Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells
Introduction
Cells are normally embedded in a complex microenvironment of specific topography and matrix elasticity [1], [2], [3], [4]. The elastic modulus of extracellular matrix (ECM), also known as Young's modulus, ranges from 0.1 kPa in brain, some few kPa in fat tissue, to several hundred MPa in calcified bone [5], [6]. In contrast, in vitro cell culture is usually performed on very rigid polystyrene surfaces of several GPa. Various studies have indicated that substrate elasticity is an important regulator for cellular function and these were mostly performed on polyacrylamide hydrogels [5], [7], [8]. However, changes in topography upon surface creasing of polyacrylamide substrates can significantly influence cell behavior [9]. Alternatively, elastomers of cross-linked polydimethylsiloxane (PDMS), a viscoelastic organic polymer, display rather smooth surfaces [10]. PDMS is widely used in biomedical engineering and cell culture due to good biocompatibility, optical transparency and tunable stiffness – it is therefore a suitable substrate for mechanotransduction experiments [11].
Effects of matrix elasticity were often addressed in mesenchymal stem cells (MSCs). These cells comprise a multipotent subset capable of differentiation potential towards osteocytes, chondrocytes and adipocytes and they are concurrently tested in a wide variety of clinical trials [12], [13]. It has been suggested that matrix elasticity alone can direct MSC faith: soft (0.1–1 kPa), intermediate (8–17 kPa), and rigid (>34 kPa) polyacrylamide hydrogels supported differentiation towards neurogenic, myogenic and osteogenic lineages, respectively [5]. MSCs reveal directed migration towards stiffer substrates, called durotaxis [14]. Cell adhesion, cell spreading, and proliferation rates are significantly increased on rigid biomaterials [8], [15], [16]. It has even been suggested that stiffness gradients, rather than stiffness alone, might be crucial for regulation of MSC behavior [14]. This is of particular relevance for tissue engineering approaches with MSCs embedded in matrix or scaffolds of specific elasticity. If matrix elasticity also entails persistent lineage conversion it would be relevant for generation of tailored cell products, too.
Cellular differentiation is reflected by the epigenetic makeup. DNA methylation (DNAm) is the best characterized epigenetic modification: cytosine guanine dinucleotides (CpGs) can be methylated at cytosine moieties and this governs chromatin structure and gene regulation [17]. It is yet unclear if matrix elasticity impacts on DNAm profiles – which would be expected if matrix elasticity induces persistent cell-intrinsic modifications.
All primary cells enter replicative senescence after a certain number of cell divisions, the so called Hayflick-Limit [18]. This process is accompanied by large-spread morphology, loss of differentiation potential, and unequivocal proliferation arrest [19], [20], [21], [22]. We have demonstrated that long-term culture of MSCs is associated with highly reproducible modifications in the DNAm pattern, particularly in developmental genes [23], [24]. Notably, these senescence-associated DNAm changes are reversed upon reprogramming of MSCs into induced pluripotent stem cells (iPSCs) – and iPSCs do not reveal replicative senescence while in pluripotent state [25]. Recently, it has been reported that tissue stiffness and stress increase lamin-A levels, which stabilizes the nucleus [26]. Furthermore, hypomethylation upon long-term culture is enriched in lamin-associated domains [27] (Hänzelmann S. et al., manuscript in preparation). Hence it might be speculated that substrate stiffness, which alters cellular and nuclear morphology, contributes to cellular senescence.
In this study, we have continuously culture expanded MSCs on PDMS of different Young's modulus until they reached replicative senescence. Thereby, we wanted to determine if matrix elasticity affects cellular aging, whether it induces persistent lineage commitment, and if this is also reflected on DNAm level.
Section snippets
Fabrication of elastomeric substrates
Polydimethylsiloxan substrates (Sylgard 184 Silicone Elastomer Kit, Dow Corning, MI, USA) were generated by mixing base and cross linker at ratios of 70:1 (1.5 kPa), 60:1 (6.5 kPa), 50:1 (15 kPa), 40:1 (50 kPa) and 30:1 (100 kPa). The mixture was then degassed and 2 ml were added in wells of 6-well plates. Cross-linking was performed at 60 °C for 16 h. Characterization of elastomer material properties were performed by stretching cylindrical test pieces and macroscopic indentation tests as
Elasticity and replicative senescence of MSCs
Mesenchymal stem cells were isolated from adipose tissue and continuously cultured in parallel either on TCP or on PDMS of different stiffness (1.5 kPa, 6.5 kPa, 15 kPa, 50 kPa and 100 kPa). Scanning electron microscopy revealed that PDMS substrates were evenly flat and cells attached on this substrate (Fig. 1A). Initial colony formation was higher on TCP than on PDMS (Fig. 1B). After 7 days (passage 0) all cell preparations displayed the typical immunophenotypic pattern of MSCs (CD14-, CD29+,
Discussion
It is commonly accepted that matrix elasticity has major impact on cellular function. So far, this has been particularly addressed by seeding of cells on different substrates and functional comparison whilst on the substrate [5]. Here, we describe that elasticity of PDMS has no sustained effect on replicative senescence, cell-intrinsic lineage commitment, or DNAm profiles of cells which are continuously cultured on these substrates.
Tissue culture plastic is biofunctionalized by plasma treatment
Conclusions
Mesenchymal stem cells which are continuously cultured on the TCP or PDMS of different elasticity reveal very similar cell-intrinsic differentiation potential, gene expression profiles, and DNAm profiles. Furthermore, we demonstrate that substrate elasticity does not affect replicative senescence and it has relatively little impact on the DNAm profiles even on the very soft hPL-gel. This indicates that neither specific subpopulations are selected by the different biomaterials – which might be
Acknowledgments
The authors would like to thank Sonja Hänzelmann (Computational Biology Lab, IZKF, RWTH Aachen University) for support in graphical presentation of gene expression results. This work was supported by the German Research Foundation (DFG; WA 1706/3-2), within the Boost-Fund Project “MechCell” of the excellence initiative of RWTH Aachen University, by the Else Kröner-Fresenius Stiftung and by the Stem Cell Network North Rhine Westphalia.
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