Effects of the calcimimetic AMG641 on cell viability of size-sieved eUCM-MSC lines.

<p>Cell viability is expressed as percentage of living (unstained) cells after Trypan blue staining. The effects of increasing doses of AMG641 in presence of 2.5 mM extracellular Ca²⁺ on the large (Panel A) and the small (Panel B) eUCM-MSC cell line are shown. Cell viability remained at high values, indicating non toxic effects of AMG641 on these cell lines. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111533#pone-0111533-g001" target="_blank">Figure 1</a>, values in cells cultured in medium with low calcium concentration are referred as negative controls (CTRL) and values observed in cells cultured in presence of 3 µM NPS R-467 are referred as positive controls (R-467). Data are mean ± standard deviation of values obtained in three replicates. Student's t-test: NS.</p

Oligonucleotide sequences used for Real Time-PCR analysis.

<p>Oligonucleotide sequences used for Real Time-PCR analysis.</p

Effects of calcium and the calcimimetic AMG641 on osteogenic and neurogenic differentiation efficiency of size-sieved eUCM-MSC lines, as assessed by quantification of extracellular matrix mineralization and Nissl bodies formation.

<p>Densitometric analysis was performed in large and small cells after von Kossa (Panel A and B) and Nissl staining (Panel C and D). For each experimental condition, areas of constant size were measured. Staining intensity was evaluated by recording: 1) the total stained area, 2) the number of stained spots and 3) the mean spot area. Data (mean ± standard deviation of values obtained in three replicates) are expressed as number of pixels. Cells induced to osteogenic differentiation showed higher mineralization levels in response to CaSR activation by the calcimimetic AMG641 (large and small cells). The addition of the CaSR antagonist NPS2390 reversed the agonists effects. In both cell lines, no statistical differences were noticed among treatments for Nissl staining intensity values. Student's t-test: comparisons AMG641-treated cells vs controls (low and high calcium) and among used AMG641 concentrations: *P<0.05; **P<0.01; ***P<0.001.</p

Relative abundance of CaSR and specific osteogenic and neurogenic differentiation biomarkers in eUCM-MSCs upon calcimimetic AMG641-induced CaSR stimulation.

<p>Panels A, B: Quantitative Real Time RT-PCR analysis of CaSR, OPN and RUNX2 transcripts inhigh [Ca²⁺]_o and (0.05, 0.1 or 1 µM) calcimimetic AMG641-treated equine UCM-MSCs (2.5 mM CaCl₂+AMG641) or cells pre-incubated with the CaSR antagonist NPS2390 (2.5 mM CaCl₂+NPS2390+AMG641) versus cells cultured in high [Ca²⁺]_o (2.5 mM CaCl₂) alone (indicated as 0 µM AMG641). In the large cell line, AMG641 increased OPN transcription (P<0.05 and P<0.01 with 0.1 and 1 µM AMG641, respectively) and had no effect on RUNX2 expression (Panel A). In the small cell line, AMG641 significantly down-regulated OPN (P<0.05, P<0.01 and P<0.05 respectively in cells stimulated with 0.05, 0.1 and 1 µM AMG641) and RUNX2 transcription (P<0.05 in cells treated with 0.1 µM AMG641) (Panel B). Upon AMG641 addition, CaSR expression was significantly down-regulated in the large cell line (P<0.01 at 0.05 and 0.1 µM AMG641), but markedly up-regulated (up to approximately 9 folds) in the small cell line, with a dose-response effect (P<0.05, P<0.01 and P<0.0001, at 0.05, 0.1 and 1 µM AMG641, respectively). In both cell lines, the addion of NPS2390 reversed the effects of AMG641 on OPN mRNA levels, with significant reduction in large cells (P<0.01) and significant increase in small cells (P<0.001) whereas it had no effects on RUNX2 mRNA levels. In both cell lines, the CaSR relative abundance remained at comparable levels after agonist or antagonist/agonist treatments. Panels C, D: Quantitative Real Time RT-PCR analysis of CaSR, Nestin and GFAP transcripts in cells cultured in the conditions as described for Panels A,B. In both cell lines, AMG641 significantly increased Nestin mRNA level at the highest tested dose (1 µM, P<0.05; Panels C and D). The GFAP transcript was never detected in any examined condition (not shown). As well, in both cell lines, AMG641 significantly increased CaSR transcription level (up to six folds in the large cell line; P<0.05 and P<0.001 at 0.05 and 1 µM AMG641 respectively; Panel C and up to approximately 4 folds in the small cell line; P<0.05 an P<0.001 at 0.05 and 0.1 µM AMG641, respectively; Panel D). In both cell line types, no repeatable reverse effects were obtained upon NPS2390 addition (data not shown). For each sample, average Ct data (mean±SD of three independent experiments in duplicate) were normalized relatively to the abundance of HPRT1 mRNA (endogenous control) and normalized values were compared among groups. Student's t-Test: a,b P<0.05; c,d P<0.01; e,f P<0.001; * P<0.01; ** P<0.001.</p

