Kiran Bhadriraju

Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

Cells lying on a bed of microneedles: An approach to isolate mechanical force

We describe an approach to manipulate and measure mechanical interactions between cells and their underlying substrates by using microfabricated arrays of elastomeric, microneedle-like posts. By controlling the geometry of the posts, we varied the compliance of the substrate while holding other surface properties constant. Cells attached to, spread across, and deflected multiple posts. The deflections of the posts occurred independently of neighboring posts and, therefore, directly reported the subcellular distribution of traction forces. We report two classes of force-supporting adhesions that exhibit distinct force–size relationships. Force increased with size of adhesions for adhesions larger than 1 μm2, whereas no such correlation existed for smaller adhesions. By controlling cell adhesion on these micromechanical sensors, we showed that cell morphology regulates the magnitude of traction force generated by cells. Cells that were prevented from spreading and flattening against the substrate did not contract in response to stimulation by serum or lysophosphatidic acid, whereas spread cells did. Contractility in the unspread cells was rescued by expression of constitutively active RhoA. Together, these findings demonstrate a coordination of biochemical and mechanical signals to regulate cell adhesion and mechanics, and they introduce the use of arrays of mechanically isolated sensors to manipulate and measure the mechanical interactions of cells.

Engineering cellular microenvironments to improve cell-based drug testing

Recent progress in the biology of cell adhesion is enabling cell culture models to better reproduce in vivo functions. Cues from adhesion to extracellular matrix and neighboring cells are important regulators of cell behaviors. The recent adaptation of semiconductor tools to spatially organize cells and their adhesions has enhanced our ability to engineer cell functions ex vivo. By using these tools to create more in vivo-like cultures, cell-based drug discovery and target validation could be improved. This review explores the biological advances made by these microfabrication tools and discusses how they could enable high-throughput cell-based assays.

Optimization of yield in magnetic cell separations using nickel nanowires of different lengths.

Ferromagnetic nanowires are shown to perform both high yield and high purity single‐step cell separations on cultures of NIH‐3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin‐mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5–35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4‐fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin‐C.

Engineering amount of cell-cell contact demonstrates biphasic proliferative regulation through RhoA and the actin cytoskeleton.

Endothelial cell-cell contact via VE-cadherin plays an important role in regulating numerous cell functions, including proliferation. However, using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation depending on the adhesive context. In this study, we used micropatterned wells combined with active positioning of cells by dielectrophoresis in order to investigate whether the number of contacting neighbors affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation; one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, and that expression of dominant negative RhoA (RhoN19) blocked the contact-mediated increase in stress fibers and proliferation. Furthermore, examination of heterotypic pairs of untreated cells in contact with RhoN19-expressing cells revealed that intracellular, but not intercellular, tension is required for the contact-mediated stimulation of proliferation. Moreover, engagement of VE-cadherin with cadherin-coated beads was sufficient to stimulate proliferation in the absence of actual cell-cell contact. In all, these results demonstrate that cell-cell contact signals through VE-cadherin, RhoA, and intracellular tension in the actin cytoskeleton to regulate proliferation.

Feel the force: using a bed of needles to map single cell generated traction forces

Understanding of how cells exert and respond to mechanical forces depends on the ability to measure the origin and magnitude of those forces at subcellular resolutions. We have developed a novel system to measure cell contractility by culturing cells on top of an array of micrometer scale elastomeric posts. Each post in the array independently bends in response to local forces exerted by the attached cell. We demonstrate that the system can consistently measure cell traction forces with a high degree of precision in real time. We use this system to measure the contractility of cells spread to different degrees. We found that cell spreading is required for cells to organize its actin cytoskeleton and exert traction force on the underlying substrate.

Lineage mapper: A versatile cell and particle tracker.

The ability to accurately track cells and particles from images is critical to many biomedical problems. To address this, we developed Lineage Mapper, an open-source tracker for time-lapse images of biological cells, colonies, and particles. Lineage Mapper tracks objects independently of the segmentation method, detects mitosis in confluence, separates cell clumps mistakenly segmented as a single cell, provides accuracy and scalability even on terabyte-sized datasets, and creates division and/or fusion lineages. Lineage Mapper has been tested and validated on multiple biological and simulated problems. The software is available in ImageJ and Matlab at isg.nist.gov.

Engineering Cell Adhesion

Cells exist within a complex and ever-changing environment, which includes soluble molecules such as growth factors, an extracellular matrix that includes adhesive proteins and carbohydrates, and other neighboring cells. They actively sense and respond to changes in this environment, existing in a state of physiological equilibrium with it. Thus, it has been said, “. . . the unit of function in higher organisms is larger than the cell itself ” [10]. The information content in the adhesive environment is encoded both in its composition and its organization on the nanometer to micrometer length scales. When taken out of this physiological context and cultured in plastic tissue culture dishes, cells lose the cues that maintain their in vivo identity or phenotype, and dedifferentiate. For example, hepatocytes— the principal cell type in the liver—perform several critical liver-specific functions such as production of bile, metabolism of urea, and the synthesis of important serum proteins such as albumin, fibrinogen, and transferrin [27]. When cultured in vitro and isolated from the liver microenvironment, they rapidly downregulate liver-specific phenotype [54]. Similarly, chondrocytes, which are required for the secretion and maintenance of cartilage, lose their differentiated function when cultured in vitro downregulating the synthesis and secretion of cartilage-specific collagens and proteoglycans [88]. Thus, tissue-specific cell function appears to be closely related to the microstructural organization of the tissue itself [11].