Differentiation alters stem cell nuclear architecture, mechanics, and mechano-sensitivity
- Perelman School of Medicine, University of Pennsylvania, United States
- University of Pennsylvania, United States
- Queen Mary University of London, United Kingdom
- Harvard University, United States
- Boston University, United States
Figures
Figure 1 with 2 supplements
MSC differentiation reduces nuclear deformation with applied stretch.
(a) On day 1, bovine MSCs on aligned nanofibrous scaffolds have elongated nuclei that are oriented in the prevailing fiber direction in both Ctrl and Diff conditions. Application of 10% scaffold …
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Figure 1—source data 1
Changes in nuclear aspect ratio and population distribution with stretch.
- https://doi.org/10.7554/eLife.18207.003
Figure 1—figure supplement 1
Type II collagen and aggrecan gene expression in bovine MSCs as a function of media condition and time.
(a) Type II collagen (Type II Col) and (b) aggrecan (AGG) gene expression in bovine MSCs as a function of media condition and time when cultured on aligned nanofibrous scaffolds. Culture in …
Figure 1—figure supplement 2
Differentiation-mediated reduction in strain transfer to the nucleus in human bone-marrow-derived MSCs and a human ES-cell line.
The overall response of human MSCs mirrored that of bovine MSCs, indicating that the reduction in nuclear strain transfer is independent of species. (a) Application of 10% scaffold stretch …
Figure 2 with 1 supplement
Decreased strain transfer to the nucleus with differentiation is a consequence of nuclear stiffening and not extracellular matrix deposition.
(a) Schematic illustrating how MSCs were cultured on aligned (AL) scaffolds in Ctrl condition or Diff condition for 7 days followed by re-seeding on fresh scaffolds (for stretch studies) or …
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Figure 2—source data 1
Peri-nuclear stiffness measurements and changes in nuclear aspect ratio with stretch.
- https://doi.org/10.7554/eLife.18207.007
Figure 2—figure supplement 1
Contractility increases in differentiated MSCs and is necessary for strain transfer to the nucleus.
(a) Traction force increases substantially over the first 16 hr of exposure to 10 ng/ml TGF-β3 (n = 20, Mean ± SD), where time t = 0 shows the baseline response in the absence of TGF-β3. (b) …
Figure 3
MSC differentiation results in marked nuclear reorganization.
(a) On day 1, Lamin A/C (LMAC) was spread diffusely throughout the nucleus in both Ctrl and Diff conditions. By day 7, Diff conditions resulted in a restriction of LMAC to the nuclear periphery …
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Figure 3—source data 1
The nuclear volume occupancy ratio of LMAC, protein levels of A-type lamin and β-actin, and HTC staining intensity.
- https://doi.org/10.7554/eLife.18207.010
Figure 4 with 2 supplements
Increases in nuclear mechanics heighten mechanosensitivity of mesenchymal stem cells.
(a) On day 7, 10% static stretch increased nuclear deformation in Ctrl conditions but did not alter nuclear shape under Diff conditions. With TSA treatment (Diff/TSA), nuclear elongation of …
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Figure 4—source data 1
Changes in nuclear aspect ratio and type II collagen and aggrecan gene expression with stretch.
- https://doi.org/10.7554/eLife.18207.014
Figure 4—figure supplement 1
TSA treatment of differentiated MSCs softens nuclei and decreases heterochromatin, but does not alter Lamin A/C amount or distribution.
(a and b) One day of TSA treatment (Diff/TSA) decreased heterochromatin (HTC) staining in differentiated MSCs (ANOVA, *p<0.05 vs. Ctrl, ‡p < 0.05 vs. Diff/TSA, n = 35, mean ± SD). Scale: 20 µm. One …
Figure 4—figure supplement 2
TSA treatment of differentiated MSCs does not alter MLCK activity, actin structure, or migration speed.
DM treatment induces chromatin reorganization but does not perturb the actin cytoskeleton.(a and b) MLCK phosphorylation increased markedly in Diff compared to Ctrl MSCs but was not altered with TSA …
Figure 5 with 1 supplement
Dynamic loading induces nuclear reorganization and increases nuclear mechanics in undifferentiated MSCs.
Dynamic loading (DL) for 5 days in the absence of soluble differentiation factors resulted in reorganization of LMAC in a manner dependent on the duration (0, 1, 3, or 6 hr/day, a–d) and magnitude …
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Figure 5—source data 1
Changes in nuclear aspect ratio with stretch, peri-nuclear stiffness, and gene expression with the application of DL.
- https://doi.org/10.7554/eLife.18207.018
Figure 5—figure supplement 1
Dynamic loading of undifferentiated MSCs induces nuclear reorganization comparable to differentiation with soluble factors.
LMAC nuclear occupancy ratio as a function of strain duration (a) and magnitude (b) in MSCs dynamically loaded in Ctrl media (ANOVA, *p<0.05 vs. 0%/0 hr, n = 25, mean ± SD). (c) LMAC was restricted …
Figure 6 with 1 supplement
Rapid alterations in MSC nuclear architecture with dynamic loading.
(a) Staining for LMAC showed little change in Ctrl conditions, and only subtle changes in Diff conditions through day 3. Conversely, DL promoted LMAC reorganization as early as day 2 (Scale: 5 µm). …
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Figure 6—source data 1
HTC staining intensity, LMAC gene expression and CCP calculation.
- https://doi.org/10.7554/eLife.18207.021
Figure 6—figure supplement 1
Rapid changes in fibrocartilaginous gene (Sox9, BMP2, TGF-β, and Aggrecan) expression with dynamic loading.
Expression of (a) SOX9, (b) BMP2, (c) TGF -β, and (d) aggrecan (AGG) was transiently upregulated under Diff conditions over the first three days of culture. DL resulted in higher expression for …
Figure 7
Altered nuclear mechanobiology with stem cell differentiation.
(a) The nucleus is mechanically linked to the extracellular environment through linker of nucleus and cytoskeleton (LINC) complex connections to a contractile cytoskeleton that interact with the …
Author response image 1
(a) Chromatin condensation parameter (CCP) as a function of treatment group (*p<0.05 vs. Ctrl, †p<0.05 vs. Ctrl/TSA, n = 24). (b) Normalized nuclear aspect ratio (NAR) as a function of treatment …
Author response image 2
PCA plot showing shifts in global gene expression in response to one loading event (DL), after a period of free swelling culture (FS) following DL, after a second loading event (2nd DL), and with another free swelling period (2nd FS).
Note that the last two points (green and grey) reside essentially on top of one another, suggesting a permanence in expression patterns across the genome following a second loading event. These …
Videos
Video 1
3D reconstruction of LMAC in the nucleus in undifferentiated bovine MSCs.
https://doi.org/10.7554/eLife.18207.011
Video 2
3D reconstruction of LMAC in the nucleus in differentiated bovine MSCs.
https://doi.org/10.7554/eLife.18207.012Additional files
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Source code 1
MATLAB code for calculation of chromatin condensation parameter (CCP).
- https://doi.org/10.7554/eLife.18207.024