Relative abundance of CaSR and specific osteogenic and neurogenic differentiation biomarkers in eUCM-MSCs upon Ca²⁺–induced CaSR stimulation.

<p>Panels A, B: Quantitative Real Time RT-PCR analysis of CaSR, OPN and RUNX2 transcripts in high [Ca²⁺]_o-treated equine UCM-MSCs (2.5 mM CaCl₂) versus low [Ca²⁺]_o controls (CTRL). In the large cell line, additional Ca²⁺ up-regulated CaSR (P<0.05) and OPN (P<0.05) mRNA expression even if it had no effect on RUNX2 (Panel A). In the small cell line, high [Ca²⁺]_o significantly increased both OPN and RUNX2 transcripts level (P<0.001 and P<0.01, respectively; Panel B) but it had no effects on CaSR expression. Panels C, D: Quantitative Real Time RT-PCR analysis of CaSR, Nestin and GFAP transcripts in high [Ca²⁺]_o-treated equine UCM-MSCs (2.5 mM CaCl₂) versus low [Ca²⁺]_ocontrols (CTRL). In both cell lines, additional Ca²⁺ up-regulated Nestin (P<0.05 in the large cell line, Panel C; P<0.01 in the small cell line, Panel D) and CaSR (P<0.05) mRNA expression whereas the GFAP transcript was not detected. For each sample, average Ct data (mean±SD of three independent experiments in duplicate) were normalized relatively to the abundance of HPRT1 mRNA (endogenous control) and normalized values were compared between groups. Student's t-Test; *P<0.05; **P<0.01; ***P<0.001.</p

Bone-like and neuron-like morphology, cytochemical and histochemical features of AMG641-treated large eUCM-MSCs as evaluated by ALP activity, von Kossa and Nissl stainings and beta III tubulin immunodetection.

<p>Photomicrographs representative of the morphological appearance of the large eUCM-MSC line induced to differentiate <i>in vitro</i> toward osteogenic (1^st and 2^nd column) or neurogenic (3^rd and 4^4h column) lineages in presence of 1µM AMG641 and observed after ALP activity assay, von Kossa and Nissl stainings, and beta III tubulin immunostaining. (A, F, M, R) control cells differentiated <i>in vitro</i> in presence of low [Ca²⁺]_o; (B, G, N, S) cells differentiated <i>in vitro</i> in presence of high [Ca²⁺]_o; (C, H, O, T) AMG641-treated cells (treated with 1 µM AMG641 in presence of of high [Ca²⁺]_o); (D, I, P, U) NPS2390/AMG641-treated cells (cells pre-incubated for 10 min in 10 mM NPS2390 and then cultured in 1 µM AMG641in presence of of high [Ca²⁺]_o); (E, L, Q, V) non-induced controls. Osteogenic differentiation was evidenced by increased ALP activity observed after 14 days <i>in vitro</i> culture and by the formation of mineralized matrix as shown by von Kossa staining after 21 days <i>in vitro</i> culture. No apparent differences can be seen for ALP activity and for the presence of mineral deposits after treatment with high [Ca²⁺]_o (B, G) or with AMG641 in high [Ca²⁺]_o (C, H) compared with controls (CTRL: A, F). ALP positivity and amount of calcium phosphate deposits appear to be reduced in antagonist/agonist-treated cells (D, I) compared with those treated with the CaSR agonist AMG641 (C, H). Neurogenic differentiation was evidenced by the presence of Nissl bodies and beta III tubulin positivity, as observed after 4 days <i>in vitro</i> culture. All cells directed toward neurogenic differentiation showed diffused discrete granular bodies after Nissl staining with no apparent differences among those cultured in presence of low (M) or high [Ca²⁺]_o (N) or high [Ca²⁺]_o plus AMG641 (O) or in cells pre-incubated with the CaSR antagonist NPS2390 and subsequently treated with AMG641 (P). Some cells showed neuron-like appearance and pancytoplasmic beta III tubulin immunopositivity in all induced conditions (R,S,T, U) with no apparent differences among treatments. Non-induced control cells were unstained for osteogenic (E, L) and neurogenic (Q, V) markers. Black scale bars indicate 10 µm; white scale bars indicate 5 µm.</p

