Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia.

Consistent with recent reports indicating that neurons differentiated in vitro from human-induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain, gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two independent discovery-based approaches-microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses-to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. These reproducible NPC phenotypes were identified through scalable assays that can be applied to expanded cohorts of SZ patients, making them a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

Surface molecular recognition.

The spatial display of cellular ligands and receptors is important for cell adhesion and communication. Two approaches that emphasize developing selective methods to dissect, modify, and control receptor-ligand interactions at the cellular interface are discussed.

Topographical and physicochemical modification of material surface to enable patterning of living cells.

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.

Direct cell adhesion to the angiopoietins mediated by integrins.

Genetic ablation of angiopoietin-1 (Ang-1) or of its cognate receptor, Tie2, disrupts angiogenesis in mouse embryos. The endothelial cells in growing blood vessels of Ang-1 knockout mice have a rounded appearance and are poorly associated with one another and their underlying basement membranes (Dumont, D. J., Gradwohl, G., Fong, G. H., Puri, M. C., Gertsenstein, M., Auerbach, A., and Breitman, M. L. (1994) Genes Dev. 8, 1897--1909; Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W., and Qin, Y. (1995) Nature 376, 70--74; Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N., and Yancopoulos, G. D. (1996) Cell 87, 1171--1180). It is therefore possible that Ang-1 regulates endothelial cell adhesion. In this study we asked whether Ang-1 might act as a direct substrate for cell adhesion. Human umbilical vein endothelial cells (HUVECs) plated for a brief period on different substrates were found to adhere and spread well on Ang-1. Similar results were seen on angiopoietin-2 (Ang-2)-coated surfaces, although cells did not spread well on Ang-2. Ang-1, but not Ang-2, supported HUVEC migration, and this was independent of growth factor activity. When the same experiments were done with fibroblasts that either lacked, or stably expressed, Tie2, results similar to those with HUVECs were seen, suggesting that adhesion to the angiopoietins was independent of Tie2 and not limited to endothelial cells. Interestingly, when integrin-blocking agents were included in these assays, adhesion to either angiopoietin was significantly reduced. Moreover, Chinese hamster ovary-B2 cells lacking the alpha(5) integrin subunit did not adhere to Ang-1, but they did adhere to Ang-2. Stable expression of the human alpha(5) integrin subunit in these cells rescued adhesion to Ang-1 and promoted an increase in adhesion to Ang-2. We also found that Ang-1 and Ang-2 bind rather selectively to vitronectin. These results suggest that, beyond their role in modulating Tie2 signaling, Ang-1 and Ang-2 can directly support cell adhesion mediated by integrins.

Using electroactive substrates to pattern the attachment of two different cell populations.

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.

The microenvironment of immobilized Arg-Gly-Asp peptides is an important determinant of cell adhesion.

This paper uses self-assembled monolayers on gold as a model system to demonstrate that the attachment and spreading of Swiss 3T3 fibroblasts depends strongly on the microenvironment of immobilized RGD peptides. This work utilized monolayers that present mixtures of Arg-Gly-Asp peptides, which are ligands for cellular integrin receptors, and oligo(ethylene glycol) groups, which resist the nonspecific adsorption of protein. The microenvironment of the peptide ligands was controlled by altering the length of the surrounding oligo(ethylene glycol) groups on the monolayer. By using thiols that present either tri-, tetra-, penta-, or hexa(ethylene glycol) units, the average distance separating the glycol groups and the peptide ligand is altered while the structure and properties of the background remain unchanged. Cell attachment to monolayers presenting a fixed density of peptide decreased as the length of the oligo(ethylene glycol) group increased. The average projected area of attached cells showed a similar trend. At lower densities of immobilized peptide, decreases in both cell attachment and projected cell area were more pronounced. Attachment and spreading did not depend on density of peptide on monolayers presenting tri(ethylene glycol) groups, but showed a high sensitivity to density of ligand on monolayers presenting longer glycol oligomers. Experiments that used a soluble peptide to inhibit the attachment of cells to monolayers demonstrated that the strength of the cell-substrate interaction decreased on monolayers presenting longer glycol groups. Together, these results suggest that the microenvironment of the peptide ligand influences the affinity of the integrin-peptide interaction and that weaker interactions display a density-dependent enhancement of binding during cell attachment and spreading. This finding is an important consideration in studies that correlate biological function with the composition of ligands on a substrate. This finding also represents an important principle for the design of biologically active materials because it illustrates the degree to which the presentation of adhesion motifs can modify the response of mammalian cells.

Using selective withdrawal to coat microparticles.

We report a method that uses the process of selective withdrawal of one fluid through a second immiscible fluid to coat small particles with polymer films. Fluid is withdrawn through a tube with its orifice slightly above a water-oil interface. Upon increasing the flow rate, there is a transition from a state where only oil is withdrawn to a state where the water, containing the particles to be coated and appropriate prepolymer reagents, is entrained in a thin spout along with the oil. The entrained particles eventually cause the spout interface to break, producing a thin coat of controllable thickness around each particle, which can be subsequently polymerized using chemical reagents, light, or heat. This method allows flexibility in the chemical composition and thickness of the conformal coatings.

Towards a fibrous composite with dynamically controlled stiffness: lessons from echinoderms.

