Guidance of stem cell fate on 2D patterned surfaces

Biomaterials 33 (2012) 6626e6633

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Review

Guidance of stem cell fate on 2D patterned surfaces Kristian Kolind a, Kam W. Leong b, Flemming Besenbacher a, c, Morten Foss a, * a

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA c Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C, Denmark b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 April 2012 Accepted 30 May 2012 Available online 28 June 2012

Stem cells possess unique abilities as they can renew themselves for extended periods of time and have the capacity to differentiate into a variety of lineages. They hold promise for treating a plethora of diseases ranging from musculoskeletal defects to myocardial infarction and to neural disorders. Understanding how to control the fate decision of these cells to self-renew or differentiate is paramount in stem cell tissue engineering. Recently, significant progress has been made in guiding stem cell differentiation in vitro, and we are beginning to understand the complex interplay of factors that control their fate. Here, we highlight the recent approaches for guidance of stem cells through patterning of surfaces at the micro- and nanoscale. Particular attention is given to chemical patterning of substrates with adhesion ligands and physical patterning with a variety of topographical features. These surfacemediated biochemical and mechanical cues have proven influential in altering a wide range of stem cell phenotypes. This approach to guide or ultimately control stem cells by surface patterning has enormous potential implications in cell therapies and regenerative medicine. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Stem cells Mechanotransduction Stem cell therapy Tissue engineering Stem cell niche 2D surfaces

1. Introduction Due to the unique ability of stem cells to proliferate and to differentiate into a variety of specialized phenotypes, immense efforts are currently put into understanding the complex interplay of intrinsic and extrinsic factors that influence their behavior. In vivo, stem cells are located within the stem cell niche, containing a multitude of complex niche factors orchestrated by the surrounding extracellular microenvironment. Here, stem cells respond to signals from neighboring cells, the extracellular matrix and soluble signaling molecules (Fig. 1). Motivated by the vision of creating a biomimetic microenvironment for directing stem cell fate, many scientists have been inspired to look for optimal combinations of the above-mentioned factors. In particular, exciting data have shown the importance of coordinating multiple extracellular factors to direct the stem cell fate; one of which being patterning of chemical and topographical cues. In this review, we highlight the recent significant advancements in directing stem cells through engineered two-dimensional (2D) surface patterning. In this context the surface patterning will refer to both chemical patterning and to the introduction of

* Corresponding author. Tel.: þ45 871 55859; fax: þ45 871 54041. E-mail address: [email protected] (M. Foss). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.05.070

topographical features such as pillars, grooves, and pits. We will focus on the most recent studies in which such substrates have been applied either alone or in conjunction with other intrinsic and/or extrinsic cues to affect stem cell behavior. In the interpretation of these and other results regarding differentiation of stem cells it is important to keep in mind the typical heterogeneity of the different stem cell populations used. As such, in the application of heterogeneous mixtures of cells such as the widely used mesenchymal stem cell (MSC), regulation may be a selective rather than an instructive process which favors a certain subpopulation of the heterogeneous mixture, instead of exerting an universal regulatory effect on cell fate [1]. This should be kept in mind when analyzing results, but does not change the promising outcomes gained from recent years showing that patterning may guide stem cell differentiation. 1.1. Native stem cell interactions with surface cues In the native environment, stem cells physically interact with the surrounding extracellular matrix (ECM) including chemical and topographical cues at the micro and nanometer length scale (microscale and nanoscale, respectively). Several cellular interaction schemes can be regulated at the microscale such as cellecell, celleECM, cell-soluble and cellemechanical stimuli interactions [2]. Specifically, micrometer range features such as grooves, ridges

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The Stem Cell Niche Extracellular Matrix Cell interactions

Cell - Cell contacts

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- Biochemical factors - Topography - Shape - Stiffness - Mechanical load

Self-renewal

Differentiation

Quiescence

Apoptosis

Fig. 1. Illustration of the multitude of different factors affecting stem cells (ECMecell interactions, cellecell interactions and soluble factors), which together determines the stem cell fate (self-renewal, differentiation, quiescence and apoptosis).

