Abstract
Distinct physical factors originating from the cellular microenvironment are crucial to the biological homeostasis of stem cells. While substrate stiffness and orientation are known to regulate the mechanical remodeling and fate decision of mesenchymal stem cells (MSCs) separately, it remains unclear how the two factors are combined to manipulate their mechanical stability under gravity vector. Here we quantified these combined effects by placing rat MSCs onto stiffness-varied poly-dimethylsiloxane (PDMS) substrates in upward (180°), downward (0°), or edge-on (90°) orientation. Compared with those values onto glass coverslip, the nuclear longitudinal translocation, due to the density difference between the nucleus and the cytosol, was found to be lower at 0° for 24 h and higher at 90° for 24 and 72 h onto 2.5 MPa PDMS substrate. At 0°, the cell was mechanically supported by remarkably reduced actin and dramatically enhanced vimentin expression. At 90°, both enhanced actin and vimentin expression worked cooperatively to maintain cell stability. Specifically, perinuclear actin stress fibers with a large number, low anisotropy, and visible perinuclear vimentin cords were formed onto 2.5 MPa PDMS at 90° for 72 h, supporting the orientation difference in nuclear translocation and global cytoskeleton expression. This orientation dependence tended to disappear onto softer PDMS, presenting distinctive features in nuclear translocation and cytoskeletal structures. Moreover, cellular morphology and focal adhesion were mainly affected by substrate stiffness, yielding a time course of increased spreading area at 24 h but decreased area at 72 h with a decrease of stiffness. Mechanistically, the cell tended to be stabilized onto these PDMS substrates via β1 integrin–focal adhesion complexes–actin mechanosensitive axis. These results provided an insight in understanding the combination of substrate stiffness and orientation in defining the mechanical stability of rMSCs.
Highlights
The stem cell niche, defined as the surrounding microenvironment of both the neighboring cells and the extracellular matrix (ECM), provides biochemical and biomechanical signals to regulate stem cell self-renewal and fate commitment (Lemischka and Moore, 2003; Moore Lemischka, 2006; Mitsiadis et al, 2007; Aichinger et al, 2012; Kordes and Haeussinger, 2013; Turksen, 2015)
We first tested nucleus longitudinal translocation in Rat bone marrow-derived stem cells (rMSCs) onto typical PDMS substrates when glass coverslip served as the control (Figure 1A)
The orientation dependence of nucleus translocation for rMSCs onto glass was presented with a higher value at 0° for 24 h (Figure 1B), consistent with that previously described for MC3T3-E1 cells (Zhang et al, 2017)
Summary
The stem cell niche, defined as the surrounding microenvironment of both the neighboring cells and the extracellular matrix (ECM), provides biochemical and biomechanical signals to regulate stem cell self-renewal and fate commitment (Lemischka and Moore, 2003; Moore Lemischka, 2006; Mitsiadis et al, 2007; Aichinger et al, 2012; Kordes and Haeussinger, 2013; Turksen, 2015). Hereinto mechanosensing is initiated from the varied cell–ECM traction force induced by different substrate stiffness levels, which alters the intracellular prestress and stem cell or nucleus stiffness and results in the mechanical remodeling of stem cells on their niches (Chowdhury et al, 2010; Swift et al, 2013; Buxboim et al, 2014; Harada et al, 2014). Directed nucleolus sedimentation inside a Xenopus oocyte is dominant over thermal fluctuation, implying that the sedimentation of a relatively dense nucleus could initiate cell gravisensing (Feric and Brangwynne et al, 2013) While these static models of orientation change are helpful in elucidating cell mechanosensing, it is still unclear in this process what a role the extracellular microenvironment, such as ECM stiffness, plays and how it is correlated with substrate orientation alteration
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