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. 2021 Jan 29;8(6):2003186.
doi: 10.1002/advs.202003186. eCollection 2021 Mar.

Precision Surface Microtopography Regulates Cell Fate via Changes to Actomyosin Contractility and Nuclear Architecture

James Carthew  1   2 , Hazem H Abdelmaksoud  3   4 , Margeaux Hodgson-Garms  1 , Stella Aslanoglou  4   5   6 , Sara Ghavamian  2   3 , Roey Elnathan  1   4   5 , Joachim P Spatz  7   8   9 , Juergen Brugger  10 , Helmut Thissen  6 , Nicolas H Voelcker  1   4   5   6 , Victor J Cadarso  2   3   4 , Jessica E Frith  1
Affiliations

Precision Surface Microtopography Regulates Cell Fate via Changes to Actomyosin Contractility and Nuclear Architecture

James Carthew et al. Adv Sci (Weinh). .

Abstract

Cells are able to perceive complex mechanical cues from their microenvironment, which in turn influences their development. Although the understanding of these intricate mechanotransductive signals is evolving, the precise roles of substrate microtopography in directing cell fate is still poorly understood. Here, UV nanoimprint lithography is used to generate micropillar arrays ranging from 1 to 10 µm in height, width, and spacing to investigate the impact of microtopography on mechanotransduction. Using mesenchymal stem cells (MSCs) as a model, stark pattern-specific changes in nuclear architecture, lamin A/C accumulation, chromatin positioning, and DNA methyltransferase expression, are demonstrated. MSC osteogenesis is also enhanced specifically on micropillars with 5 µm width/spacing and 5 µm height. Intriguingly, the highest degree of osteogenesis correlates with patterns that stimulated maximal nuclear deformation which is shown to be dependent on myosin-II-generated tension. The outcomes determine new insights into nuclear mechanotransduction by demonstrating that force transmission across the nuclear envelope can be modulated by substrate topography, and that this can alter chromatin organisation and impact upon cell fate. These findings have potential to inform the development of microstructured cell culture substrates that can direct cell mechanotransduction and fate for therapeutic applications in both research and clinical sectors.