Sea urchins and sea cucumbers, like other echinoderms, control the tensile properties of their connective tissues by regulating stress transfer between collagen fibrils. The collagen fibrils are spindle-shaped and up to 1 mm long with a constant aspect ratio of approx. 2000. They are organized into a tissue by an elastomeric network of fibrillin microfibrils. Interactions between the fibrils are regulated by soluble macromolecules that are secreted by local, neurally controlled, effector cells. We are characterizing the non-linear viscoelastic properties of sea cucumber dermis under different conditions, as well as the structures, molecules and molecular interactions that determine its properties. In addition, we are developing reagents that will bind covalently to fibril surfaces and reversibly form cross-links with other reagents, resulting in a chemically controlled stress-transfer capacity. The information being developed will lead to the design and construction of a synthetic analogue composed of fibres in an elastomeric matrix that contains photo- or electro-sensitive reagents that reversibly form interfibrillar cross-links.

Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates.

Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 microm2 spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 microm2 failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-microm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 microm2. Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-microm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 microm2), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis-growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.

Tailored substrates for studies of attached cell culture.

Substrates for studies of the interactions of attached cells with extracellular matrix components are often prepared by allowing a protein to adsorb from solution onto a glass or polystyrene substrate. This method is simple and effective for many studies, but it can fail in cases that require rigorous control over the structure and composition of adsorbed protein. Self-assembled monolayers formed by the spontaneous ordering of terminally functionalized alkanethiols onto a gold substrate are a class of well-ordered substrates and provide a convenient method for tailoring substrates with ligands, proteins and other groups. Methods that can pattern the monolayers provide a general strategy to create substrates that control the size, shape and spacing of attached cells. This review illustrates recent work that has used these methods of surface chemistry to create tailored substrates for studies in cell biology.

Micropatterned surfaces for control of cell shape, position, and function.

The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering. Using microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer-scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells. In addition, the size and geometry of the islands were shown to control cell shape. Traditional approaches to modulate cell shape, either by attaching suspended cells to microbeads of different sizes or by plating cells on substrates coated with different densities of ECM, suggested that cell shape may play an important role in control of apoptosis as well as growth. Data are presented which show how micropatterned substrates were used to definitively test this hypothesis. Progressively restricting bovine and human endothelial cell extension by culturing cells on smaller and smaller micropatterned adhesive islands regulated a transition from growth to apoptosis on a single continuum of cell spreading, thus confirming the central role of cell shape in cell function. The micropatterning technology is therefore essential not only for construction of biosurface devices but also for the investigation of the fundamental biology of cell-ECM interactions.

Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver.

This paper describes a convenient methodology for patterning substrates for cell culture that allows the positions and dimensions of attached cells to be controlled. The method uses self-assembled monolayers (SAMs) of terminally substituted alkanethiolates (R(CH2)11-15S-) adsorbed on optically transparent films of gold or silver to control the properties of the substrates. SAMs terminated in methyl groups adsorb protein and SAMs terminated in oligo(ethylene glycol) groups resist entirely the adsorption of protein. This methodology uses microcontact printing (microCP)-an experimentally simple, nonphotolithographic process-to pattern the formation of SAMs at the micrometer scale; microCP uses an elastomeric stamp having at its surface a pattern in relief to transfer an alkanethiol to a surface of gold or silver in the same pattern. Patterned SAMs having hydrophobic, methyl-terminated lines 10, 30, 60, and 90 microm in width and separated by protein-resistant regions 120 microm in width were prepared and coated with fibronectin; the protein adsorbed only to the methyl-terminated regions. Bovine capillary endothelial cells attached only to the fibronectin-coated, methyl-terminated regions of the patterned SAMs. The cells remained attached to the SAMs and confined to the pattern of underlying SAMs for at least 5-7 days. Because the substrates are optically transparent, cells could be visualized by inverted microscopy and by fluorescence microscopy after fixing and staining with fluorescein-labeled phalloidin.

On-line detection of nonspecific protein adsorption at artificial surfaces.

A detailed understanding of the interaction of proteins with artificial surfaces is essential for many applications in medicine and biochemistry. The affinity of surfaces toward proteins may, for instance, remove pharmacological proteins from media or control the adherence of pathogenic bacteria to protheses. Only a few analytical techniques now exist that can be used to study the binding process in real time, using unlabeled proteins. By investigating the adsorption kinetics of fibrinogen at differently terminated self-assembled monolayers (SAMs) of alkanethiols on thin gold films, it is demonstrated that acoustic plate-mode sensors are a promising analytical tool for studying the adsorption of proteins. In agreement with previous studies for fibrinogen, it is shown in situ that hexa(ethylene glycol)-terminated SAMs (HS(CH2)11 (OCH2CH2)6OH) exhibit very low protein adsorption and that methyl-terminated SAMs (HS(CH2)11CH3) tend to absorb large amounts of protein nonspecifically. The observed adsorption kinetics deviate from classical Langmuir behavior; these kinetics are compatible with a mechanism that involves an unfolding of fibrinogen after adsorption. Film quality is controlled by IR, XPS, and contact angle measurements.

Geometric control of cell life and death.

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.

Self-assembled monolayers (SAMs) formed on the adsorption of long-chain alkanethiols to the surface of gold or alkylsilanes to hydroxylated surfaces are well-ordered organic surfaces that permit control over the properties of the interface at the molecular scale. The ability to present molecules, peptides, and proteins at the interface make SAMs especially useful for fundamental studies of protein adsorption and cell adhesion. Microcontact printing is a simple technique that can pattern the formation of SAMs in the plane of the monolayer with dimensions on the micron scale. The convenience and broad application offered by SAMs and microcontact printing make this combination of techniques useful for studying a variety of fundamental phenomena in biointerfacial science.