Karina Kulangara

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Youngs moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Nanotopography as Modulator of Human Mesenchymal Stem Cell Function

Nanotopography changes human mesenchymal stem cells (hMSC) from their shape to their differentiation potential; however little is known about the underlying molecular mechanisms. Here we study the culture of hMSC on polydimethylsiloxane substrates with 350 nm grating topography and investigate the focal adhesion composition and dynamics using biochemical and imaging techniques. Our results show that zyxin protein plays a key role in the hMSC response to nanotopography. Zyxin expression is downregulated on 350 nm gratings, leading to smaller and more dynamic focal adhesion. Since the association of zyxin with focal adhesions is force-dependent, smaller zyxin-positive adhesion as well as its higher turnover rate suggests that the traction force in focal adhesion on 350 nm topography is decreased. These changes lead to faster and more directional migration on 350 nm gratings. These findings demonstrate that nanotopography decreases the mechanical forces acting on focal adhesions in hMSC and suggest that force-dependent changes in zyxin protein expression and kinetics underlie the focal adhesion remodeling in response to 350 nm grating topography, resulting in modulation of hMSC function.

Dynamic Topographical Control of Mesenchymal Stem Cells by Culture on Responsive Poly(ϵ‐caprolactone) Surfaces

There is clear, emerging evidence in the literature supporting the influence of surface topography on various cell phenotypes.[1–5] Recent advancements in mechanobiology have relied heavily on synthetic extracellular matrix (ECM) mimics to investigate how cellular phenomena are dependent upon surface geometry. Concurrent developments in micro/nano-fabrication techniques have enabled the construction of well-defined surface arrays which aim to emulate the extracellular microenvironment.[6] Numerous patterns of different sizes and shapes including grooves, posts, and pits have been used to study the in vitro response of various cell types such as: fibroblasts, osteoblasts, epithelial cells, neuronal cells, and more recently stem cells.[7–14]

Substrate topography shapes cell function

Influencing cell behavior from proliferation to differentiation using substrate or implant topography is an attractive strategy for regenerative medicine applications. Substrate topography at the submicron range is of particular interest because the size range is comparable to extracellular matrix structures. Emerging literature presents many interesting findings on how nanotopography enhances cell adhesion, alters cell morphology, affects proliferation, initiates intracellular signaling, provides contact guidance and mediates stem cell differentiation. Incorporating topographical consideration into the design of a biomimetic microenvironment for cell culture will become increasingly important in light of these studies and practical with advances in nanofabrication technologies. This Highlight underscores the promise of and the unknown information about topographical effects in manipulating cell-substrate interaction and advancing tissue engineering.

Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells.

Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm−2 doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1+ (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP+ cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.

Effects of topographical and mechanical property alterations induced by oxygen plasma modification on stem cell behavior.

Polymeric substrates intended for cell culture and tissue engineering are often surface-modified to facilitate cell attachment of most anchorage-dependent cell types. The modification alters the surface chemistry and possibly topography. However, scant attention has been paid to other surface property alterations. In studying oxygen plasma treatment of polydimethylsiloxane (PDMS), we show that oxygen plasma treatment alters the surface chemistry and, consequently, the topography and elasticity of PDMS at the nanoscale level. The elasticity factor has the predominant effect, compared with the chemical and topographical factors, on cell adhesions of human mesenchymal stem cells (hMSCs). The enhanced focal adhesions favor cell spreading and osteogenesis of hMSCs. Given the prevalent use of PDMS in biomedical device construction and cell culture experiments, this study highlights the importance of understanding how oxygen plasma treatment would impact subsequent cell-substrate interactions. It helps explain inconsistency in the literature and guides preparation of PDMS-based biomedical devices in the future.

Deformation of stem cell nuclei by nanotopographical cues

Cells sense cues in their surrounding microenvironment. These cues are converted into intracellular signals and transduced to the nucleus in order for the cell to respond and adapt its function. Within the nucleus, structural changes occur that ultimately lead to changes in the gene expression. In this study, we explore the structural changes of the nucleus of human mesenchymal stem cells as an effect of topographical cues. We use a controlled nanotopography to drive shape changes to the cell nucleus, and measure the changes with both fluorescence microscopy and a novel light scattering technique. The nucleus changes shape dramatically in response to the nanotopography, and in a manner dependent on the mechanical properties of the substrate. The kinetics of the nuclear deformation follows an unexpected trajectory. As opposed to a gradual shape change in response to the topography, once the cytoskeleton attains an aligned and elongation morphology on the time scale of several hours, the nucleus changes shape rapidly and intensely.

Nanotopography alters nuclear protein expression, proliferation and differentiation of human mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells respond to physical cues present in their microenvironment such as substrate elasticity, geometry, or topography with respect to morphology, proliferation, and differentiation. Although studies have demonstrated the role of focal adhesions in topography-mediated changes of gene expression, information linking substrate topography to the nucleus remains scarce. Here we show by two-dimensional gel electrophoresis and western blotting that A-type lamins and retinoblastoma protein are downregulated in mesenchymal stem/stromal cells cultured on 350 nm gratings compared to planar substrates; these changes lead to a decrease in proliferation and changes in differentiation potential.

Stem cell differentiation indicated by noninvasive photonic characterization and fractal analysis of subcellular architecture

We hypothesised that global structural changes in stem cells would manifest with differentiation, and that these changes would be observable with light scattering microscopy. Analysed with a fractal dimension formalism, we observed significant structural changes in differentiating human mesenchymal stem cells within one day after induction, earlier than could be detected by gene expression profiling. Moreover, light scattering microscopy is entirely non-perturbative, so the same sample could be monitored throughout the differentiation process. We explored one possible mechanism, chromatin remodelling, to account for the changes we observed. Correlating with the staining of HP1α, a heterochromatin protein, we applied novel microscopy methods and fractal analysis to monitor the plastic dynamics of chromatin within stem cell nuclei. We showed that the level of chromatin condensation changed during differentiation, and provide one possible explanation for the changes seen with the light scattering method. These results lend physical insight into stem cell differentiation while providing physics-based methods for non-invasive detection of the differentiation process.