Keywords: mechanotransduction; mesenchymal stem/stromal cells; microtopography; osteogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Fabrication of high resolution micropatterned substrates. a) Schematic representation of microfabricated grid platforms containing micropillar designs; i) Blank (flat controls), 1 × 5 (yellow), 2.5 × 5 (purple) and 5 × 5 (orange), or ii) Blank (flat control), 5 × 5 (orange), 7.5 × 5 (blue), and 10 × 5 (red). b) Scanning electron micrographs of micropatterned designs. Grid designs are described in the format of micropillar width and spacing × micropillar height. c) Scanning electron micrographs of micropillars in cross‐section. Heights range from 1 to 10 µm. d) Representative false coloured scanning electron micrographs highlighting phenotypic differences between MSCs cultured on i) flat (control) substrates and ii) micropatterned substrates. Scale bar, 5 µm unless otherwise stated.
Figure 2
Figure 2
Characterization of MSC phenotype in response to substrate microtopography. a) Representative images of MSCs showing actin (red), microtubules (green), and nuclei (blue) on flat (control) and micropatterned substrates. Scale bar, 20 µm. b) Cell area and c) cell aspect ratio quantification on each micropattern design tested. d,e) Gene expression levels for vinculin and PTK2 (FAK) respectively as determined via RT‐PCR. f) Western blots of cytoskeletal and focal adhesion components in MSCs cultured on flat and micropatterned substrates. g) Representative immunofluorescent images of focal adhesions on 5 × 5 µm substrate designs, depicted with actin (red), vinculin (green), and nuclei (blue). Scale bars, 10 µm. All micropattern designs represent consistent micropillar heights of 5 µm. All graphs show mean ± SD for three independent MSC donors relative to control samples. Samples were analysed by one‐way ANOVA with Tukey post hoc testing. Statistically different samples are denoted by *p < 0.05, and **p < 0.005.
Figure 3
Figure 3
Characterization of nuclear indentation on micropatterned surfaces. a) Representative fluorescence microscopy images of MSC nuclear phenotypes on 5 × 5 micropatterned substrates, depicting actin (green) and nuclei (blue). Scale bar, 20 µm. b) Confocal z‐stacks demonstrating nuclear “hole” extending through the nuclear compartment. c) Schematic representation of two potential types of nuclear deformation, either nuclear indentation (in which the micropillar deforms the nuclear envelope, thus displacing genetic material), or nuclear perforation (in which a new nuclear envelope is formed around the micropillar to produce a doughnut shaped nucleus). Cytosol is shown in red, genetic material in blue, and nuclear envelope in green. d). Representative images of lamin A/C (green), DNA (blue), and actin (red). Asterisks demonstrate regions of lamin A/C accumulation on the surface of micropillars and corresponding regions of DNA displacement respectively. Arrows denote regions of peripheral nuclear envelope accumulation of lamin A/C. Scale bar, 5 µm. e) Quantification of lamin A/C staining on flat and the 5 × 5 substrates, respectively. f) Representative false‐coloured FIB/SEM image of MSCs cultured on 5 × 5 substrates. Micropillars are colored red, with nuclei colored blue. Scale bar, 5 µm g) RT‐PCR and h) Western blotting analysis of LMNA and lamin A/C expression profiles respectively across flat, 1 × 5, 5 × 5 and 10 × 5 micropillars. Data was collected from three independent replicates across three MSC donors. RT‐PCR data is presented as mean ± SD relative to TCP control samples.
Figure 4
Figure 4
Micropattern‐defined nuclear indentation regulates heterochromatin expression and DNMT activity. a) Representative lamin A/C (green) and nuclei (blue) staining demonstrating phenotypes resulting from varied micropatterned spacing and width. Scale bar, 5 µm. b) Nuclear phenotype categories and associated quantification with changing c) micropillar height or d) micropillar width/spacing. e) Fluorescence staining of actin (red), nuclei (blue), and H3K9 (heterochromatin marker (green)). Scale bar, 5 µm. f) Mean fluorescent intensity quantification of H3K9 expression with changing micropillar width and spacing. g) Western blotting of H3K9 in MSCs cultured on control substrates and micropatterns with a constant height of 5 µm. h,i,j) RT‐PCR analysis of DNMT1, DNMT3a, and DNMT3b, respectively for each pattern at a constant 5 µm micropillar height. All graphs show mean ± SD for three independent MSC donors relative to TCP samples. Samples were analyzed by one‐way ANOVA with Tukey post hoc testing. Statistically different samples are denoted by *p < 0.05, **p < 0.01, and ****p < 0.001.
Figure 5
Figure 5
Micropatterned substrates enhance osteogenic capacity of MSCs. a) Representative mineralization images of MSCs, depicting nuclei (blue) and calcium deposition (red) on flat (control) and micropatterned substrates. Scale bar, 50 µm. b) Quantification of mineral generation per cell for 5 µm micropillar heights. c) Quantification of total cell number following 21 day osteogenic differentiation. d,e) Representative YAP fluorescence staining and quantification on flat and micropatterned substrates maintained at a constant micropillar height of 5 µm. Scale bar, 10 µm. f) Calculated force required to bend each micropillar by 1 µm, depicting forces applied at the top and middle of the micropillar. Graphs (b), (c), and (d) show mean ± SD from 300 cells for three hMSC donors. Samples were analyzed by one‐way ANOVA with Tukey post hoc testing. Statistically different samples are denoted by ****p < 0.001.
Figure 6
Figure 6
Nuclear indentations and associated enhanced osteogenic capacity of MSCs are determined by myosin II‐dependent actin contractility. a) Representative fluorescence microscopy images of MSCs treated with actin and microtubule inhibitory drugs during culture on the 5 × 5 micropatterned design, with actin (red), microtubules (green), and nuclei (blue). Arrows denote regions of nuclear indentation and DNA displacement around the micropillars. Scale bar, 10 µm. b) Cell area and c) associated nuclear phenotype quantification of MSCs following drug treatment on the 5 × 5 micropillars. d) Quantification of mineralization at 21 days of osteogenesis in MSCs treated with actin and microtubule inhibitory drugs during culture on the control and 5 × 5 micropatterned design. All graphs show mean ± SD from across 300 cells for three independent MSC donors relative to control samples. Samples were analyzed by one‐way ANOVA with Tukey post hoc testing for comparisons within either Flat or 5 × 5 conditions. For comparisons between flat and 5 × 5 conditions, t‐tests were performed. Statistically different samples are denoted by **p < 0.01, ****p < 0.001.
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