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Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration.

Kohler J, Popov C, Klotz B, Alberton P, Prall WC, Haasters F, Müller-Deubert S, Ebert R, Klein-Hitpass L, Jakob F, Schieker M, Docheva D - Aging Cell (2013)

Bottom Line: Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained.Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments.Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell-matrix interactions and the ROCK kinase pathway might be key players in TSPC aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Experimental Surgery and Regenerative Medicine, Ludwig Maximilians University Munich, Nussbaumstr. 20, 80336, Munich, Germany.

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Related in: MedlinePlus

Investigation of TSPC migration potential. (A) Time-lapse experiment for 18 h. Representative images at the beginning and at the end of the experiment are shown. Tracked cells and migratory paths are indicated with stars and black lines. (B) Quantification of migration distance and cell velocity. Two independent experiments with three donors per group were performed (180 cells per group). Scratch assays on collagen I (C and D) and fibronectin (E and F). Representative images at 0 h and 7 h are shown, and the cellular fronts are outlined with black lines. Cell velocity was calculated from four different scratches per donor.
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fig03: Investigation of TSPC migration potential. (A) Time-lapse experiment for 18 h. Representative images at the beginning and at the end of the experiment are shown. Tracked cells and migratory paths are indicated with stars and black lines. (B) Quantification of migration distance and cell velocity. Two independent experiments with three donors per group were performed (180 cells per group). Scratch assays on collagen I (C and D) and fibronectin (E and F). Representative images at 0 h and 7 h are shown, and the cellular fronts are outlined with black lines. Cell velocity was calculated from four different scratches per donor.

Mentions: We carried out time-lapse experiments for monitoring of random cell migration and an in vitro scratch assay mimicking wound closure. Quantifications of migratory distance revealed that A-TSPC migration speed and distances were significantly slower compared with Y-TSPC (Fig.3A,B). To estimate the effect of matrix proteins, scratch assay experiments were performed on collagen I or fibronectin and also revealed a decelerated migration and longer wound closure time in the aged cells (Fig.3C–F). In addition, pronounced morphological differences were noticed between Y- and A-TSPC; cells from aged donors exhibited a star-like flattened cell appearance, while cells from young donors were smaller in size and spindle-shaped (Fig.4A,B). It is known that cell shape and cell migration strongly dependent on actin cytoskeleton organization and the rate of actin filament turnover (Rottner & Stradal, 2011). Therefore, we performed phalloidin stainings for F-actin and compared the actin filament dynamics by treating the TSPC with latrunculin A (LatA) in a time-dependent manner. LatA inhibits actin polymerization by sequestering monomeric G-actin and thereby disrupts the turnover of actin filaments. Our results showed that A-TSPC have more robust actin stress fibers (Fig.4C) and a higher actin content than Y-TSPC (Fig.4D,E). In conclusion, the smaller effect of LatA on the A-TSPC indicated a slower actin turnover in these cells. Taken together, our results clearly demonstrate a dramatic decrease in the migratory capacity of TSPC during aging and suggested that distorted actin dynamics might be a core reason.


Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration.

Kohler J, Popov C, Klotz B, Alberton P, Prall WC, Haasters F, Müller-Deubert S, Ebert R, Klein-Hitpass L, Jakob F, Schieker M, Docheva D - Aging Cell (2013)

Investigation of TSPC migration potential. (A) Time-lapse experiment for 18 h. Representative images at the beginning and at the end of the experiment are shown. Tracked cells and migratory paths are indicated with stars and black lines. (B) Quantification of migration distance and cell velocity. Two independent experiments with three donors per group were performed (180 cells per group). Scratch assays on collagen I (C and D) and fibronectin (E and F). Representative images at 0 h and 7 h are shown, and the cellular fronts are outlined with black lines. Cell velocity was calculated from four different scratches per donor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225469&req=5

fig03: Investigation of TSPC migration potential. (A) Time-lapse experiment for 18 h. Representative images at the beginning and at the end of the experiment are shown. Tracked cells and migratory paths are indicated with stars and black lines. (B) Quantification of migration distance and cell velocity. Two independent experiments with three donors per group were performed (180 cells per group). Scratch assays on collagen I (C and D) and fibronectin (E and F). Representative images at 0 h and 7 h are shown, and the cellular fronts are outlined with black lines. Cell velocity was calculated from four different scratches per donor.
Mentions: We carried out time-lapse experiments for monitoring of random cell migration and an in vitro scratch assay mimicking wound closure. Quantifications of migratory distance revealed that A-TSPC migration speed and distances were significantly slower compared with Y-TSPC (Fig.3A,B). To estimate the effect of matrix proteins, scratch assay experiments were performed on collagen I or fibronectin and also revealed a decelerated migration and longer wound closure time in the aged cells (Fig.3C–F). In addition, pronounced morphological differences were noticed between Y- and A-TSPC; cells from aged donors exhibited a star-like flattened cell appearance, while cells from young donors were smaller in size and spindle-shaped (Fig.4A,B). It is known that cell shape and cell migration strongly dependent on actin cytoskeleton organization and the rate of actin filament turnover (Rottner & Stradal, 2011). Therefore, we performed phalloidin stainings for F-actin and compared the actin filament dynamics by treating the TSPC with latrunculin A (LatA) in a time-dependent manner. LatA inhibits actin polymerization by sequestering monomeric G-actin and thereby disrupts the turnover of actin filaments. Our results showed that A-TSPC have more robust actin stress fibers (Fig.4C) and a higher actin content than Y-TSPC (Fig.4D,E). In conclusion, the smaller effect of LatA on the A-TSPC indicated a slower actin turnover in these cells. Taken together, our results clearly demonstrate a dramatic decrease in the migratory capacity of TSPC during aging and suggested that distorted actin dynamics might be a core reason.

Bottom Line: Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained.Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments.Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell-matrix interactions and the ROCK kinase pathway might be key players in TSPC aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Experimental Surgery and Regenerative Medicine, Ludwig Maximilians University Munich, Nussbaumstr. 20, 80336, Munich, Germany.

Show MeSH
Related in: MedlinePlus