CaSR transcript relative abundance in eUCM-MSCs upon Ca²⁺- or calcimimetic-induced CaSR stimulation.

<p>Quantitative Real Time RT-PCR analysis of the CaSR transcript in Ca²⁺ (2.5 mM CaCl₂) and calcimimetic-treated (2.5+R-467) equine UCM-MSCs versus controls (CTRL) or cells pre-incubated with the CaSR antagonist (2.5+2390+R-467). Cells were examined on day 4^th after treatments. For each sample, data (mean ± SD of three independent experiments in duplicate, CaSR average Ct) were normalized relatively to the abundance of HPRT1 mRNA (endogenous control) and normalized values were compared among groups. In the large cell line (panel A), CaSR transcription was strongly down-regulated in presence of additional Ca²⁺ (2% of control value; vs b: P<0.001) and NPS R-467 (13%; a vs b: P<0.001). In the small cell line (panel B), CaSR transcription was again down-regulated by additional Ca²⁺ (25%; a vs b: P<0.001) and NPS R-467 (72%; a vs b: P<0.001) even at a lesser extent. In both cell lines, the CaSR antagonist NPS 2390 reversed the effects of NPS R-467 (38% and 97%, for the large and the small cell line, respectively; c vs d: P<0.05).</p

CaSR protein expression and subcellular localization in eUCM-MSCs upon Ca²⁺- or calcimimetic-induced CaSR stimulation.

<p>Detection of CaSR expression in equine UCM-MSCs in the >8 µm cell line (A, C) and <8 µm cell line (B, D) by immunofluorescence with a primary antibody against a 20 amino acid peptide sequence near the C-terminus of human CaSR and observation by confocal laser scanning microscopy. In both cell lines, cells showing CaSR labeling either predominantly evident whitin the cytoplasm (A, B) or on the plasma membrane (C, D) were present. For each cell, scanning was conducted with 12 optical series from the top to the botton of the cell with a step size of 0.45 µm and images were taken to the equatorial plane. Representative photomicrograph of equine UCM-MSC as observed after thawing, staining with Hoechst 33258 and observed under phase contrast microscopy merged with UV light epifluorescence (E). In this cell, regular round shape morphology and an eccentric nucleus can be seen. Negative minus primary control (F). Scale bar represent 20 µm (A, C, E, F) or 10 µm (B, D).</p

Effects of Ca²⁺, the calcimimetic NPS R-467 and the CaSR antagonist NPS2390 on eUCM-MSC growth.

<p>Cells were plated in 6-wells plates and treated as described in materials and methods. Effects of increasing doses of extracellular Ca²⁺ on both, large (A) and small (B), cell lines. In the large cell line (A'), increasing Ca²⁺ concentration to 2.5 mM was not effective on cell growth. In the presence of 2.5 mM Ca²⁺, the allosteric activator of the CaSR, NPS R-467 (3 µM) significantly increased the cell number/well from day 7^th after treatment (P<0.001). In the small cell line (B'), increasing Ca²⁺ concentration induced cell growth on day 9^th (P<0.05). In the presence of 2.5 mM Ca²⁺, the allosteric activator of the CaSR, NPS R-467 significantly increased the cell number/well on day 9^th (P<0.05). In both cell lines, 30 min preincubation in presence of the CaSR antagonist NPS 2390 (10 µM) significantly inhibited the agonist effect. Results are mean ± SD of 2 data points. Student's t Test: a vs b: P<0.05; a vs e: P<0.01; c vs d P<0.001.</p