Modulation of cellular polarization and migration by ephrin/Eph signal-mediated boundary formation.

Compartment boundaries are essential for ensuring proper cell organization during embryo development and in adult tissues, yet the mechanisms underlying boundary establishment are not completely understood. A number of mechanisms, including (i) differential adhesion, (ii) differential tension, and (iii) cell signaling-mediated cell repulsion, are known to contribute and likely a context-dependent balance of each of these dictates boundary implementation. The ephrin/Eph signaling pathway is known to impact boundary formation in higher animals. In different contexts, ephrin/Eph signaling is known to modulate adhesive properties and migratory behavior of cells. Furthermore it has been proposed that ephrin/Eph signaling may modulate cellular tensile properties, leading to boundary implementation. It remains unclear however, whether, in different contexts, ephrin/Eph act through distinct dominant action modes (e.g. differential adhesion vs. cell repulsion), or whether ephrin/Eph signaling elicits multiple cellular changes simultaneously. Here, using micropatterning of cells over-expressing either EphB3 or ephrinB1, we assess the contribution of each these factors in one model. We show that in this system ephrinB1/EphB3-mediated boundaries are accompanied by modulation of tissue-level architecture and polarization of cell migration. These changes are associated with changes in cell shape and cytoskeletal organization also suggestive of altered cellular tension.

Tissue Patterning: Translating Design Principles from In Vivo to In Vitro.

Recapitulating the architecture of native tissue remains a significant challenge, impeding the progress of engineering tissues. Imposing appropriate organization is especially challenging in tissues that contain multiple cellular components in complex structural units. One solution is to mimic developmental processes in embryos. In an embryo, cells are organized by tissue patterning, whereby induction of fate-determining genes is spatially controlled to generate patterns of cell differentiation and maturation. Following patterning, the imposed cell organization is further reinforced by implementation of compartment boundaries, which prevent intermingling of cells from distinct phenotypic domains, thereby ensuring preservation of proper cell organization in growing and reorganizing tissues. Both morphogenic processes utilize a conserved set of fundamental principles, the implementation of which leads to highly regulated cell organization. In this article, we review these patterning principles in vivo and reflect on the progress made by tissue engineers in mimicking tissue patterning ex vivo.

An in vitro model of tissue boundary formation for dissecting the contribution of different boundary forming mechanisms.

During development and in adult tissues separation of phenotypically distinct cell populations is necessary to ensure proper organization and function of tissues and organs. Various phenomena, such as differential adhesion, differential mechanical tension and cell-cell repulsion, are proposed to cause boundary formation. Moreover, emerging evidence suggests that interplay between multiple such phenomena can underlie boundary formation. Boundary-forming mechanisms are commonly studied in vivo in complex embryo models or in vitro using simple model systems not reflective of in vivo boundary complexity. To better elucidate the interplay between multiple boundary formation mechanism, there is therefore a need for more relevant in vitro model systems that allow quantitative and concomitant studies of the multiple changes in cell/tissue behaviour that lead to boundary establishment. Here, we develop such a model using patterned co-cultures of two cell populations. Using a set of quantitative tools, we demonstrate that our approach allows us to study the mechanisms underlying boundary formation. We demonstrate that in our specific system differential mechanical tension and modulation of migratory behavior of cells accompany boundary formation. The design of our in vitro model system will allow researchers to obtain quantitative, integrative mechanistic data facilitating a faster and more thorough understanding of the fundamental principles underlying boundary formation.

A microgroove patterned multiwell cell culture plate for high-throughput studies of cell alignment.

Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1 µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200 µm. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues.

Micropatterning cells on permeable membrane filters.

Epithelium is abundantly present in the human body as it lines most major organs. Therefore, ensuring the proper function of epithelium is pivotal for successfully engineering whole organ replacements. An important characteristic of mature epithelium is apical-basal polarization which can be obtained using the air-liquid interface (ALI) culture system. Micropatterning is a widely used bioengineering strategy to spatially control the location and organization of cells on tissue culture substrates. Micropatterning is therefore an interesting method for generating patterned epithelium. Enabling micropatterning of epithelial cells however requires micropatterning methods that are designed to (i) be compatible with permeable membranes substrates and (ii) allow prolonged culture of patterned cells, both of which are required for appropriate epithelial apical-basal polarization. Here, we describe a number of methods we have developed for generating monoculture as well as coculture of epithelial cells that are compatible with ALI culture.

A simple and rapid method for generating patterned co-cultures with stable interfaces.

In native tissues, different cell types are organized into defined structures and architectures that are critical for correct tissue function. In vitro cellular patterning methods enable control over the spatial organization of cells, permitting, to some extent, the reproduction of native tissue structures and the generation of a more "in vivo-like" culture platform. While this is advantageous for applications such as drug screening, existing patterning methods are time-consuming, labor-intensive, and low-throughput. Here, we describe a novel medium-throughput patterning strategy for generating spatially controlled co-cultures of two cell types based on differential deposition of BSA solution in a tilted plate. Our method allows generation of homotypic and heterotypic co-cultures that are stable for at least seven days in culture. The reproducibility and consistency of this patterning technique, together with its low cost and ease of use, make it a promising cell culture platform for medium- to high-throughput screening using high-content imaging.

Design principles for generating robust gene expression patterns in dynamic engineered tissues.