and pits, as well as chemical motifs, affect cell behavior. Likewise, it has been shown that structures on the nanometer length scale influence cell-ECM interactions through receptor (integrin) binding. Well-characterized examples include the fibers of the ECM and basement membrane (10e300 nm in diameter), the ECMs interconnecting nanopores and the hydroxyapatite crystals (4 nm) found in bone [3,4]. Thus, it is not surprising that the composition of the ECM has decisive regulatory and structural consequences on cells, affecting their adhesion, orientation, motility, cytoskeletal arrangement, signal transduction and gene expression [5]. This influence of the ECM on cells is dictated by their interaction through focal adhesions, which are sites of integrin binding and clustering, connecting the intracellular cytoskeleton of the cell with the surrounding ECM. As such, the patterning of the matrix influences the size, number and organization of focal adhesions, rearranges the cytoskeleton and alters the morphology of cell body and cell nucleus. A key concept to describe this influence of surface cues on stem cell behavior is mechanotransduction [6], which is the process of transmitting external mechanical stimuli to the cell nucleus via the cytoskeleton, thereby inducing adaptive changes in gene and protein expression. In describing the transmission a distinction is made between indirect and direct mechanotransduction. Indirect mechanotransduction involves signaling cascades induced at focal adhesions, leading to indirect downstream effects on gene expression. Direct mechanotransduction, in contrast, involves physical pulling of the cytoskeleton on the nucleus to exert mechanical forces on the nuclear components and potentially altering gene transcription. Both of these effects are involved in cellesurface interactions that can influence such processes as stem cell differentiation. It would therefore be fruitful to incorporate proper micro- and nanoscale surface design of biomaterials in the creation of an optimal microenviroment for stem cell tissue engineering. 1.2. Stem cell interaction with 2D substrate patterns Given the complexity of the native three-dimensional (3D) niche described above, two-dimensional (2D) surfaces provide a convenient configuration for deconstructing the niche in a reductionistic approach and analyzing the effects of individual

components on stem cell fate. The role of synthetic 2D substrate patterning on cells was first discussed in detail by Curtis et al. in 1964 [7]. They analyzed the influence of topographical patterning on cells such as endothelia, fibroblasts, and epithelia, and demonstrated that cells strongly aligned along the direction of a few micrometer wide grooves. Recently, the field has grown rapidly, and it is now widely accepted that patterning of surfaces on the micro- and nanometer scale influences different cellular processes, including proliferation and differentiation [8e10]. 1.3. Chemically micropatterned surfaces for control of stem cell shape The improved ability to fabricate precisely defined chemical patterns on surfaces is a consequence of advances in UV- and more recently soft- and colloidal lithographic techniques. This has led to new possibilities to address fundamental questions related to cellesubstrate interactions. The patterning of substrates aims principally at positioning chemical/biological components of interest in a defined manner for regulation of cell function. If surface patterning at the microscale (1e500 mm) is introduced, the substrates are typically used for control of cell/colony shape and positioning, while nanoscale patterning (1e100 nm) involves regulation of the cellesubstrate interaction through control of integrin binding sites. The focus of this review is on patterning at the micro- and nanoscale for control of stem cell differentiation. More detailed descriptions of the actual fabrication of both microand nanoscale patterns can be found from several other excellent reviews [11e17]. The most widely used technique for fabrication of micrometer patches is microcontact printing utilizing a polydimethylsiloxane (PDMS) stamp to pattern self-assembled monolayers (SAMs) of chemistries and a range of cell binding proteins such as fibronectin, vitronectin, etc. [18]. Such technique provides lateral dimensions ranging from tens of micrometer down to hundreds of nanometers making it ideal for control of cell shape. Chen and co-workers applied microcontact printing to analyze the effect of stem cell shape on differentiation. They observed that human MSCs (hMSCs) that adhere, flatten and spread differentiate along the osteogenic lineage, while a restriction in cell size by means of denser culture density or smaller micro-island size

