Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on "rigid" glass or "stiff" gels than on "soft" gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.

Two important goals in stem cell research are to control the cell proliferation without differentiation and to direct the differentiation into a specific cell lineage when desired. Here, we demonstrate such paths by controlling only the nanotopography of culture substrates. Altering the dimensions of nanotubular-shaped titanium oxide surface structures independently allowed either augmented human mesenchymal stem cell (hMSC) adhesion or a specific differentiation of hMSCs into osteoblasts by using only the geometric cues, absent of osteogenic inducing media. hMSC behavior in response to defined nanotube sizes revealed a very dramatic change in hMSC behavior in a relatively narrow range of nanotube dimensions. Small (Ϸ30-nm diameter) nanotubes promoted adhesion without noticeable differentiation, whereas larger (Ϸ70-to 100-nm diameter) nanotubes elicited a dramatic stem cell elongation (Ϸ10-fold increased), which induced cytoskeletal stress and selective differentiation into osteoblast-like cells, offering a promising nanotechnology-based route for unique orthopedics-related hMSC treatments.differentiation ͉ mesenchymal ͉ nanotopography ͉ osteogenesis ͉ proliferation N anostructures are of particular interest because they have the advantageous feature of a high surface-to-volume ratio, and they elicit a higher degree of biological plasticity compared with conventional micro-or macrostructures. In the field of biomaterial development and in vivo implant technology, the nanoscale structure and morphologenic factor of the surface have played a critical role in accelerating the rate of cell proliferation and enhancing tissue acceptance with a reduced immune response (1, 2). In terms of in vitro cell biology, there has also been much attention placed on cellular responses to their structural surroundings (3). In fact, it has been observed that macro-, micro-and nano-sized topographical factors stimulate behavioral changes in both cells and tissues. Recent studies related to the effect of nanotopography on cellular behavior indicated that osteoblast adhesion and functionality was enhanced by 30% when cultured on a nanograined Al 2 O 3 and TiO 2 substrate (4-6) compared with those cultured on a micrograined surface, and nanostructures such as TiO 2 nanotubes with Ͻ100-nm spacing showed superior characteristics in bone mineral synthesis (5). However, most of the previous studies on nanostructures and cell responses have mainly used oriented, patterned, or semiordered polymer arrays (7-9) and alumina/ polymer hybrid patterned arrays (10).The material and mechanical characteristics of titanium (Ti) metal, which has a thin native oxide layer of TiO 2 , make it an ideal orthopedic material that bonds directly to the adjacent bone surface (11,12). Fabrication of the nanostructured titanium dioxide (TiO 2 ) nanotube arrays has been a primary subject of investigation lately because of the wide range of TiO 2 applications in the fields of solar cells (13-16), photocatalysis (17-19), photoelectrolysis (20), sensors (21,22), and b...

The modulus of elasticity of the extracellular matrix (ECM), often referred to in a biological context as “stiffness,” naturally varies within the body, e.g., hard bones and soft tissue. Moreover, it has been found to have a profound effect on the behavior of anchorage‐dependent cells. The fabrication of matrix substrates with a defined modulus of elasticity can be a useful technique to study the interactions of cells with their biophysical microenvironment. Matrix substrates composed of polyacrylamide hydrogels have an easily quantifiable elasticity that can be changed by adjusting the relative concentrations of its monomer, acrylamide, and cross‐linker, bis‐acrylamide. In this unit, we detail a protocol for the fabrication of statically compliant and radial‐gradient polyacrylamide hydrogels, as well as the functionalization of these hydrogels with ECM proteins for cell culture. Included as well are suggestions to optimize this protocol to the choice of cell type or stiffness with a table of relative bis‐acrylamide and acrylamide concentrations and expected elasticity after polymerization. Curr. Protoc. Cell Biol. 47:10.16.1‐10.16.16. © 2010 by John Wiley & Sons, Inc.

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E ∼12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.

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Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A `cysteine shotgun' method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development.

The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine.