Structure of CS Chains in cartilage of newborn mice.

<p><i>A</i>, CS extracted from proximal humeral cartilage of newborn mice was labeled with [³H]-sodium borohydride, applied to a Superose 6 column with effluent fractions of 0.5 ml each, and analyzed for radioactivity. The elution profiles of the samples obtained from CSS2^−/− (CSS2KO) (×) mice and wild-type (WT) (•) littermates are shown. Three independent experiments (n = 3) showed the same elution profile. <i>Numbered arrow 3.3</i>, <i>10</i>, and <i>30</i> indicate the eluted positions of chondroitin polysaccharides of known sizes (molecular size, 3,300, 10,000, and 30,000, respectively). <i>B</i>, Each total radioactivity of peaks of CSS2KO mice and WT littermates in Fig. 4A is shown. <i>C</i>, CS isolated from proximal humeral cartilage of newborn mice were digested with chondroitinase ABC and subjected to reverse-phase ion pair chromatography with postcolumn fluorescence labeling as described in “Experimental Procedures”. The histogram shows the amount and compositions of unsaturated disaccharide in the CS isolated from cartilage of CSS2KO (▪) mice and WT (□) littermates. All experiments were performed three times independently, and bars in the graphs were shown as a mean ± S.D. *, <i>p</i><0.01. <i>0S</i>, <i>4S</i>, and <i>6S</i>, represent ΔDi-0S, ΔDi-4S, and ΔDi-6S, respectively.</p

Expression level of CSSs in cartilage of CSS2^−/− (CSS2KO) mice and their wild-type (WT) littermates.

<p>Realtime RT-PCR using cDNA synthesized from humerus of newborn mice were performed to investigate relative expression levels of CSS1, CSS2, ChSy3, Csgalnact1, and Csgalnact2. The amount of expression was normalized by GAPDH. All experiments were performed three times independently, and bars in the graphs were shown as a mean ± S.D. *; p<0.05.</p

Histology of the articular cartilage of knee joints from CSS2^−/− (CSS2KO) mice and their wild-type (WT) littermates.

<p>Sections were stained with Safranin-O, which stains sulfated glycosaminoglycans, and fast green for counter staining. Each image shown is one representative section selected from 50 sections from an experimental group (10 sections from each knee joint and 5 knee joint in each experimental group). There were no obvious degradations of articular surface in knee joints shown and no morphologic differences among the groups at ages 1 and 6 months (1 M and 6 M).</p

Generation of CSS2^−/− mice.

<p>A, Genomic structure of CSS2 gene, the targeting vector, and the genome with homologous recombination and that whose fragments were excised by flippase and Cre systems. <i>A</i>, The genomic structure of CSS2 gene is depicted on the top line (a). CSS2 targeting vector (b) was constructed by flanking exon 1 with <i>loxP</i> sites, and flanking a Neo^R cassette with FRT sites. ES cell clones with homologous recombination of the vector segment (c) were obtained by positive selection, and used for generation of chimera mice, followed by germ line transmission. The FRT-flanked Neo cassette was subsequently deleted from the recombinant allele by crossing with CAG-Flp Tg mice (d). Then, a genomic fragment containing exon 1, flanked by loxP sites, was excised from the recombinant allele by crossing with CAG-Cre Tg mice (e). <i>B</i>, Genomic PCR. Genotyping was performed by PCR using tail DNA and primers shown in black arrows in Fig. 1A. The PCR product of wild-type allele and CSS2 mutant allele was 2.1 kb and 420 bp respectively. <i>C</i>, Immunoprecipitation of the CSS2, followed by western blot. The cell lysates obtained from WT MEFs were subjected to western blot analysis as described in “Experimental Procedures”. Western blot analysis shows bands of CSS2 (85 kDa) and the CSS2 variant (68 kDa).</p

Effects of CSS1 siRNA in CSS2^−/− (CSS2KO) and wild-type (WT) chondrocytes on CS synthesis.