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induces adipogenesis [19]. Such switch in lineage commitment required the generation of cytoskeletal tension through RhoA dependent acto-myosin motors. Upon inhibition of the contractile acto-myosin machinery through an actin depolymerizing agent (cytochalasin) or a Rho kinase inhibitor (Y-27632), an adipogenic phenotype similar to that of less spread cells, was observed. Even in the presence of only osteogenic medium, an inhibition of the RhoA pathway induced adipogenesis, whereas an activated RhoA promoted osteogenesis despite the culture in an adipogenic medium. Thus, the studies provide evidence of RhoA activity as essential for guiding differentiation, and points to its importance of both mechanical and soluble factor signaling. In addition to control of cell spreading, chemical patterning of more complex shapes also has an impact on stem cell differentiation. When culturing hMSCs on microcontact-printed substrates with similar-sized shapes and only small geometric differences, osteogenesis is promoted on shapes with local regions of curvature that cause an increase in cytoskeletal tension (Fig. 2) [20]. However, upon disruption of cytoskeletal function the stem cell loses this ability to sense and respond to substrate shape. Together these studies show that cell shape, RhoA activity and cytoskeletal tension have essential roles in guiding the response of stem cells to its physical surroundings. Nevertheless, it remains to be elucidated whether these factors represent independent regulators, or whether they essentially modulate stem cell response through the same mechanosensitive compartment or molecule within the cell. Given their importance in guiding differentiation, identifying such morphological characteristics as cell shape and cytoskeletal tension may represent an easily applicable method to help predict differentiation of stem cells. It is especially early (within the first 24 h) in culture upon initial attachment and spreading that stem cells undergo significant changes in their cell shape and cytoskeletal distribution as they adapt to the surrounding geometry. In a study by Treiser et al. they investigated whether there was a possible correlation between such early morphological characteristics and stem cell differentiation potential after several weeks in culture [21]. By chemically patterning glass slides with fibronectin in order to control cell spreading, they found that it was not possible to predict lineage commitment based purely on an analysis of traditional cell shape [19]. Instead, higher-order variations in shape and cytoskeletal organization (such as actin fluororeporter shapes, intensities, textures, and spatial distributions), as reflected in image processing and by computational analysis, did provide

distinct expression patterns that correlated with stem cell differentiation commitment (Fig. 3). Their work confirmed the importance of a functional cytoskeleton in guiding shape-dependent differentiation and it provided new insight describing the close correlation between cytoskeleton and osteogenesis. As such, the transient upregulation of a glycerophosphodiester phosphodiesterase molecule localized in close proximity to actin triggered a morphological change in cell shape and an increase in alkaline phosphatase activity indicating that the osteogenic lineage commitment may be encoded within the cytoskeleton itself [21]. Such computer-aided technique helping to foresee and to trace cell proliferation and differentiation has gained significant popularity in recent years [22e25]. Along with advancements in imaging tools, several groups have developed computational methods to analyze dynamic features of cells over time. The methods are able to predict self-renewal or terminal division of stem cells with very high accuracy and have made the data acquisition less tedious and less manually heavy. As such, it is possible to capture not just one but the whole range of cell dynamics in a single frame, and at the same time monitor critical events such as mitosis and cell death. Also, the more advanced computational analysis would minimize the errors associated with currently existing automated tools due to the elimination of visual ambiguity inherent in the captured frames [25]. The new techniques hold promise to provide important insight into developmental biology, and offers a potential tool for precise early detection of specific lineage commitment. This will facilitate a more efficient screening of biomaterials and culture conditions to control stem cell differentiation. Clearly cells cultured on substrates with different chemically patterned shapes can be used to promote the differentiation of stem cells to distinct lineages, this commitment depending on the adhesive area, aspect ratio, and subcellular curvature. These modifications to cell shape affect cytoskeletal tension, which ultimately leads to changes in gene and protein expression. Many other molecular factors affected by size, and involved in regulating differentiation, are still to be identified. 1.4. Topographical patterning for directing stem cell lineage commitment Adopting from fabrication methods developed in the microelectronics industry, researchers have increasingly used sophisticated methods to produce micrometer and more recently

