Jianping Fu

Mechanical regulation of cell function with geometrically modulated elastomeric substrates

We report the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility predicted later stem cell fate decisions in single cells.

A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins

Microfabricated regular sieving structures hold great promise as an alternative to gels to improve the speed and resolution of biomolecule separation. In contrast to disordered porous gel networks, these regular structures also provide well defined environments ideal for the study of molecular dynamics in confining spaces. However, the use of regular sieving structures has, to date, been limited to the separation of long DNA molecules, however separation of smaller, physiologically relevant macromolecules, such as proteins, still remains a challenge. Here we report a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array. The designed structural anisotropy causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow size-based separation of DNA and proteins, as well as electrostatic separation of proteins, was achieved, demonstrating the potential use of this device as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system.

Forcing Stem Cells to Behave: A Biophysical Perspective of the Cellular Microenvironment

Physical factors in the local cellular microenvironment, including cell shape and geometry, matrix mechanics, external mechanical forces, and nanotopographical features of the extracellular matrix, can all have strong influences on regulating stem cell fate. Stem cells sense and respond to these insoluble biophysical signals through integrin-mediated adhesions and the force balance between intracellular cytoskeletal contractility and the resistant forces originated from the extracellular matrix. Importantly, these mechanotransduction processes can couple with many other potent growth-factor-mediated signaling pathways to regulate stem cell fate. Different bioengineering tools and microscale/nanoscale devices have been successfully developed to engineer the physical aspects of the cellular microenvironment for stem cells, and these tools and devices have proven extremely powerful for identifying the extrinsic physical factors and their downstream intracellular signaling pathways that control stem cell functions.

Nanotopography Influences Adhesion, Spreading, and Self-Renewal of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.

Cell Shape and Substrate Rigidity Both Regulate Cell Stiffness

Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. For soft substrates, the influence of substrate stiffness on cell cortical stiffness is more prominent than that of cell shape, since increasing adherent area does not lead to cell stiffening. On the other hand, for cells constrained to a small area, cell shape effects are more dominant than substrate stiffness, since increasing substrate stiffness no longer affects cell stiffness. These results suggest that cell size and substrate stiffness can interact in a complex fashion to either enhance or antagonize each others effect on cell morphology and mechanics.

Molecular sieving using nanofilters: Past, present and future

Filtration of molecules by nanometer-sized structures is ubiquitous in our everyday life, but our understanding of such molecular filtration processes is far less than desired. Until recently, one of the main reasons was the lack of experimental methods that can help provide detailed, microscopic pictures of molecule-nanostructure interactions. Several innovations in experimental methods, such as nuclear track-etched membranes developed in the 70s, and more recent development of nanofluidic molecular filters, played pivotal roles in advancing our understanding. With the ability to make truly molecular-scale filters and pores with well-defined sizes, shapes, and surface properties, now we are well positioned to engineer better functionality in molecular sieving, separation and other membrane applications. Reviewing past theoretical developments (often scattered across different fields) and connecting them to the most recent advances in the field would be essential to get a full, unified view on this important engineering question.

Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity

We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. This protocol describes how to fabricate the silicon micropost array masters (∼2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1–14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation.

Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive1. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons (MNs) derived from hPSCs within 23 days of culture using soft PMAs were improved more than 4- and 10-fold, respectively, compared to coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo-YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

How vinculin regulates force transmission

Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (VH) and tail domains. VH enhances adhesion strength by increasing ECM-bound integrin–talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; VH has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or VH, correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin’s active conformation to promote force transfer.