MOESM1 of Culture expansion of adipose derived stromal cells. A closed automated Quantum Cell Expansion System compared with manual flask-based culture

Additional file 1: Fig S1. Differentiation of ASCs after culture in either T75 flasks or quantum system towards adipogenic, osteogenic, and chondrogenic lineage. Differentiation was evaluated by cytochemically staining with Oil Red O for adipogenic, Alizarin Red S for osteogenic, and Alcian Blue for chondrogenic

Stabilisation of HIF-1 in hASCs after trypsin and 1% oxygen exposure alone or in combination.

<p>(<b>A</b>) HIF-1 activation/stabilization during 6 hours culture after trypsin exposure in combination with 24 hours in hypoxic/normoxic conditions was analysed by ELISA. All cells were harvested <i>in situ</i>. Values are represented as the mean and SEM (n = 12). Asterisks denote statistical difference between this and all other groups (p<0.05). (<b>B</b>) Analysis of HIF-1α induction at 4 and 12 hours following 5 min trypsin exposure was done by immunoblotting. All cells were harvested <i>in situ</i>. HIF-1α positive controls are ASCs subjected to 48 hours of 1% oxygen.</p

Trypsin-activated PAR2 intracellular signaling in hASCs.

<p>(<b>A</b>) Schematic rendition of signal-transduction pathways linking PAR2 and <i>VEGF</i>. (<b>B</b>) The effect of specific kinase inhibitors on suppressing trypsin-induced <i>VEGF</i> activation after 5 min trypsin exposure was assessed by real-time RT-PCR (n = 6). Expression levels were normalized to the levels induced by trypsin (Ctrl). (<b>C</b>) The effect of PI3K and Mek inhibitors on phosphorylation of Akt and Erk1/2, respectively, as a result of 5 min trypsin exposure was determined by immunoblotting. PI3K and Mek inhibitors were added 2 hours prior to trypsin exposure. Cells after a 4-day culture at 20% oxygen were used as controls (Ctrl). Representative data obtained from ASC12 cells are presented. Values are represented as the mean and SEM. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control.</p

Expression of PAR2 in hASCs and its association with transcriptional activation of <i>VEGF</i>.

<p>(<b>A</b>) Detection of PAR2 in hASC lines by immunoblotting. (<b>B</b>) Expression of PAR2 by indirect immunofluorescence using SAM11 antibody. Representative pattern as detected on the surface of ASC12 cells is presented. (<b>C</b>) SAM11 antibody blocked trypsin-induced PAR2 activation, measured using real-time RT-PCR to determine <i>VEGF</i> expression levels 12 hours after trypsin exposure (n = 15). Expression levels were corrected for basal <i>VEGF</i> activity in hASCs cultured at 20% oxygen and normalised to the levels induced by trypsin. Values are represented as the mean and SEM. Scale bar indicates 200 µm. Abbreviations: PAR2, protease-activated receptor 2; VEGF, vascular endothelial growth factor; Ctrl, control (NIH 3T3 cells).</p

Immunophenotypical analysis of hASC lines at passage 2.

<p>(<b>A</b>) Representative distributions of positive markers expressed on the ASC12 cells are presented. (<b>B</b>) Surface markers profile was obtained as an average from ASC12, 21, and 23 lines.</p

Blocking cys-LT signaling reduces myocardial hypoxic load.

<p>Hypoxic myocardial load is significantly upregulated under acute hypoxic stress in Apoe^−/− (#, p<0.05 versus Apoe^−/− normoxia); pretreatment with Montelukast (Hypoxia+MTK) reduces hypoxic myocardial load to levels equal to those observed in hearts of Apoe^−/− mice under normoxia (**, p<0.01 versus Apoe^−/− hypoxia) (Panel A). Panel B is a representative immunostaining for negative control (1) and positive staining (2) with 2 different magnifications. Hypoxyprobe signal was detected and quantified as described in the methods section. Results of Panel A are expressed as mean±SD of C57BL/6J normoxia n = 4; C57BL/6J hypoxia n = 5; Apoe^−/− normoxia n = 6; Apoe^−/− hypoxia n = 7; Apoe^−/− hypoxia with montelukast pre-treatment n = 7.</p

LTC₄S and CysLT1 are upregulated in cardiac tissue of Apoe^−/− mice.

<p>In the Apoe^−/− heart, levels of LTC₄S and CysLT1 are significantly upregulated as compared to control C57BL/6J mice (Panel A). Acute hypoxic stress in Apoe^−/− mice increases the cardiac expression of LTC₄S (p<0.05) and CysLT1 (p = 0.06 for two sided t-test, p<0.05 for one sided t-test) compared to normoxic conditions (Panel B). Each experiment was run in duplicate and changes in mRNA levels were expressed as ΔΔCt values and presented as relative to the mean of C57BL/6J mice (Panel A) or normoxia (Panel B). Values are mean±SD of C57BL/6J normoxia n = 4; Apoe^−/− normoxia n = 6; Apoe^−/− hypoxia n = 7. *, p<0.05; **, p<0.01.</p

CysLT1 receptor is expressed in cardiac mouse tissue.

<p>Immunohistochemistry analysis confirms CysLT1 receptor expression in mouse heart tissue notably in endothelial cells. The panels show representative immunostaining for CysLT1 receptor expression in (A) Apoe−/− without hypoxia; (B) Apoe−/− following hypoxic stress and (C) negative controls. The arrows point to positive staining cells. The results are representative of staining performed in n = 6 per group.</p

LTC₄S enzyme activity is increased in cardiac tissue of Apoe^−/− mice.

<p>LTC₄S enzymatic activity is enhanced in Apoe^−/− mice compared to C57BL/6J mice under both normoxic (not statistically significant) and hypoxic conditions (#, p<0.05 versus C57BL/6J hypoxia). LTC₄S enzymatic activity was enhanced by 10-fold in Apoe^−/− mice under acute hypoxia compared to normoxia condition (*, p<0.05 versus Apoe^−/− normoxia). Results expressed as LTC₄ (pg/mg microsomal pellet) are mean ± SD of C57BL/6J normoxia n = 3; C57BL/6J hypoxia n = 5; Apoe^−/− normoxia n = 4; Apoe^−/− hypoxia n = 3.</p

Additional file 2: Figure S2. of Cardiovascular magnetic resonance imaging of myocardial oedema following acute myocardial infarction: Is whole heart coverage necessary?

Correlation coefficient between 3-slice MM and 10-slice MM. Association between 3-slice MM quantification and 10-slice MM quantification assessed by CMR. (TIFF 114 kb