A list of primers used for gene expression analysis.

<p>A list of primers used for gene expression analysis.</p

Stem cell marker expression in TSCs from differently aged mice (2.5m–2.5 months; 5m–5 months; 9m–9 months and 24m–24 months).

<p><b>A–P:</b> Immunocytochemical staining for the indicated stem cell markers was performed using stem cell marker specific antibodies. <b>Q–T:</b> Semi-quantitation of stem cell marker expression after immunocytochemical staining. Increase in the mouse age decreased the number of TSCs expressing Oct-4, NS, Sca-1, and SSEA-1. In 24 months old mice, except for a few NS-expressing TSCs, expression of the remaining three stem cell markers (Oct-4, Sca-1 and SSEA-1) was very low. Semi-quantitation data are expressed as mean ± SD. Student’s <i>t</i>-test was used to determine statistical significance (*<i>P</i> < 0.05) in comparison with data from the 2.5-months old mice. Bar—100 μm.</p

Morphology and proliferation of TSCs from aging mice after moderate treadmill running (MTR).

<p><b>A.</b> Control mice without MTR. <b>B</b>. Mice after MTR, <b>C.</b> Semi-quantitation of the number of cobblestone-shaped TSCs. <b>D.</b> TSC proliferation, measured as PDT, from mice in the control and MTR groups. Aging TSCs in the control group were large and round, a typical phenotype of senescent cells, but after MTR most cells exhibited a cobblestone shape (arrows point to the two remaining large TSCs as in the control group), which also proliferated at a higher rate. Data are mean ± SD, and *<i>P</i> < 0.05, in comparison with data from the control group. Bar—100 μm.</p

The expression of tenocyte and non-tenocyte related genes in patellar (A) and Achilles (B) TSCs in response to mechanical loading <i>in vitro</i>.

<p>Total RNA were collected from TSCs stretched to 4% or 8% for qRT-PCR analysis. In PTSCs under low mechanical loading (green, 4% stretching), only those genes related to tenocytes (Coll. I, or collagen type I; Tenom or tenomodulin) were highly expressed, but under high mechanical loading (red, 8% stretching), both tenocyte and non-tenocyte related genes increased their expression. Similar results were obtained for ATSCs in response to low (4%) and high (8%) mechanical loading. Note the different scale in gene expression by PTSCs and ATSCs between the two loading conditions (*p<0.05, with respect to non-loaded cells; ^#p<0.05 with respect to 4% stretching).</p

The population doubling time (PDT) of TSCs isolated from mouse tendons.

<p>TSCs were seeded in a 6-well plate and cultured to determine PDT. Mouse treadmill running enhanced proliferation of TSCs from patellar tendons (<b>A</b>) and Achilles tendons (<b>B</b>), as indicated by decreased PDT compared to cage controls. In fact, ITR stimulated cell growth more quickly than MTR (<b>A, B</b>) (*p<0.05 with respect to cage control; ^#p<0.05 with respect to MTR).</p

Expression of the stem cell marker SSEA-4 by hTSCs cultured <i>in vitro</i> in various concentrations of PGE₂.

<p><b>A</b>: without PGE₂ treatment; <b>B</b>: 0.01 ng/ml PGE₂; <b>C</b>: 0.1 ng/ml PGE₂; <b>D</b>: 1 ng/ml PGE₂; <b>E</b>: 10 ng/ml PGE₂; and <b>F</b>: 100 ng/ml PGE₂. hTSCs were seeded in 12-well plates, cultured with six different concentrations of PGE_2, incubated with mouse anti-human SSEA-4 primary antibody, and detected with Cy3-conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst (Blue). Expression of SSEA-4 (red) is dose-dependent, with more robust expression seen in hTSCs treated with low levels of PGE₂ (<b>A–D</b>) than expression levels seen in those treated with high levels (<b>E, F</b>). Positively stained cells were also counted to calculate percentage staining (<b>G</b>) (*p<0.05 with respect to hTSCs not treated with PGE₂). Bar: 100 µm.</p

The expression of tenocyte and non-tenocyte related genes in patellar (A) and Achilles (B) mouse tendons in response to treadmill running.

<p>Total RNA collected from the tendons of controls and mice in the MTR and ITR groups were subjected to qRT-PCR. As shown, MTR only increased the expression of tenocyte related genes in the two types of tendons (Coll. I, or collagen type I; Tenom or tenomodulin), whereas ITR increased the expression of both tenocyte and non-tenocyte related genes (LPL: a marker for adipocyte; Sox9: a marker for chondrocyte; Runx2 and Osterix: two markers for osteoblasts). (*p<0.05 with respect to the corresponding controls; ^#p<0.05 with respect to MTR. In A, however, # in Coll. I represents p<0.05 with respect to ITR).</p

Population doubling time (PDT) of hTSCs treated with various concentrations of PGE₂.

<p>hTSCs were seeded in 6-well plates and cultured for six days on medium containing six different concentrations of PGE₂. PDT increased with increasing concentration of PGE₂, meaning that increased PGE₂ resulted in decreased cell proliferation (*p<0.05 when compared to control cells without PGE₂ treatment).</p

Number of TSCs cultured from Achilles tendons of mice subjected to treadmill running.

<p>Number of TSCs cultured from Achilles tendons of mice subjected to treadmill running.</p

Immunostaining of nucleostemin (NS) (A–C), semi-quantitation (D) and qRT-PCR analysis of gene expression (E, F) in aging TSCs subjected to mechanical loading <i>in vitro</i>.

<p>Patellar TSCs isolated from aging mice (9 months old) were subjected to no stretching (Control), 4% stretching or 8% stretching followed by the analyses. Immunostaining (<b>A–C</b>) and semi-quantitation (<b>D</b>) showed that compared to the control without stretching, 4% stretching increased the number of NS-expressing TSCs, whereas 8% stretching further decreased it. qRT-PCR analysis of the expression of three stem cell and tenocyte-related genes, Nanog, collagen I and tenomodulin (<b>E</b>) and three non-tenocyte related genes, LPL, Sox-9 and Runx-2 (<b>F</b>) showed that 4% stretching effectively up-regulated Nanog, collagen I and tenomodulin, but 8% was less effective (<b>E</b>). Moreover, while 4% stretching did not alter LPL and Runx-2 expression, and slightly up-regulated Sox-9 level, 8% stretching up-regulated the expression of all three genes (<b>F</b>). Data are mean ± SD, and *<i>P</i> < 0.05, compared to the respective controls. Bar—100 μm.</p