Recapitulating native tissue organization is a central challenge in regenerative medicine as it is critical for generating functional tissues. One strategy to generate engineered tissues with predictable and appropriate organization is to mimic the gene expression patterning process that organizes tissues in the developing embryo. In a developing embryo, correct organization is accomplished by tissue patterning via the generation of temporal and spatial patterns of gene expression coupled with, and leading to, extensive cellular re-organization. Methods to pattern gene expression in vitro could therefore provide both better models for understanding the cellular and molecular events taking place during tissue morphogenesis and novel strategies for engineering tissues with more realistic and complex architectures. While a few attempts have been made to genetically pattern tissues in vitro, these do not produce sharp predictable patterning. In both the embryo and an in vitro tissue, patterning often occurs during extensive cell re-organization but how the dynamics of gene induction and cell re-distribution interact to impact the final outcome of patterning and ultimately tissue organization is not known. Understanding this relationship and the system parameters that dictate robust pattern formation is critical for engineering genetic patterning in vitro to organize artificial tissues. We set out to identify key requirements for pattern formation by patterning gene expression in vitro in sheets of re-distributing cells using a drug-inducible gene expression system and patterned drug delivery to mimic morphogen gene induction. Based on our experimental observations, we develop a mathematical model that allows us to identify and experimentally verify the conditions under which generation of sharp gene expression patterns is possible in vitro. Our results highlight the importance of coordinating gene induction dynamics and cellular movement in order to achieve robust pattern formation.

Tools for micropatterning epithelial cells into microcolonies on transwell filter substrates.

Despite the importance of epithelial tissue in most major organs there have been limited attempts to tissue engineer artificial epithelium. A key feature of mature epithelium is the presence of an apical-basal polarization, which develops over 7-20 days in culture. Currently, the most widely used 2D system to generate polarized epithelium in vitro involves the filter insert culture system, however this system is expensive, laborious and requires large numbers of cells per sample. We have developed a set of micropatterning techniques to spatially control the organization of epithelial cells into microsheets on filter inserts under the culture conditions necessary to induce epithelial cell polarization. Micropatterning improves cell uniformity within each microsheet, allows multiple sheet analysis on one filter insert, and reduced cell number requirements. We describe an agarose patterning method that allows maintenance of cell patterns for over 15 days, the time necessary to induce apical-basal polarization. We also describe a Parafilm™ patterning method that allows patterning for 5 to 15 days depending on cell type and only allows the generation of stripes and circular microsheets. The parafilm™ method however is extremely straightforward and could be easily adopted by any laboratory without the need of access to specialized microfabrication equipment. We also demonstrate that micropatterning epithelial cells does not alter the localization of the apical-basal marker ZO-1 or the formation of cilia, a marker of epithelium maturation. Our methods provide a novel tool for studying epithelial biology in polarized epithelium microsheets of controlled size.

A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm™ inserts.

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm™, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm™ insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm™ insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells.

Selection of aptamers for a non-DNA binding protein in the context of cell lysate.

Aptamer-facilitated Protein Isolation from Cells (AptaPIC) is a recently introduced method that allows, in particular, generation of aptamers for a protein target in a context of a crude cell lysate. The approach enables efficient, tag-free, affinity purification of target proteins which are not available in a pure form a priori, and for which no affinity ligands are available. In the proof-of-principle work, AptaPIC was used to develop aptamers for and purify MutS, a DNA mismatch repair protein. The DNA-binding nature of MutS raised concerns that AptaPIC was not a generic technique and could be inapplicable to protein targets that do not possess native nucleic acid-binding properties. Here we prove that these concerns are invalid. We used AptaPIC to generate pools of aptamers for human Platelet-Derived Growth Factor chain B (PDGF-B) protein, a non-DNA binding protein, in the context of a bacterial cell lysate, and subsequently purify it from the same lysate. Within a small number of rounds, the efficiencies of aptamer selection were similar in conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for pure protein and in AptaPIC for protein in the cell lysate. The conventional selection approach resulted in an aptamer pool with an EC(50) value of 2.0±0.1 µM, while the AptaPIC selection approach resulted in a pool with an EC(50) value of 3.9±0.4 µM. Our results clearly demonstrate that selection of aptamers for proteins in the cell lysate is not only realistic but also efficient.

Selection of aptamers for a protein target in cell lysate and their application to protein purification.

Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.

Diffusion as a tool of measuring temperature inside a capillary.

Application of capillary electrophoresis (CE) to temperature-sensitive biomolecular interactions requires knowledge of the temperature inside the capillary. The simplest approach to finding temperature in CE employs a molecular probe with a temperature-dependent parameter. Up until now only spectral parameters of molecular probes were utilized for temperature measurements in CE. The arbitrary nature of spectral parameters leads to several inherent limitations that compromise the accuracy and precision of temperature determination. This paper introduces the concept of finding temperature in CE through the measurement of a nonspectral parameter of the molecular probeits diffusion coefficient. Diffusion is a fundamental property of molecules that depends only on the molecular structure of the probe, the nature of the environment, and the temperature. It is ideally suited for temperature measurements in CE if an approach for measuring the diffusion coefficient in a capillary with high precision is available. This work first develops an approach for measuring the diffusion coefficient in a capillary with a relative standard deviation of as low as 2.1%. It is then demonstrated that such precise measurements of the diffusion coefficient could facilitate accurate temperature determination in CE with a precision of 1 degrees C. This new method was used to study the effect on temperature of different amounts of joule heat generated and different efficiencies of heat dissipation. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, for example, electrochemical and mass spectrometric detection.