification (VE-cadherin, green; actin, red; cell nuclei, blue). Scale bars, 250 µm (<b>a–d</b>), 50 µm (<b>e</b>).</p

Microfluidic device design and fabrication.

<p>(<b>a</b>) The microfluidic-based cell culture platform directs vascular growth along an alginate bead scaffold to form a 3D capillary bed. The optically transparent PDMS is bonded to a glass coverslip, and the gel region is flanked by two microfluidic channels with inlet and outlet ports for medium renewal. (<b>b</b>) Dimensions of the microfluidic device in µm. The gel region (1,750 µm wide, 1,250 µm long, 240 µm high) is surrounded by six square micropillars (250×250×240 µm) for gel containment. (<b>c</b>) Microfluidic devices cultured under static and flow conditions. (<b>d</b>) Schematic of 10 mm pressure drop set-up for flow analysis studies.</p