190 Profile of MicroRNAs of Equine Amniotic Mesenchymal Cell-Derived Microvesicles in Different Time-Span of In Vitro Culture

Abstract

Mesenchymal stromal cells (MSC) were found to secrete many factors with therapeutic relevance for their antioxidants, antiapoptotic, antifibrotic, angiogenic, immunomodulatory, and chemiotactic activities. The culture supernatant of MSC (in our case, of equine amniotic-derived cells) is defined secretome or conditioned medium (CM) and it is composed of soluble and insoluble factors secreted by cells. Soluble factors are represented by cytokines and growth factors, whereas microvesicles (MV), recently demonstrated to be an integral component of cell-to-cell communication during tissue regeneration, represent insoluble factors (Bruno et al. 2009 J. Am. Soc. Nephrol. 20, 1053-1067). Our previous data showed that equine amniotic-derived CM administered in vivo in equine spontaneous tendon lesion is able to regenerate the injured tissue. overlapping the results obtained by using in vivo the cells of origin in the same pathology (Lange-Consiglio et al. 2013 Stem Cells Devel. 22, 3015-3024). We also studied the amniotic-derived MV and found that they are involved in down-regulation of pro-inflammatory genes in in vitro LPS-stressed equine tendon and endometrial cells (Lange-Consiglio et al. 2016 Stem Cells Devel. 25, 610-621; Perrini et al. 2016 Stem Cell Res. Ther. 7, 169). Usually, protocols to produce CM and MV are different: CM is collected after 48 to 96 h of culturing cells without renewal of tissue culture medium, whereas MV are collected after culturing cells overnight. In future comparative studies of regenerative medicine aiming to understand the efficacy of CM and MV, understanding the quality of secretion of cells according to culturing time is crucial. The aim of the present study was to evaluate whether different times of culture can influence the micro-RNA (miRNA) cargo of equine amniotic-derived cells and their MV. Biological triplicates of a pool of amniotic-derived cells from 2 amniotic membranes and their MV, collected after overnight or after 4 days, were used. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisSEqn 2000 (Illumina Inc., San Diego, CA, USA). Mirdeep2 on Illumina high-quality trimmed sequences was used to detect known miRNAs and to support the individuation of novel miRNAs. The analysis identified 335 Equus caballus miRNA, which were quantified in both amniotic cells and MV. Hierarchical clustering of 83 miRNA that were observed in all 12 samples clearly discriminated amniotic cells and MV. The profile of miRNA in AMC and MV was similar between overnight and 4 days of culture. Further functional studies on the predicted target genes and pathways involved in the biological effect of equine amniotic secretome will be performed.