Geometrically defined environments direct cell division rate and subcellular YAP localization in single mouse embryonic stem cells.

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell-cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

Actomyosin contractility and RhoGTPases affect cell-polarity and directional migration during haptotaxis.

Although much is known about chemotaxis- induced by gradients of soluble chemical cues - the molecular mechanisms involved in haptotaxis (migration induced by substrate-bound protein gradients) are largely unknown. We used micropatterning to produce discontinuous gradients consisting of µm-sized fibronectin-dots arranged at constant lateral but continuously decreasing axial spacing. Parameters like gradient slope, protein concentration and size or shape of the fibronectin dots were modified to determine optimal conditions for directional cell migration in gradient patterns. We demonstrate that fibroblasts predominantly migrate uphill towards a higher fibronectin density in gradients with a dot size of 2 × 2 µm, a 2% and 6% slope, and a low fibronectin concentration of 1 µg ml-1. Increasing dot size to 3.5 × 3.5 µm resulted in stationary cells, whereas rectangular dots (2 × 3 µm) orientated perpendicular to the gradient axis preferentially induce lateral migration. During haptotaxis, the Golgi apparatus reorients to a posterior position between the nucleus and the trailing edge. Using pharmacological inhibitors, we demonstrate that actomyosin contractility and microtubule dynamics are a prerequisite for gradient recognition indicating that asymmetric intracellular forces are necessary to read the axis of adhesive gradients. In the haptotaxis signalling cascade, RhoA and Cdc42, and the atypical protein kinase C zeta (aPKCζ), but not Rac, are located upstream of actomyosin contractility.

Hierarchical Micro-Nano Surface Topography Promotes Long-Term Maintenance of Undifferentiated Mouse Embryonic Stem Cells.

Understanding of stem cell-surface interactions and, in particular, long-term maintenance of stem cell pluripotency on well-defined synthetic surfaces is crucial for fundamental research and biomedical applications of stem cells. Here, we show that synthetic surfaces possessing hierarchical micro-nano roughness (MN-surfaces) promote long-term self-renewal (>3 weeks) of mouse embryonic stem cells (mESCs) as monitored by the expression levels of the pluripotency markers octamer-binding transcription factor 4 (Oct4), Nanog, and alkaline phosphatase. On the contrary, culturing of mESCs on either smooth (S-) or nanorough polymer surfaces (N-surfaces) leads to their fast differentiation. Moreover, we show that regular passaging of mESCs on the hierarchical MN-polymer surface leads to an increased homogeneity and percentage of Oct4-positive stem cell colonies as compared to mESCs grown on fibroblast feeder cells. Immunostaining revealed the absence of focal adhesion markers on all polymer substrates studied. However, only the MN-surfaces elicited the formation of actin-positive cell protrusions, indicating an alternative anchorage mechanism involved in the maintenance of mESC stemness.

Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, ß1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

Subcellular stretch-induced cytoskeletal response of single fibroblasts within 3D designer scaffolds.

In vivo, cells are exposed to mechanical forces in many different ways. These forces can strongly influence cell functions or may even lead to diseases. Through their sensing machinery, cells are able to perceive the physical information of the extracellular matrix and translate it into biochemical signals resulting in cellular responses. Here, by virtue of two-component polymer scaffolds made via direct laser writing, we precisely control the cell matrix adhesions regarding their spatial arrangement and size. This leads to highly controlled and uniform cell morphologies, thereby allowing for averaging over the results obtained from several different individual cells, enabling quantitative analysis. We transiently deform these elastic structures by a micromanipulator, which exerts controlled stretching forces on primary fibroblasts grown in these scaffolds on a subcellular level. We find stretch-induced remodeling of both actin cytoskeleton and cell matrix adhesions. The responses to static and periodic stretching are significantly different. The amount of paxillin and phosphorylated focal adhesion kinase increases in cell matrix adhesions at the manipulated pillar after static stretching whereas it decreases after periodic stretching.

Multifunctional polymer scaffolds with adjustable pore size and chemoattractant gradients for studying cell matrix invasion.

Transmigrating cells often need to deform cell body and nucleus to pass through micrometer-sized pores in extracellular matrix scaffolds. Furthermore, chemoattractive signals typically guide transmigration, but the precise interplay between mechanical constraints and signaling mechanisms during 3D matrix invasion is incompletely understood and may differ between cell types. Here, we used Direct Laser Writing to fabricate 3D cell culture scaffolds with adjustable pore sizes (2-10 µm) on a microporous carrier membrane for applying diffusible chemical gradients. Mouse embryonic fibroblasts invade 10 µm pore scaffolds even in absence of chemoattractant, but invasion is significantly enhanced by knockout of lamin A/C, a known regulator of cell nucleus stiffness. Nuclear stiffness thus constitutes a major obstacle to matrix invasion for fibroblasts, but chemotaxis signals are not essential. In contrast, epithelial A549 cells do not enter 10 µm pores even when lamin A/C levels are reduced, but readily enter scaffolds with pores down to 7 µm in presence of chemoattractant (serum). Nuclear stiffness is therefore not a prime regulator of matrix invasion in epithelial cells, which instead require chemoattractive signals. Microstructured scaffolds with adjustable pore size and diffusible chemical gradients are thus a valuable tool to dissect cell-type specific mechanical and signaling aspects during matrix invasion.