The Chen Lab


Research Projects

Cells are the fundamental units of life. Their behavior is dictated by both genetics and environment. In vivo, the local tissue structure defines the cellular environment, constraining how cells interact with surrounding extracellular matrix substrates, neighboring cells, soluble growth factors, and physical forces. These "microenvironmental" cues not only cooperate to regulate the behaviors of individual cells - including cell proliferation, differentiation, and gene expression - but also govern emergent properties of the multicellular community. Yet, although understanding these interactions between cells and their surroundings is a fundamental aspect of both biology and tissue engineering, few experimental models exist to control these interactions at the cellular length scale, making it difficult to study the structure-function relationships of cells and tissues.

To address these questions in novel ways, we are pursuing several research programs: 1) to explore and develop new ways to use materials, microfabrication, and nanotechnologies to interact with, probe, and manipulate cells; and 2) to use these systems to understand the underlying mechanisms by which cells sense and respond to the physical, chemical, and structural cues in their surrounding microenvironment. Our laboratory is investigating the molecular mechanisms through which the local microenvironment regulates several aspects of cell behavior including cell migration, proliferation, differentiation, and apoptosis. We are actively studying how stem cells choose to commit to different lineages, and the biology of endothelial cells and smooth muscle cells in the context of angiogenesis, wound healing, atherosclerosis, and hypertension.

Specific projects include:

               
   


Stem cell differentiation
We are investigating how adult mesenchymal stem cells choose to commit and differentiate into specific lineages. Human mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into many of the cells resident in musculoskeletal and stromal tissues of the human body, including fibroblasts, chondrocytes, osteoblasts, myocytes, and adipocytes. While differentiation of the MSCs into appropriate lineages may enhance healing of injured tissues, inappropriate lineage specification may be responsible for numerous pathophysiologic processes, including the decreased bone mass and increased fat in osteoporotic bones, and the calcification of atherosclerotic vessel walls. Regulation of the lineage commitment of MSCs by local microenvironmental cues therefore may be critical to our fundamental understanding of numerous degenerative as well as healing processes. The long term objective of this research is to characterize the cues within the local surrounding microenvironment that drive the lineage specification and differentiation of human mesenchymal stem cells (MSCs), and the molecular pathways involved. We have recently discovered that adhesion of MSCs to fibronectin regulates a commitment switch in the MSCs between adipogenic and osteogenic lineage specification, through a mechanism involving RhoA signaling and cytoskeletal tension (McBeath et al., 2004). We are now pursuing several questions involving the role of cytoskeletal tension and signaling in MSC biology. These studies will provide a deeper understanding the role of stem cells in disease, and a foundation for establishing approaches for engineering stem cells for therapeutic applications.

               
   


Angiogenesis
The vascularization of engineered tissues is critical to the ultimate success of tissue engineering as an organ replacement therapy. The formation of new capillary vessels in vivo, or angiogenesis, is linked to the pathogenesis of numerous diseases including cancer, and is regulated by local cues within the tissue microenvironment. The general goal of our studies is to understand the mechanism by which local extracellular matrix (ECM) properties, integrins, and cadherins regulate initial signal transduction, subsequent gene expression pathways, capillary endothelial cell proliferation and capillary tube morphogenesis required in angiogenesis. We have found that adhesion to ECM cooperates with growth factors to generate not only biochemical, but also mechanical signals that are important to driving capillary endothelial cell function. We have demonstrated that the spatial organization and geometric presentation of ECM affects the degree to which cells attach and spread against the substrate. These changes in cell shape appear to regulate proliferation and capillary tube formation by modulating contractile tension generated by the actin cytoskeleton (Chen et al., 1997; Chen et al., 1998; Dike et al,. 1999). Interestingly, modulating cell-cell contact and engagement of VE-cadherin also appears to modulate endothelial cell proliferation via changes in cytoskeletal tension (Nelson et al., 2002; Nelson et al., 2003). The interactions between cell adhesion, mechanics, and cell function appear to be mediated via the Rho family of small GTPases (Chen et al., 2003; Tan et al., 2003; Nelson et al., 2004). Based on these findings, we are now investigating how angiogenic factors regulate changes in cell mechanics, and whether these changes critical for the regulation of angiogenic processes. Together, these studies will define the mechanisms by which microenvironmental cues modulate endothelial cell function and capillary morphogenesis, and establish new strategies to promote angiogenesis in native ischemic tissues as well as in ex-vivo engineered tissues.

               
   


Engineering Extracellular Matrix
Many behaviors of cells are regulated by adhesion to an extracellular matrix, including cell proliferation, differentiation, apoptosis, and gene expression. Cell adhesion involves binding and clustering of integrin receptors to immobilized extracellular matrix, and active spreading of the cells against the substrate. We have used self-assembled monolayers of alkanethiolates on gold to present micrometer-scale patches of ECM on a substrate, and thereby decouple the local surface density of extracellular matrix from the degree to which cells can spread against the substrate. We have demonstrated that integrin signaling, cell shape, and cytoskeletal structure coordinate to control endothelial cell proliferation, differentiation, and apoptosis (Chen et al., 1997; Roberts et al., 1998; Dike et al., 1999; Chen et al., 2003). These findings provide a foundation for recognizing that cell adhesion is not a simple signaling event determined by binding of integrin to its ligand, but instead a complex interplay between the biochemical signals of integrins and structural changes associated with cell spreading. We are now investigating how these changes in cell shape exert their effects at the molecular and transcriptional level, and continuing to explore numerous technological opportunities to address other questions about how ECM structure can regulate cell function.