Fig. 2. (A) Myosin IIa, a primary msotorprotein responsible for cell contractility, was immunofluorescently stained and heatmaps from >80cells demonstrated a higher degree of actomyosin contractility on the curved edges of the hollow shape as compared to the circular shape. (B) Corresponding differentiation of cells in the two shapes after exposure to bipotential adipogenic and osteognic media, p < 0.005. Reprinted by permission from Proceedings of the National Academy of Sciences of the United States of America [20].

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B

24-96 h Cells (hMSCs) Gauss Filtered Image

Substrate

Contrast Enhancement

Thresholding to Select AOI’s

Individual Cell Segmentation and Descriptor Calculation

Confocal Imaging Direct Data Entry of Descriptors Based on Cytoskeletal Morphology

D Multi-Dimensional Scaling to Reduce Dimensionality F1= f(d1,d2,d3 ...dn) Dimension 1 F2= f(d1,d2,d3 ...dn) Dimension 2 Dimension 3 F3= f(d1,d2,d3 ...dn)

C

Quantitative Descriptors Based on Actin Morphology Descriptor 1 d1 Descriptor 2 d2 Descriptor 3 d3 . . . . Descriptor n

dn

Fig. 3. The general setup for predicting longer term cell behavior based on early morphometric descriptors of cells. (A) hMSCs were cultured under various media conditions and on different substrates and (B) tile scanned confocal images of cells were obtained. These were subjected to image processing steps (C) providing a list of 43 descriptors of whole cell shape and cytoskeletal distribution. (D) Using multidimensional scaling key combinations of the 43 descriptors were reduced into three dimensions to visualize data. Reprinted with permission of Proceedings of the National Academy of Sciences of the United States of America [21].

nanometer-scale topographical (micro and nanotopography) patterns with features such as pillars, grooves and pits [3,17] for cellular studies. Common techniques include photolithography and electron beam lithography (EBL), the latter allowing for the production of feature sizes down to approximately 5 nm [26]. Injection molding and nanoimprint lithography provide methods for replication of master topographies in a fast and inexpensive manner. Yet other techniques include polymer phase separation and colloidal lithography, although both producing more random topographies [27]. Photolithography has been used to pattern microwells for control of ESC culture size. ESCs provide a powerful source of stem cells due to their ability to renew and differentiate into lineages of all three germ layers. However, current culturing conditions, such as growing them in 3D clusters forming embryoid bodies (EBs), are deemed suboptimal because their differentiation pattern is largely heterogenic. It is speculated that one major cause of variability in differentiation pattern is inconsistency in the initial size of ESC aggregates. In an attempt to overcome this, microwells of different dimensions to control ESC aggregate size have been fabricated [28e31]. Such constraint allows for control of EB size and shape, which in turn has a major impact on differentiation. In a study by Hwang et al. microwells of 150, 300 and 450 mm in diameter were fabricated by a micromolding procedure in order to restrict EB size. Cardiogenesis was enhanced in larger EBs (450 mm in diameter), while endothelial cell differentiation was increased in smaller EBs (150 mm in diameter) [28]. Between the two different well sizes a significant differential expression of WNT signaling family molecules was found. The smaller EBs expressed high levels of WNT5a but no WNT11, while larger EBs expressed WNT11 and highly reduced levels of WNT5a. Such differential expression of specific WNT molecules supports previous studies that have identified the WNT signaling pathway as having an essential role in