<p>Primary chondrocytes obtained from CSS2KO and WT mice were downregulated of CSS1 using siRNA, as described in “Experimental Procedures”. All experiments were performed three times independently, and bars in the graphs were shown as a mean ± S.D. <i>A</i>, Relative expression level of CSS1 and CSS2 in WT and CSS2^−/− chondrocytes were shown. siRNA, cells transfected with CSS1 siRNA; control, cells transfected with control siRNA; mock, untransfected cells. <i>B</i>, The total radioactivity of the elution profiles in each sample. <i>C</i>, Chain length of CS in chondrocytes. Three independent experiments (n = 3) showed the same elution profile. <i>Numbered arrow 3.3</i>, <i>10</i>, and <i>20</i> indicate the eluted positions of chondroitin polysaccharides of known sizes (molecular size, 3,300, 10,000, and 20,000, respectively).</p

Skeletal analysis of CSS2^−/− (CSS2KO) mice and their wild-type (WT) littermates at newborn and age of 1 month.

<p><i>A</i>, skeletal structure of CSS2KO and WT newborn mice stained with alcian blue and alizarin red. Whole body (a, b), upper extremity (c, d), and lower extremity (e, f) of WT littermates and CSS2KO mice, respectively, were shown. Scale bar: 500 µm. <i>B</i>, Proximal humerus of WT and CSS2KO newborn mice stained with hematoxylin-eosin is shown. The area size, the number and orientation of chondrocytes are similar between WT littermates (g) and CSS2KO (h) mice. The lengths of proliferative (P), prehypertrophic (PH), and hypertrophic (H) zones of WT (□) littermates and CSS2KO (▪) mice are shown in the histogram. Bar graph shown in the graphs indicate a mean ± S.D. of 10 samples’ measurements (n = 10). Large scale bar: 50 µm, small scale bar: 5 µm. <i>C</i>, skeletal structure of CSS2KO and WT 1-month-old mice stained with alcian blue and alizarin red. Whole body (i, j), upper extremity (k, l), and lower extremity (m, n) of WT littermates and CSS2KO mice, respectively, were shown. Scale bar: 5 mm. <i>D</i>, The histogram shows comparison of the length of humerus, ulna, femur, and tibia of WT (□) littermates and CSS2KO (▪) mice. Bar graph shown in the graphs indicate a mean ± S.D. of 10 samples’ measurements (n = 10). *; p<0.005.</p

Primers for realtime PCR.

<p>Primers for realtime PCR.</p

Growth factor secretion and the cytoprotective effect of amnion and chorion MSCs.

<p>(A–D) Cytoprotective effect of FM MSC-derived conditioned medium was analyzed by the MTS assay (A, B) and caspase-3 activity (C, D) in HUVECs (A, C) and cardiomyocytes (B, D). Values are mean ± SEM. *p<0.05 vs. serum-free. (E–H) Conditioned medium obtained from FM-derived MSCs was collected after incubation for 24 h. The concentration of HGF (E), IGF-1 (F), bFGF (G), and VEGF (H) in serum free conditioned medium was measured by ELISA. *p<0.05 and ***p<0.001.</p

Characterization of human amnion- and chorion-derived MSCs.

<p>(A) Representative photographs of human amnion and chorion. (B) Photographs of cultured MSCs obtained from human amnion and chorion at passage 3. Scale bars = 500 µm. (C) Relative cell number of amnion- and chorion-derived MSCs at each passage. (D) FACS analysis of amnion and chorion MSCs. (E, F) Differentiation of amnion and chorion MSCs into adipocytes (E) and osteocytes (F). Scale bars = 100 (E) and 50 (F) µm.</p

Immunosuppressive property of amnion and chorion MSCs.

<p>(A) Inhibition of human CD4+ T cell proliferation upon co-culture with human amnion, chorion, and bone marrow MSCs. (B) The concentration of PGE2 in FM-MSC-conditioned medium was measured by ELISA. Amnion MSCs secreted a significant amount of PGE2 compared with chorion MSCs. (C, D) Effect of human amnion (C) or chorion (D) MSC transplantation in a murine GVHD model. Treatment with amnion MSCs significantly reduced recipient weight loss in a mouse model of GVHD. *p<0.05, **p<0.01 and ***p<0.001.</p