               
   


Controlling Cell-to-Cell Adhesion
Signaling between neighboring cells is the hallmark of multicellular systems, and is critical to many processes. However, these signals traditionally have been exceedingly difficult to isolate, because current experimental approaches cannot uncouple cell-cell communication by direct cell-cell contact (juxtacrine), by diffusible factors (paracrine), and changes in cell-substrate adhesion. For example, seeding increasing density of cells on a surface to promote cell-cell contact also increases paracrine signaling and decreases the amount of cell adhesion to the substrate. By engineering substrates containing bowtie-shaped wells, we could introduce cell-cell contact while simultaneously controlling cell spreading against the substrate. Using this system, we discovered that engagement of cadherin receptors upon direct contact between endothelial cells elicits a complex system of previously unknown signals that cause changes in integrin signaling, cell shape, cytoskeletal structure, and cell behavior (Nelson and Chen, 2002; Nelson and Chen, 2002; Nelson et al., 2004). We are currently identifying the specific molecular pathways that mediate this coupling. These efforts are providing early indications of how multicellular organization may exert previously unknown mechanical and functional effects on cells residing in tissues. We are now beginning to establish our next generation of these techniques to examine the interaction between different types of cells (heterotypic interactions), and more complex multicellular aggregates.

               
   


Mechanotransduction and the Study of Cellular Forces
Mechanical forces play a critical role in nearly all aspects of cell biology, from cell migration to morphogenesis to cell proliferation. These forces are ubiquitous to the interactions between cells and their substrates (such as shear stress in the vascular tree). Even in the absence of applied external forces, cells themselves apply forces against their surroundings by actively contracting their actin-myosin cytoskeletal networks. Our studies suggest that integrins and their associated focal adhesions are critical to how cells transduce mechanical stresses into biochemical signals. In additions, growth factor signaling and cell spreading appear to affect how cells contract, and these contractions are in turn thought to modulate how growth factors, integrins, and cell shape alter cell behaviors. To characterize these forces, we built a device containing an array of elastomeric posts to measure the traction forces generated by cells as they adhere and spread against the substrate. Using the elastomeric force sensors, we have found that the magnitude of forces generated at adhesions affects adhesion size, and that progressively increasing cell spreading caused an increase in cytoskeletal tension, through changes in G-protein signaling (Tan et al., 2003). We also demonstrated that changes in substrate stiffness could alter cell adhesiveness (Gray et al., 2002). By manipulating and measuring the mechanical interactions between cells and surfaces, my laboratory continues to characterize this mechano-chemical control system in which cells appear to simultaneously integrate physical and biochemical cues within their signal transduction networks in order to navigate in their microenvironment. In particular, we are examining how these forces are important in numerous processes, such as in the regulation of proliferation required in angiogenesis, and in stem cell lineage commitment. Understanding the mechanism of this integration may provide the key to how multicellular organisms can organize into structurally ordered systems.

               
   


Organizing Principles in Multicellularity
We have established several tools to control the microenvironment at a cellular length scale, and characterized some of the molecular mechanisms of how a cell coordinates biochemical and physical cues to regulate its function. We have begun to use what we have learned in order to connect additional relevant length scales - from single cells to small groups of cells to larger tissues - to understand how single cell behavior translates to tissue organization and function. It is our hypothesis that between single cells and large populations of cells there exists a regime of ‘mesoscale’ multicellular structures that give rise to phenomena that are important in tissue development and function. We have begun to study how progressively larger groups of cells, and groups of different types of cells, coordinate with each other. To this end, we are developing next-generation approaches to organize multiple cell types in specific arrangements within a multicellular structure (Tien et al., 2002), and using magnetic and electrical forces to actively and dynamically position and assemble cells onto surfaces (Hultgren et al., 2003; Gray et al., 2003). We are beginning to explore approaches to control multicellular architecture in a three-dimensional context. Collectively, these tools provide several handles to investigate how the organization of multiple cells might itself lead to emergent cellular signals. Emergence of new control systems at these larger length scales should provide us with additional understanding of how to translate single cell behavior to tissue-scale function.

Summary
Cells integrate not only the chemical but also the structural and mechanical aspects of their environment - including soluble growth factors, extracellular matrix scaffolds, neighboring cells, and mechanical forces - to regulate cell function. To understand and manipulate these control systems, my laboratory has developed substantial expertise in microsystems technologies that allow us to integrate solid-state and cellular systems. Combining these approaches with traditional molecular tools, we will continue to enrich the understanding of the dynamic interactions between cells and their surroundings. By embracing the complexities of the in vivo cellular environment within our in vitro systems, we hope to understand how cells coordinate in progressively more complex environments, thereby allowing us to 1) provide a fundamental cellular basis for understanding a wide variety of physiologic and pathologic processes; 2) engineer artificial tissue constructs; and 3) build hybrid living-nonliving devices for both medical and non-medical applications.