Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia A [Abstract]

Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia A [Abstract

HemAcure: Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia A

HemAcure: Application of combined gene and cell therapy within an implantable therapeutic device for the treatment of severe haemophilia

Immunophenotype of hCPC-derived spheroids.

<p>(<b>A</b>) Confocal microscopy images of spheroids for mesenchymal/stromal, stemness, extracellular matrix, and cardiomyogenic markers, YAP and HGF receptor expression using specific antibodies followed by secondary FITC-labelled antibodies or TRITC-phalloidin. One representative out of the three experiments performed with similar results is shown. Scale bar, 50 μm. (<b>B</b>) For comparison, images of hCPCs kept as monolayers at phase contrast microscope (top; scale bar 100 μm) or in immunofluorescence after staining for some of the same markers are shown (20 x magnification).</p

Migratory activity of hCPCs kept as monolayers or of cells migrated from seeded spheroids (wound healing assay).

<p>(<b>A</b>) Quiescent cell monolayers were “wounded” with a pipette tip and incubated for 24 hours in the absence (C-) or presence of 20% FBS (C+) or of different concentrations of the HGF receptor agonist mAb DO-24 (5, 20, 80 nM respectively). (<b>B</b>) Quantitative analysis calculated as % variation of cell numbers in the rectangles depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137999#pone.0137999.g005" target="_blank">Fig 5A</a> relative to untreated samples. The assay was repeated three times with triplicates and one representative assay is reported. Images were taken at 5x magnification. After 24 hours healing was significantly induced by 20% FBS and mAb DO-24 treatments. Cells derived from spheroids (dark grey bars) were more efficient than cells from 2D cultures at any condition (light grey bars).</p

Antibody array scheme.

<p>Antibody array scheme.</p

<i>In vivo</i> transplantation of spheroids in myocardium-injured mice.

<p>(<b>A</b>) Evaluation of cardiotoxin-induced injury in the myocardium wall at two magnifications (right) compared to healthy myocardium (left) in Hematoxylin-Eosin staining. (<b>B</b>) Myocardium sections from myocardium injured mice transplanted with spheroids were stained with TO-PRO3 (blue) to show all nuclei, anti-human nuclei antibody (green), and connexin-43 or phalloidin (red) for actin and analysed by confocal microscopy. Representative experiments out of three performed are shown. The circle and arrows show the engrafted spheroid 1 day after the injection and the dispersed hCPCs 7 days after the injection, respectively.</p

Primary antibodies used for immunofluorescence.

<p>Primary antibodies used for immunofluorescence.</p

Protein expression of different growth factors, their receptors, and MMP activities.

<p>(<b>A</b>) Antibody-array analysis of protein expression in supernatants (<b>A</b>) from 2D monolayers (left) and 3D spheroids (centre) and in cell extract (<b>B</b>) from 2D monolayers (left) and 3D spheroids (centre). Graphs on the right represent fold variation of protein levels in spheroids normalized <i>vs</i> monolayers (line). Data are expressed as mean ± SD of at least three experiments. Significant difference of p≤0.05 is indicated as * and p≤0.0001 is indicated as **. (<b>C</b>) Activities of MMP-2 and MMP-9 present in culture supernatants and cell extracts from monolayers 2D or 3D spheroids of hCPCs in a Gelatin-substrate zymography. Arrows indicate location of enzymatic activity corresponding to pro- and active forms of MMP-9 and MMP-2. Molecular weight markers (kDa) are shown on the left.</p

Morphology and viability of hCPC spheroids.

<p>(<b>A</b>) hCPC spheroids obtained from 5x10³, 10x10³ or 25x10³ cells before and after their passage through a 27G needle (scale bar, 100 μm). (<b>B</b>) Viability of spheroids as evaluated in a Live/Dead assay (viable cells in green, PI-positive dead cells in red). Scale bar, 100 μm. Four optical sections of the spheroids taken at different levels of the z-axis are reported. Live/Dead assay was performed also on 5x10³ cells spheroids after passage through the needle (A, bottom). One representative out of the three experiments performed with similar results is shown.</p

<i>In vivo</i> transplantation of spheroids in healthy mice.

<p>(<b>A</b>) Myocardium sections from mice after transplantation with hCPCs in suspension or as spheroids, at different time points, were stained with TO-PRO3 (blue) to show all nuclei, anti-human nuclei antibody (green), and phalloidin (red) for actin and analysed by confocal microscopy. (<b>B</b>) Myocardium sections from mice transplanted with spheroids were also stained at different time points with TO-PRO3 (blue), anti-human nuclei antibody (green), and anti-connexin 43 (red). Representative experiments out of three performed are shown.</p