Rapid Chromatin Condensation Increases Stem Cell Nuclear Mechanics and Mechanosensitivity

Transcription

1 Rapid Chromatin Condensation Increases Stem Cell Nuclear Mechanics and Mechanosensitivity Su-Jin Heo, MS 1, Tristan P. Driscoll, BS 1, Stephen Thorpe, PhD 2, Woojin M. Han, MS 1, Dawn M. Elliott, PhD 3, David A. Lee, PhD 2, Robert L. Mauck, PhD 1. 1 University of Pennsylvania, Philadelphia, PA, USA, 2 Queen Mary, University of London, London, United Kingdom, 3 University of Delaware, Newark, DE, USA. Disclosures: S. Heo: None. T.P. Driscoll: None. S. Thorpe: None. W.M. Han: None. D.M. Elliott: None. D.A. Lee: None. R.L. Mauck: None. Introduction: Mesenchymal stem cells (MSCs) interpret biophysical cues emanating from their microenvironment to inform lineage specification and commitment [1]. Previously, we showed that fibrochondrogenic differentiation of MSCs was accompanied by an increase in chromatin condensation and nuclear stiffness, resulting in a loss of nuclear deformation when stretch was applied to the nanofibrous scaffolds on which they were seeded [2]. Interestingly, this increased nuclear stiffness appeared to sensitize the differentiated MSCs, enhancing response to applied stretch in terms of both intracellular Ca 2+ and matrix gene expression levels [2, 3]; this heightened response could be blocked by softening the differentiated cell nucleus. While these findings suggest that nuclear stiffness plays a role in mechanosensitivity, many cellular processes change during differentiation beyond just the nucleus, and factors that decondense the nucleus can have unwanted side effects. As an alternative to nuclear stiffening through differentiation, it has recently been reported (in chondrocytes) that a step increase in osmolality can elicit rapid chromatin condensation [4], which correlates strongly with nuclear stiffness [5, 6]. In the present study, we used transient osmotic shock to elicit a rapid increase in chromatin condensation in undifferentiated MSCs, absent the attendant changes that come with differentiation, and tested the hypothesis that the stiffness of the nucleus serves as a rheostat, regulating and modulating how MSCs sense and respond to mechanical perturbation. Methods: Bovine bone marrow derived MSCs (50,000 cells) were seeded onto aligned poly(ε-caprolactone) nanofibrous scaffolds (5 60 mm 2 ) fabricated via electrospinning [2]. Constructs were cultured in a chemically defined media (CM) for 2 days. To condense nuclear chromatin and stiffen nuclei, undifferentiated MSCs were treated with a hypertonic solution (DM, 500mM D-mannitol) for 30 mins prior to application of strain [4]. The degree of nuclear condensation was evaluated using a chromatin condensation parameter (CCP) calculated from DAPI stained mid-section nuclear confocal microscopy images (Zeiss, LSM 510) using a gradient-based Sobel edge detection algorithm [4]. Additional constructs were stretched (at 3% increments) up to 15% grip-to-grip strain (1%/sec) on a custom tensile device, and the degree of nuclear deformation (defined by the nuclear aspect ratio (NAR)) was measured by tracking individual DAPI stained nuclei at each strain step [3]. In other samples, MSCs were labeled with Cal-520AM (AAT Bioquest), which stains intracellular calcium and stretched to 10% (at 1%/sec) on a confocal microscope (Zeiss 5 LIVE DUO High Speed Confocal). Intracellular calcium levels were determined for single cells at the end of stretch, and for 600 seconds thereafter (at a capture rate of 0.25frames/sec). Signal intensity was determined on a cell-by-cell basis. Actin stress fibers were stained with Alexa Fluor 488 phalloidin (Invitrogen). Statistical analysis was performed by t-test or one-way ANOVA with Fisher s post-hoc tests (p<0.05). Results: Consistent with previous findings, hyperosmotic shock (DM conditions) increased chromatin condensation in MSCs, as evidenced by numerous chromatin-free spaces and edges detected in the nuclei, resulting in a 3-fold increase in the CCP (Fig. 1 A, B, p<0.05). DM treatment did not change actin cytoskeletal appearance, spread area, or baseline nuclear aspect ratio (NAR) (not shown). When MSC-seeded scaffolds were subjected to stretch, the nuclei of MSCs cultured in CM conditions increased in NAR, while those in DM conditions deformed significantly less (Fig. 2 A, B, p<0.05). When MSCs were exposed to a more rapid stretch to 10%, intracellular Ca 2+ levels increased by 29% in CM conditions, while pre-treatment with DM resulted in a significantly greater (65%) increase in Ca 2+ (Fig. 3 A, B, p<0.05). In CM conditions, the stretch-induced increase in intracellular Ca 2+ returned to baseline within 15 sec after stretch, while in DM conditions intracellular Ca 2+ levels remained higher for a much longer duration (~80 sec, not shown). Discussion: The nucleus is the largest and stiffest element of the cell, and is subject to marked remodeling and stiffening as progenitor cells undergo differentiation. The nucleus is connected to the extracellular environment through the contractile cytoskeleton, and can serve as a direct mediator of mechanical transduction. As such, we posited that nuclear mechanics act as a rheostat within the cell, altering force transduction and potentially modulating mechanosensitivity. Our previous studies showed that, with differentiation, MSC nuclei as stiffen the cells become sensitized to stretch. However, interpretation of these findings was complicated by the many attendant changes that occur with differentiation. Here, we directly stiffened the MSC nucleus by inducing a rapid condensation of the chromatin via hyperosmotic shock. With this treatment, MSC nuclei became resistant to strain transfer when the scaffold was stretched, with no marked changes in organization of the actin cytoskeleton. When a step stretch was applied to these cells, higher levels of intracellular calcium were observed, and these changes persisted over a

2 longer duration. Together with our previous findings, these results suggest that a stiffer nucleus sensitizes MSCs to respond to mechanical perturbation. Future work will explore the signaling pathways that mediate these changes in MSC mechanosensitivity, and further define how such changes influence differentiation and tissue regeneration. Significance: This study suggests that an increase in nuclear stiffness sensitizes undifferentiated MSCs to mechanical perturbation. These results provide new insight into the role of nuclear stiffness in regulating how progenitor cells interpret and respond to their biophysical environment to direct lineage specification and commitment. Acknowledgments: This work was supported by the NIH (AR056624) and the Human Frontiers in Science Program. References: [1] Pittenger+, Science [2] Heo+, ORS [3] Heo+, ORS [4] Irianto+, Biophys J [5] Li+, Biophys J [6] Deguchi+, J Biomech [7] Hu+, Biochem Biophys Res Commun 2005.

3 Figure 1. (A) Representative DAPI stained MSC nuclei (top row) and corresponding edge detection (bottom row) in control (CM) and condensed (DM) conditions (bar = 3 μm). (B) Chromatin condensation parameter (CCP) for each treatment group (n=30, *: p<0.05 vs. CM, mean ± SD).

4 Figure 2. (A) DAPI stained nuclei at 15% scaffold stretch under control (CM) or condensed (DM) conditions (bar = 20 μm). (B) ).

5 Figure 3. (A) Intracellular Ca2+ signal under free swelling (0%) and stretch (10%) conditions for control (CM) and condensed (DM) treated MSCs (bar = 100 µm). (B) Quantification of calcium signal intensity at the end of stretch (n>40, *: p<0.05 vs. CM, mean ± SD). ORS 2014 Annual Meeting Poster No: 0457

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