vascular and cardiac tissue development [32]. Across these experiments, WNT5a also supported endothelial cell proliferation and differentiation [33,34], and WNT11 promoted cardiac development [35,36]. Despite the strong correlation between lineage commitment and specific WNT molecule expression, it remains to be elucidated how WNT5a and WNT11 cooperate and how they interact with other WNT molecules. As such, differentiation could likely be modulated through the same size-dependent signaling molecule, with WNT5a and WNT11 not representing two separate regulators of differentiation. In a similar study with microwells ranging in diameter between 100 and 500 mm, Mohr et al. investigated its effect on the differentiation of hESCs to cardiomyocytes. They showed that 300 mm microwells had the highest percentage of spontaneously contracting EBs, while smaller microwells of 100 mm had fewer EBs, but with each containing a larger fraction of differentiated cardiomyocytes [31]. This influence of well size on differentiation likely involved a plethora of different factors. Hwang et al. identified the importance of the WNT signaling pathway [28], while present authors suggested that limitation to passive diffusion could well be a major factor influencing differentiation. Hence, the decreased fraction of cardiomyocytes found as EB size increased could be due to a reduction in cardiac promoting molecules reaching the inner cell mass. Another possible explanation for the altered cardiac differentiation may be due to a change in gradients of signaling factors across the germ layers of the EBs. Thus, from embryonic development of the human heart with germ layers similar to those of cultured EBs, tightly controlled gradients have proven essential for normal growth. Here, WNT signals from the adjoining neuroectodermal germ layer inhibits cardiac growth, while in contrast BMPs released from the anterior endoderm promote its development [37e40]. From a simple geometric consideration the relative size of such germ layers is altered as EB size is changed, likely affecting growth factor gradients and

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Fig. 4. (aec): GFP expressing adult neural stem cells (ANSCs) localized in a random fashion on the planar control (a) and on random fibers (c), while they oriented specifically in the direction of the aligned fibers (b). (def): After five days in neurogenic inducing media cell nuclei were immunostained with SYTOX Green and early neurons with the immature neuronal marker anti-Tuj1 antibody (red). Most Tuj1þ cells on the aligned fibers (e) had neurites extending along the axis of the fiber that were several times the length of the cell body, while cells on the random fibers (f) showed no specific orientation of the neurite esxtensions. No significant neurite growth was seen on the planar surface (d). Also, all aligned substrates yielded greater fractions of Tuj1 positive cells compared with the random fibers and the planar substrate (see [58], Fig. 3, for details). Scale bar ¼ 50um. Reprinted by permission from Biomaterials Journal [58].

ultimately cardiogenesis. The influence of both limitations to diffusion and altered growth factor gradients needs to be investigated. Utilizing EBL and hot embossing to produce nanoscale imprints in polymethylmethacrylate (PMMA) samples Dalby et al. provided evidence that stem cells also sense and respond to topographical patterns with nanometer dimensions [41], findings which have been extended in more recent studies [42e46]. Specifically, substrates composed of nanopits with a controlled disorder resulted in increased osteogenesis, relative to substrates consisting of either highly ordered or randomly placed nanopits with otherwise similar dimensions. Such increase in substrate-induced stem cell differentiation is likely a result of strength of attachment to the surface. For cells on surfaces with a controlled disorder of nanopits, more mature long fibrillar-like adhesions were found, leading to an increased cytoskeletal tension relative to that induced by the shorter adhesions formed by cells on the other surfaces. This cytoskeletal tension has likely mediated mechanical forces to the nucleus and altered the mechanosensitive pathways, which together promoted osteogenesis resulting in the observed upregulation of bone markers osteopontin, osteocalcin and alkaline phosphatase. However, the underlying mechanism behind topography-mediated cellular behavior (here osteogenesis) has only been studied by few. Seo et al. investigated the role of the rhoassociated kinase (ROCK) and downstream myosin II. When culturing cells on microstructured surfaces, they observed an increase in focal adhesion size relative to cells cultured on a flat substrate. In order to reduce the focal adhesion maturation known to regulate numerous intracellular signals, a ROCK inhibitor was added [47]. This resulted in reduced focal adhesion maturation,

