Efficient Route to Label Mesenchymal Stromal Cell-Derived Extracellular Vesicles

Recent research results report that extracellular vesicles (EVs) have a central role in both physiological and pathological processes involving intercellular communication. Herein, a simple EVs labeling procedure based on the metabolic labeling of secreting cells (mesenchymal stroma cells, MSCs) with a fluorescein-containing bio-orthogonal dye is described. This procedure was carried out by incubating cells for 72 h with tetraacetylated <i>N</i>-azidoacetyl-d-mannosamine (Ac₄ManNAz), a modified sugar containing an azido group that, upon incorporation on the external surface of the cytoplasmatic cell membrane, is specifically conjugated with cyclooctyne-modified fluorescein isothiocyanate (ADIBO-FITC). MSCs released fluorescent EVs did not need any further purification. Finally, cellular uptake and tracking of the fluorescein-labeled EVs were successfully assessed by targeting experiments with MSCs. The method appears of general applicability and it may be very useful opening new horizon on diagnostic and therapeutic protocols exploiting EVs

Additional 1: Table S1.

Mean Fluorescence Intensity (MFI) of monocyte-derived cells cultured in presence or absence of renal cancer cells (CD105+ CSCs and CD105- TCs). (DOCX 13 kb

Additional 2: Table S2.

Mean Fluorescence Intensity (MFI) of monocyte-derived cells stimulated with or without EVs shed by CD105+ CSCs and CD105- TCs. (DOCX 14 kb

List and function of genes that significantly differ between cisplatin-treated human tubular cells stimulated or not with MVs.

<p>List and function of genes that significantly differ between cisplatin-treated human tubular cells stimulated or not with MVs.</p

MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury.

<p>Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.</p

Body weight, survival, renal function and morphology in SCID mice injected with cisplatin and different regimens of MVs.

<p>Results are expressed as mean±SD; ANOVA with Dunnet’s multicomparison test: </p><p>* <i>p<</i>0.05 siMV and miMV treatments <i>vs</i> cisplatin (CIS); </p><p>† <i>p<</i>0.05 miMV <i>vs</i> siMV.</p><p>CIS = cisplatin injection; CIS+siMV = cisplatin treated with single injection of MVs; CIS+RNase-MV = cisplatin treated with injection of MV pre-treated with RNase; CIS+miMV = cisplatin treated with multiple injection of MVs.</p

Additional 3: Figure S1.

EVs characterization. A. Representative size distribution of EVs shed by CD105+ CSCs and CD105- TCs obtained using NanoSight LM10 instrument equipped with the nanoparticle tracking analysis (NTA) 2.0 analytic software. B. Representative cytofluorimetric analysis performed by Guava easyCyte Flow Cytometer of EVs shed by CD105+ CSCs and CD105- TCs and analyzed with InCyte software. The following markers were evaluated: CD44, CD105, ι5 integrin, ι6 integrin, CD73, CD29, CD90 and CD146. (DOCX 451 kb

Schematic representation of the protocol of cisplatin induced AKI and MV administration regimens and survival curves.

<p>A) Graph showing time-points of cisplatin administration, siMVs or miMVs and the time-points of sacrifice. B) Survival curves of SCID mice with cisplatin induced AKI treated with different regiments of MVs administration. All mice receiving vehicle alone died within 5 days. Mice that received siMVs or miMVs injections survived significantly longer than control mice treated with vehicle alone or with a si(RNase-inactivated)MVs. Data was analysed via a log-rank test: * <i>p</i><0.05 siMV <i>vs</i> CIS; ** <i>p</i><0.05 miMV <i>vs</i> siMV. Abbreviations: vehicle = cisplatin treated mice injected with vehicle alone; siMV = cisplatin treated mice with single injection of MVs; miMV = cisplatin treated mice with multiple injection of MVs; RNase MV = cisplatin treated mice injected with a single dose of MVs pre-treated with RNase.</p

Renal cell apoptosis and proliferation in cisplatin-AKI mice untreated or treated with different regiments of MVs.

<p>A) Quantification of Tunel-positive cells/high power field (hpf) at different time points. Data are expressed as mean ±SD of 8 different mice for each experimental condition. ANOVA with Dunnet’s multicomparison test was performed: * <i>p</i><0.05 siMVs or miMVs <i>vs</i> CIS; ** <i>p</i><0.05 miMVs <i>vs</i> siMVs. Representative micrographs of Tunel staining of renal sections of cisplatin mice given vehicle alone (day 4) and of cisplatin mice treated with different regiments of MVs at different time points (4, 14 and 21 days). Original magnification: ×400. B) Quantification of PCNA positive cells/hpf and of BrdU positive cells/hpf at different time points. BrdU was injected intraperitoneally for 2 successive days before mice being killed. Data are expressed as mean ±SD of 8 different mice for each experimental condition. ANOVA with Dunnet’s multicomparison test was performed: * <i>p</i><0.05 siMVs versus CIS. Abbreviations: Ctrl = healthy mice; CIS = cisplatin treated mice injected with vehicle alone; MV = cisplatin treated mice with single injection of MVs.</p

RNase treatment does not modify MV size, but reduces RNA content of MVs.

<p>A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of <i>SUMO-1</i>, <i>POLR2</i> and <i>Act B</i> transcripts in MVs treated or not with RNase, express as 2^-δCt, as described in material and methods.</p