Differences in mRNA expression between KD-MSCs and HC-MSCs by RT-PCR analysis.

<p>*p<0.05 between HC-MSCs (n = 6) and KD-MSCs (n = 9).</p

Real-time PCR array, quantitative PCR, and western blot analyses of MSCs.

<p>(A) Downregulation of multiple stem cell-relevant transcription factors in KD-MSCs (<i>n</i> = 9) compared with HC-MSCs (<i>n</i> = 6). The fold change [2∧(−ΔΔCt)] is the normalized gene expression [2∧(−ΔCt)] in KD-MSCs relative to that in HC-MSCs. <i>P</i>-values were calculated based on the Student's <i>t</i>-test of replicate 2∧(−ΔCt) values for each gene in HC-MSCs and KD-MSCs. <i>P</i><0.05 is indicated with black arrows. (B) Quantitative PCR was performed to measure the levels of gene expression in HC-MSCs (<i>n</i> = 6) and KD-MSCs (<i>n</i> = 6). Data are expressed as the mean ± SE. ^*<i>P</i><0.05. (C) Western blot analysis of PCAF in KD-MSCs and HC-MSCs. PCAF expression was decreased in KD-MSCs (<i>n</i> = 9) compared with HC-MSCs (<i>n</i> = 6). PCAF protein levels are expressed relative to β-actin. Data are expressed as the mean ± SE. ^*<i>P</i><0.05.</p

Differentiation capacities of HC-MSCs and KD-MSCs.

<p>(A) Adipogenic differentiation of HC-MSCs (top and left) and KD-MSCs (top and right) was examined after 2 weeks of culture under adipogenic conditions by Sudan III staining (original magnification, ×100). Osteogenic differentiation of HC-MSCs (second from top and left) and KD-MSCs (second from top and right) was examined after 4 weeks of culture under osteogenic conditions by von Kossa staining (original magnification, ×100). Chondrogenic differentiation of HC-MSCs (bottom and left) and KD-MSCs (bottom and right) was examined after 3 weeks of culture under chondrogenic conditions by Safranin O/Fast green staining (original magnification, ×100). (B) GPDH activity of cells was measured to compare the adipogenic differentiation capacities of HC-MSCs (<i>n</i> = 5) and KD-MSCs (<i>n</i> = 5). Data are expressed as the mean ± standard error (SE). ^*<i>P</i><0.05. (C) ALP activity of the cells was measured to indicate their osteogenic differentiation capacity (<i>n</i> = 4). Data are expressed as the mean ± SE. ^*<i>P</i><0.05.</p

Proliferation and senescence of HC-MSCs and KD-MSCs.

<p>(A) Representative images of HC-MSCs and KD-MSCs (magnification, ×40). Left columns show assessment of senescence using the senescence biomarker SA-β-gal (green) in HC-MSCs and KD-MSCs. Black scale bars represent 50 µm. Right columns show DAPI staining of senescence-associated heterochromatic foci (SAHF) in MSC DNA foci. White scale bars represent 10 µm. Insets show an enlargement of DAPI staining (white scale bars represent 5 µm). Early passage: P5; late passage: P10. (B) Quantitative assessment of SA-β-gal positive cells. Data are the mean ± SE (<i>n</i> = 4). ^*<i>P</i><0.05. (C) Cumulative population doublings (PDs) of HC-MSCs (<i>n</i> = 5) and KD-MSCs (<i>n</i> = 5) from passage 5–10. Data are expressed as the mean ± SE. ^*<i>P</i><0.05. Experiments were performed in triplicate.</p

Analysis of the toxic effects of feline serum on feline DAF (fDAF)-expressing cells.

<p>(A–D) Control swine endothelial cell (sEC) line (fDAF⁻) or fDAF-expressing sEC clone (fDAF⁺) incubated with 80% feline serum. Time zero (0 h) of incubation of the control sEC line (A) and of the fDAF-expressing sEC clone (C). After a 6-h incubation, cells of the control sEC line appear small, round, and detached from the dish (B), whereas cells of the fDAF-expressing sEC clone remain attached to the dish (D). (E and F) A lactate dehydrogenase (LDH) assay of the control cells (fDAF⁻, white box) and the fDAF-expressing sEC clone (fDAF⁺, gray box) incubated with 40% feline serum (E) or 80% feline serum (F). Each data point represents mean ± SEM of five independent experiments (*<i>P</i> < 0.05; **<i>P</i> < 0.01).</p