hBMSCs were cultured until 60% confluence. Then, the medium was discarded and, after three PBS 1X washes, replaced with α-MEM-GlutaMax medium with only 100 U/ml of Penicillin-100 mg/ml Streptomycin (basal medium). Cells were maintained under normoxic (20% O2 and 5% CO2 at 37 °C) or hypoxic (1% O2 and 5% CO2 at 37 °C in a hypoxic incubator) conditions, for 72 h. Conditioned media (CM) were collected and centrifuged at 300 g for 10 min at 4 °C to eliminate dead cells and debris, and at 2,000 g for 20 min at 4 °C to get rid of apoptotic bodies. Clarified CM was then ultracentrifuged at 10,000 g for 40 min at 4 °C, and finally at 100,000 g for 2 h at 4 °C, to pellet small-size EVs (sEVs).
Growth protocol
Cells were cultured in Alpha-MEM-GlutaMAX, supplemented with 100 U/ml penicillin-100 mg/ml streptomycin mixture and (i) 10% FBS, 1 ng/ml Fibroblast Growth Factor-2 for standard culture conditions, (FBS-samples) or (ii) Xeno-free Purstem supplement (XFS-samples).
Extracted molecule
total RNA
Extraction protocol
Extracellular Vesicles (EVs) were suspended in 1 ml of Qiazol reagent (Qiagen); EV-RNA was extracted with the miRNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions and stored at -80 °C. MiRNA content was evaluated by the Qubit® 2.0 Fluorometer using the Qubit® microRNA Assay Kit (Thermo Fisher Scientific) and small RNA quality and size were assessed by Agilent 2100 Bioanalyzer using the Small RNA chip (Agilent Technologies, Santa Clara, CA, USA).
Label
Cyanine 3-pCp
Label protocol
EV-RNA samples were processed using the Agilent miRNA complete labeling and hybridization kit (part number 5190-0456) and Agilent miRNA spike-in kit (part number 5190-1934). The Agilent miRNA microarray was performed using 50 ng of purified EV- RNA.
Hybridization protocol
After adding Hyb Spike-In, each sample was hybridized at 55°C for 20 hours on G4872A-070156 Human miRNA Microarray, Release 21.0, 8 x 60K (Agilent Technologies). The supplement of a commercial synthetic DNA poly-A oligonucleotide 3’ labeled pCp-Cy3, was added to the final hybridization mix. The 30-mer synthetic oligonucleotide (HPLC purified) was synthesized by TIB Molbiol s.r.l. (Genova, Italy) at 50 amol/μL.
Scan protocol
Microarrays were scanned (3 microns, 20 bit) on an Agilent microarray scanner (part number G2505C) and data extracted using Agilent feature extraction v.12.0.0.7. QC metrics from feature extraction were used to evaluate the success of the labeling and hybridization.
Description
miRNA profiling
Data processing
Pre-processing, filtering and differential expression analysis were carried out using the Limma package, available within Bioconductor. Background correction and between array normalization were carried out using the normexp method, with an offset = 20, and the quantile method, respectively. Replicated probes were then averaged.
