IL-17 does not affect SAMD2 activation, but attenuates SOX9 phosphorylation induced by chondrogenic culture.

<p>Human MSCs were cultured in monolayer (DMEM containing 5% FBS) with indicated concentrations of IL-17 throughout the culture period. After 16 hr culture with serum-starved medium (0% FBS), cells were stimulated with TGF-β3 (10 ng/mL) for 15 min and A, whole cell lysates or B, cytoplasmic (upper panel) and nuclear (lower panel) fraction were analyzed for SMAD2 and phospho-SMAD2 expression by western blotting. β-actin and TBP were used as loading controls. C, Human MSCs were cultured on cover-glass slides and SMAD2 localization was determined by immunofluorescence microscopy. Original magnification×20. Scale bars represent 100 µm. D, Human MSCs were cultured as aggregates in chondrogenic induction medium supplemented with TGF-β3 and aggregate lysates were evaluated at the indicated time points by western blot analysis for total and phosphorylated SOX9. β-actin was used as a loading control. (A, B, C and D) Data are representative of two independent experiments with similar findings. E, IL-17 or 10 µM H89 was added at the indicated concentrations and analysis carried out at day 2 and 7 by western blotting (top: day 2, bottom: day 7). F, Densitometric analysis on day 7 was performed with CS Analyzer, version 3.0 (bottom). Values represent the mean±SD of three independent experiments. *<i>P<</i>0.05, compared to no cytokine, by Dunnett’s multiple comparison test. NS: not significant.</p

IL-17 suppressed the expression of chondrogenic marker genes.

<p>Human MSCs were cultured as aggregates in TGF-β3-containing medium with the indicated concentrations of IL-17. After 14 days, type II collagen (<i>COL2A1</i>), aggrecan (<i>ACAN</i>), type X collagen (<i>COL10A1</i>), and alkaline phosphatase (<i>ALP</i>) mRNA levels were determined by real-time PCR and expressed relative to β-actin expression level. Values are mean±SD of 3 aggregates from 1 of 3 independent experiments with similar findings.</p

IL-17 treatment reduces PKA activity during chondrogenesis.

<p>A, Human MSCs were cultured as aggregates with TGF-β3 in the presence of IL-17 (100 ng/mL) or H89 (10 µM). After 7 days, 3 aggregates were pooled and lysed in each group, and PKA activity within the soluble protein fraction was determined using PepTag non-radioactive PKA assay (top). Recombinant PKA catalytic subunit (2 µg/mL) was used as a positive control and water was used as a negative control. Densitometric analyses of the band intensities were performed and results were expressed as the test band intensity relative to that of the “no TGF-β3” sample (bottom). B, The DNA content of three aggregates in each group was measured after 7-day culture. Values shown in A and B are mean±SD of three independent experiments. **<i>P<</i>0.01 compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test.</p

IL-17 inhibits TGF-β3-induced chondrogenic differentiation.

<p>A, Human mesenchymal stem cells (MSCs) were cultured in pellets, with or without TGF-β3 (10 ng/mL). Macro-images of the aggregates cultured with IL-17, TNF-α or IL-1β at the indicated concentrations for 14 days. Scale bar represents 1 mm. B, Aggregates cultured in the presence of the indicated cytokines for 21 days were fixed and paraffin embedded, then sections were stained with Safranin O and anti-type II collagen antibody. Original magnification×10. Scale bars represent 500 µm. (A and B) Data are representative of two independent experiments with similar findings. C, Wet weight of aggregates treated with IL-17 for 14 days. Values are mean±SD of 6–7 aggregates per group from three independent experiments with similar tendencies. ^##<i>P<</i>0.01, compared to no cytokine (−TGF-β3), by Student’s <i>t</i>-test. *<i>P</i><0.05; **<i>P<</i>0.01, compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test. D, Sulfated glycolaminoglycan (sGAG) content in aggregates treated with IL-17 for 21 days. Values are mean±SD of 3–4 aggregates per group from two independent experiments with similar findings. ^##<i>P<</i>0.01, compared to no cytokine (−TGF-β3), by Student’s <i>t</i>-test. **<i>P<</i>0.01, compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test. E, IL-17 receptor A (<i>IL-17RA</i>) mRNA levels in aggregates were determined by real-time PCR for the indicated time points. Values are normalized to β-actin expression and expressed as mean±SD of 6 aggregates per group from three independent experiments with similar tendencies. **<i>P<</i>0.01, compared to day 0 (undifferentiated MSC), by Dunnett’s multiple comparison test.</p

PKA activation is required for chondrogenic differentiation of human MSCs.