actin activation and a decrease in FAK phosphorylation, ultimately altering the gene response induced by the topography. The mechanosensitive FAK, along with Src and ERK, present the best studied kinases which intact function seems essential in order to induce a topography-mediated cell response [33,48,49]. This ability to alter stem cell differentiation through nanoscale topography also accounts for substrates consisting of nanotubes with varying diameter. Oh et al. showed that culturing human MSCs (hMSCs) on a patch of TiO2 nanotubes with a diameter of 30 nm promoted their proliferation, while 100 nm nanotubes selectively differentiated the hMSCs along the osteogenic lineage [45]. Contrary to this, Park et al. showed that nanotubes of the same dimensions promoted osteogenic differentiation of rat MSCs (rMSCs) on small 15 nm diameter nanotubes in the presence of osteoinductive medium, while any differentiation was inhibited on 100 nm nanotubes [46]. These apparently conflicting results highlight the general challenge found in the pursuit to control stem cell differentiation and emphasize the importance of establishing common standard lab protocols, as even small variations in the experimental parameters may result in opposing results. Besides osteogenic differentiation, micro and nanotopographical patterns have been widely studied for their ability to promote neurogenesis. This lineage specification was initially thought to be restricted to ESCs and neural stem cells until work published by Woodbury et al., which suggested that rat and human bone marrow-derived MSCs might have the ability to differentiate into neuron-like cells, a non-mesenchymal derivative [50]. Such transdifferentiation has been shown to be induced by either neuronal induction medium [50e53] and/or cell contact with neurons [54]. However, the exact mechanism is not well

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understood and both cell source and culture conditions appear to be crucial to reproduce the result. Nevertheless, it has been found that grating structures are especially beneficial in promoting neurogenesis of ESCs and neural stem cells [43,55], as well as mesenchymal stem cells [42,56]. Typically, cells on these surfaces align along the grating, with the degree of elongation strongly dependent on feature depth and width. This alters the cytoskeletal distribution and tension, ultimately leading to a general increase in neurogenesis. Another substrate type with a similar effect is electrospun nanofiber meshes consisting of aligned fibers. Upon use of uniaxially aligned poly(3-caprolactone) (PCL) nanofibers an upregulation of neuronal markers has been observed, and the fibers were found to support and direct neural outgrowth (Fig. 4). Interestingly, no differentiation or guidance of neural growth was observed on randomly oriented fibers [57,58]. On grating structures both the cytoskeleton and the connected nucleus is strongly elongated. This is found to induce an increase in cytoskeletal tension mediated by integrin dependent cell adhesion, and likely causes direct changes to the organization of the nuclear components. Such reorganization affects gene expression, which then favors the observed neurogenesis [42,59,60]. Also, tension indirectly affects a complex cascade of intracellular signaling events. However, only little is presently known about the detailed molecular changes involved. These are, as already discussed, known to include a range of kinases such as FAK, Src and ERK, which affect downstream molecules and ultimately gene expression. Other studies have provided evidence that certain actin-associated proteins, colocalized at the cell adhesion complex, are mechanosensitive [61e63]. For example, zyxin increases its unbinding rate constant when a tensional force is applied to a cell (such as that caused by grating structures), and when released translocates to the nucleus and alters transcription of genes [61]. Future research is needed to identify more of such intracellular molecular interactions. In summary, research has shown that both precise ordering and the dimensions of the micro and nanoscale topographical patterns influence stem cell differentiation. For example, controlling the size of EBs by growing them in microwells with specific dimensions provides a more homogenous differentiation of ESCs. The specification of size alters the regulation of molecules in the WNT signaling pathway and directs the stem cell lineage commitment. Likewise, the alignment of nanofibers seems more beneficial than randomly organized nanofibers for directing neurogenic differentiation. Such results are likely caused by the ability and the organization of the nanofeatures to influence focal adhesion density and distribution, altering the cytoskeletal arrangement and ultimately introducing changes to gene and protein expression. The cascade of molecular mechanisms, and the combined effect of multiple factors that may affect differentiation, are only beginning to be understood. 1.5. High throughput screening of biomaterials for control of stem cells It is clear that the micro and nanoscale patterning of surfaces has a significant influence on stem cells. Nevertheless, it still remains challenging to obtain guidelines to design a biomaterial that will provide the necessary biological cues for directing stem cells. This is due to the multidimensional space of possible patterns and also the influence of other physical and chemical signals on the cellematerial interaction. A beneficial direction to elucidating these effects may be through a more systematic screening approach [64]. Such high-throughput methods have been used for drug discovery in the pharmaceutical industry for almost a decade, and are just recently being applied in biomaterials research. In an early study utilizing such method Anderson et al. identified the proper polymer composition for stem cell growth and differentiation out of 576

