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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: These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton.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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Short- and long-term proliferation analyses and investigation of cellular senescence. (A and B) Growth curves of three different donors per TSPC group were assembled by calculating cumulative population doubling time. (C) Colony-forming unit (CFU) assay. Crystal violet-stained colonies at day 14. CFU efficiency shown as mean ± SD of three Y-TSPC and three A-TSPC from two independent experiments. (D) Proliferation analysis prior and after stimulation with FGF-2 (1 ng mL−1, 7 days) and TGF-β1 (50 ng mL−1, 3 day). Two independent WST-1 measurements with three different donors per group were performed. (E) Senescence-associated β-gal staining at passage 12 (arrows indicate senescence cells). (F) Quantification of senescent cell number at passage 4, 12, and 14. Three Y- and three A-TSPC donors were analyzed as approx. 600 cells per donor were evaluated. (G) Semi-quantitative PCR analysis of cell cycle gene regulators (p16INKA4, p14ARF, p21WAF1, and p53) in TSPC in passages 1, 10, and 14. PCR was carried out twice independently with three different donors per group. Staining for p16INKA4 (in green) and CD44 (in red). (I) Quantification of p16INKA4-positive cells. Two different donors (200 cells/donor) per group were analyzed at two different passages.
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fig01: Short- and long-term proliferation analyses and investigation of cellular senescence. (A and B) Growth curves of three different donors per TSPC group were assembled by calculating cumulative population doubling time. (C) Colony-forming unit (CFU) assay. Crystal violet-stained colonies at day 14. CFU efficiency shown as mean ± SD of three Y-TSPC and three A-TSPC from two independent experiments. (D) Proliferation analysis prior and after stimulation with FGF-2 (1 ng mL−1, 7 days) and TGF-β1 (50 ng mL−1, 3 day). Two independent WST-1 measurements with three different donors per group were performed. (E) Senescence-associated β-gal staining at passage 12 (arrows indicate senescence cells). (F) Quantification of senescent cell number at passage 4, 12, and 14. Three Y- and three A-TSPC donors were analyzed as approx. 600 cells per donor were evaluated. (G) Semi-quantitative PCR analysis of cell cycle gene regulators (p16INKA4, p14ARF, p21WAF1, and p53) in TSPC in passages 1, 10, and 14. PCR was carried out twice independently with three different donors per group. Staining for p16INKA4 (in green) and CD44 (in red). (I) Quantification of p16INKA4-positive cells. Two different donors (200 cells/donor) per group were analyzed at two different passages.

Mentions: TSPC were successfully isolated from human Achilles tendon biopsies (Table S1) and characterized in vitro to validate their stem/progenitor character. We used FACS and immunocytochemistry to examine the expression of surface antigens and stem cell markers in TSPC based on the studies by Bi et al. (2007), Tempfer et al. (2009), and Mienaltowski et al. (2013). More than 98% of Y-TSPC and A-TSPC were positive for MSC-related surface antigens CD73, CD90, and CD105 (Fig. S1A). They were negative for hematopoietic and endothelial cell antigens CD19, CD34, CD45, and HLA-DRA, thus excluding possible contamination with such cells (Table S2 and S3). TSPC were also positive for STRO-1 (MSC marker), CD146 (pericyte marker), Musashi-1 (muscle, neural, and pericyte markers) (Fig. S1B), and CD44 (MSC marker, Fig.1H). Next, we performed quantitative and semi-quantitative PCR analyses of tendon-related genes, which confirmed the expression of the transcription factors scleraxis, Eya1, and Six1, the tendon marker gene tenomodulin and several extracellular matrix proteins abundant in tendon (collagen types I and III, COMP, decorin, and tenascin C) in both TSPC types (Fig. S1C-E).


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)

Short- and long-term proliferation analyses and investigation of cellular senescence. (A and B) Growth curves of three different donors per TSPC group were assembled by calculating cumulative population doubling time. (C) Colony-forming unit (CFU) assay. Crystal violet-stained colonies at day 14. CFU efficiency shown as mean ± SD of three Y-TSPC and three A-TSPC from two independent experiments. (D) Proliferation analysis prior and after stimulation with FGF-2 (1 ng mL−1, 7 days) and TGF-β1 (50 ng mL−1, 3 day). Two independent WST-1 measurements with three different donors per group were performed. (E) Senescence-associated β-gal staining at passage 12 (arrows indicate senescence cells). (F) Quantification of senescent cell number at passage 4, 12, and 14. Three Y- and three A-TSPC donors were analyzed as approx. 600 cells per donor were evaluated. (G) Semi-quantitative PCR analysis of cell cycle gene regulators (p16INKA4, p14ARF, p21WAF1, and p53) in TSPC in passages 1, 10, and 14. PCR was carried out twice independently with three different donors per group. Staining for p16INKA4 (in green) and CD44 (in red). (I) Quantification of p16INKA4-positive cells. Two different donors (200 cells/donor) per group were analyzed at two different passages.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Short- and long-term proliferation analyses and investigation of cellular senescence. (A and B) Growth curves of three different donors per TSPC group were assembled by calculating cumulative population doubling time. (C) Colony-forming unit (CFU) assay. Crystal violet-stained colonies at day 14. CFU efficiency shown as mean ± SD of three Y-TSPC and three A-TSPC from two independent experiments. (D) Proliferation analysis prior and after stimulation with FGF-2 (1 ng mL−1, 7 days) and TGF-β1 (50 ng mL−1, 3 day). Two independent WST-1 measurements with three different donors per group were performed. (E) Senescence-associated β-gal staining at passage 12 (arrows indicate senescence cells). (F) Quantification of senescent cell number at passage 4, 12, and 14. Three Y- and three A-TSPC donors were analyzed as approx. 600 cells per donor were evaluated. (G) Semi-quantitative PCR analysis of cell cycle gene regulators (p16INKA4, p14ARF, p21WAF1, and p53) in TSPC in passages 1, 10, and 14. PCR was carried out twice independently with three different donors per group. Staining for p16INKA4 (in green) and CD44 (in red). (I) Quantification of p16INKA4-positive cells. Two different donors (200 cells/donor) per group were analyzed at two different passages.
Mentions: TSPC were successfully isolated from human Achilles tendon biopsies (Table S1) and characterized in vitro to validate their stem/progenitor character. We used FACS and immunocytochemistry to examine the expression of surface antigens and stem cell markers in TSPC based on the studies by Bi et al. (2007), Tempfer et al. (2009), and Mienaltowski et al. (2013). More than 98% of Y-TSPC and A-TSPC were positive for MSC-related surface antigens CD73, CD90, and CD105 (Fig. S1A). They were negative for hematopoietic and endothelial cell antigens CD19, CD34, CD45, and HLA-DRA, thus excluding possible contamination with such cells (Table S2 and S3). TSPC were also positive for STRO-1 (MSC marker), CD146 (pericyte marker), Musashi-1 (muscle, neural, and pericyte markers) (Fig. S1B), and CD44 (MSC marker, Fig.1H). Next, we performed quantitative and semi-quantitative PCR analyses of tendon-related genes, which confirmed the expression of the transcription factors scleraxis, Eya1, and Six1, the tendon marker gene tenomodulin and several extracellular matrix proteins abundant in tendon (collagen types I and III, COMP, decorin, and tenascin C) in both TSPC types (Fig. S1C-E).

Bottom Line: These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton.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