<p>A, Paraffin sections from aggregates cultured in the presence of 10 µM H89 for 21 days were stained with Safranin O and anti-type II collagen antibody. Original magnification×10. Scale bars represent 500 µm. B, <i>COL2A1, ACAN, COL10A1</i>, and <i>ALP</i> mRNA levels in aggregates treated with the indicated concentrations of H89 for 21 days were determined by real-time PCR. Values are mean±SD of three aggregates from 1 of 2 independent experiments with similar findings. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079463#pone-0079463-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079463#pone-0079463-g002" target="_blank">2</a> for the definition of other symbols.</p

Nano-fiber plugs induce osteogenesis of human MSCs.

<p>Healthy donor-derived MSCs were seeded onto plastic plates (A-C) or injected into the nano-fiber plugs (D-I), and then cultured in MSCGM for 1 day (D), 7 days (E, G), 14 days (F, H) and 28 days (A-C, I). Then, the expression levels of RUNX2 (A, D, G), osteocalcin (B, E, H), and DMP-1 (C, F, I) were evaluated immunohistochemically. Representative results of three experiments are shown. (J) Healthy donor-derived MSCs were cultured in MSCGM onto plastic plates (two-dimensional) or nano-fiber plugs (three-dimensional) for 28 days. After RNA extraction, <i>MEPE</i> expression in the 4 groups of healthy donor-derived MSCswas evaluated by real-time PCR. *p<0.05 vs. two-dimensional culture by paired <i>t</i>-test. Scale bars, 50 μm.</p

Characteristics of hMSCs derived from healthy donors or patients with RA/OA.

<p>Expanded bone marrow cells collected from healthy donors (n = 6) or patients with RA (n = 3), OA (n = 3) were analyzed as for their characteristics. Representative histograms of the cell surface markers on bone marrow cells derived from healthy donors (A), patients with RA (B), OA (C) were shown. Red lines show isotype controls. Gene expression of <i>RUNX2</i> (D) and <i>SOX9</i> (E) in hMSCs was analyzed by real-time PCR. n.s.: not significant by ANOVA.</p

The hierarchy of gene expression in MSCs during differentiaion into osteoblasts, chondrocytes, and osteocytes.

<p>MSCs differentiate into osteoblasts or chondrocytes under the regulation of their master regulators, RUNX2 or SOX9, respectively. A part of chondrocytes also differentiate into osteoblasts.</p

Nano-fiber plugs induce morphological changes in MSCs.

<p>Healthy donor-derived MSCs were seeded onto nano-fiber plugs, and then cultured in MSCGM. Scanning electron micrographs taken on Days 7 (A) and 28 (B) are shown. All samples were tested three times, and representative results are shown. Scale bars, 50 μm.</p

Local Delivery of Mesenchymal Stem Cells with Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold Suppress Arthritis in Rats

<div><p>Mesenchymal stem cells (MSC) have been used recently for the treatment of autoimmune diseases in murine animal models due to the immunoregulatory capacity. Current utilization of MSC requires cells in certain quantity with multiple courses of administration, leading to limitation in clinical usage. Here we efficiently treated collagen-induced arthritis rats with a single local implantation with reduced number of MSC (2∼20% of previous studies) with nano-fiber poly-lactic-co-glycolic acid (nano-fiber) scaffold. MSC seeded on nano-fiber scaffold suppressed arthritis and bone destruction due to inhibition of systemic inflammatory reaction and immune response by suppressing T cell proliferation and reducing anti- type II collagen antibody production. <i>In vivo</i> tracing of MSC demonstrated that these cells remained within the scaffold without migrating to other organs. Meanwhile, <i>in vitro</i> culture of MSC with nano-fiber scaffold significantly increased TGF-β1 production. These results indicate an efficient utilization of MSC with the scaffold for destructive joints in rheumatoid arthritis by a single and local inoculation. Thus, our data may serve as a new strategy for MSC-based therapy in inflammatory diseases and an alternative delivery method for bone destruction treatment.</p></div