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synthesized polymers in nanoliter volumes [65]. Later, Bhat et. al. and Mei. et al. fabricated gradient structures altering the composition of the material blend to identify the biological responses of cells along a gradually changing surface [66,67]. Acknowledging the importance of the physical environment surrounding the stem cells, researchers have also evaluated a range of substrate topographical patterns. Washburn et al. investigated the influence of a range of nanoroughness on osteoblast proliferation and found that cells grew at a higher rate on smooth compared to rough regions [68]. Lovmand et al. demonstrated how a combinatorial approach enabled the identification of optimal surface micrometer topography for osteogenic differentiation by a preosteoblastic murine cell line [69]. Following the same approach, Markert et al. found that increasing the vertical feature dimension (pillar height) from 0.6 to 2.4 mm and setting the lateral dimensions to a feature width of 1 mm and a feature gap size of 2 or 4 mm, increased the number of undifferentiated colonies of murine ESCs [70]. It is well established that the nano- and micrometer topography exerts its effect on cells through different mechanisms. The nanoroughness explored by Washburn et al. influences protein adsorption, which in turn contributes to differences in cell behavior. Conversely, such micrometer topographies investigated by Lovmand et al. and Market et al. does not affect protein conformation, but more likely enforces differences in focal adhesion assembly and maturation, known to play a role in cell behavior. Such uses of high-throughput approaches allows us to quickly screen a variety of surface types, and track trends in how cells react to small changes in topography. The identified structures may then be explored further by, e.g. qPCR and proteomics approaches [71]. A future advance to increase the power of high-throughput screens would likely involve more computer-aided designs. Jan de Boer and co-workers recently developed a platform with thousands of topographies to screen for cellular response, which is practically impossible by conventional means [72]. With their computer-based method they performed high-content imaging of cells on all surfaces and correlated their behavior with parameters of the mathematical algorithms. This allowed them to extract novel design criteria for surface topographies that induced MSC proliferation and osteogenic differentiation. Such approach may further accelerate the discovery of optimal combinations of cues to direct stem cell fate decision. 2. Conclusion Micro and nanoscale surface patterning provides a valuable tool to examine how cellesubstrate interactions might influence stem cell expansion and differentiation, and holds promise as a novel modality to direct stem cell behavior. Studies have shown that surfaces can be chemically patterned in order to control cell shape, and that topographical features can act on cell alignment, adhesion and cytoskeletal organization. Such factors change the cell behavior through mechanical interaction, inducing a range of signaling cascades. Accompanied by a physical pulling of the cytoskeleton that subsequently rearranges the nuclear components, such indirect and direct mechanotransduction alters gene and protein level response of the cells to the patterns. This approach for directing stem cell lineage commitment may provide a stable and reliable scheme of culturing cells over extended periods of time. However, the molecular interactions and signaling cascades involved in substrate-induced differentiation are only just beginning to be unraveled. Advancements in bioimaging and bridging of cell and molecular biology with computational analysis hold promise to accelerate progress in mapping lineage tracing and cell dynamics to direct cell fate. Such understanding will allow for research to carefully design substrates to direct stem cell differentiation either alone or in combination with other intrinsic and/or extrinsic cues.

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