Anti-inflammatory and immunomodulatory miRNA cargo of microvesicles obtained from equine amniotic progenitor cells in different time-span of in vitro culture

Mesenchymal stromal cells (MSCs) were found to secrete many factors with therapeutic relevance for their anti-oxidants, anti-apoptotic, anti-fibrotic, angiogenic, immunomodulatory and chemiotactic activities. The culture supernatant of MSCs (in our case namely of equine amniotic derived cells) is defined secretome or conditioned medium (CM) and it is composed of soluble and no soluble factors secreted by cells. Soluble factors are represented by cytokines and growth factors, while microvesicles (MVs), that it has recently been demonstrated to be an integral component of cell-to-cell communication during tissue regeneration, represent no soluble factors (Bruno et al., 2009 Journal of the American Society of Nephrology 20, 1053–1067). Our previous data showed that equine amniotic derived CM, administrated 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 Development 22, 3015–3024). We also studied the amniotic derived MVs and we found that they are involved in downregulation of pro-inflammatory genes in in vitro LPS stressed equine tendon and endometrial cells (Lange-Consiglio et al,. 2016 Stem Cells Development 25, 610–621; Perrini et al,. 2016 Stem Cell Research and Therapy 7, 169). Usually, protocols to produce CM and MVs are different: CM can be collected after 12-96 hours of culturing cells without renewal of tissue culture medium, while MVs are usually collected culturing cells overnight. Future comparative study of regenerative medicine using CM and MVs will be difficult to interpret because there are not information about the quality of secretion of cells and the difference in micro-RNA cargo in MVs isolated after only 1 night of culture or isolated from CM that usually required a longer culture time. In this context, the aim of this relation is to comparatively report the miRNA content in AMCs and their MVs in two time points chosen between 1 night and 4 nights (the longer time of production of CM identified in literature and used in our previous studies) to evaluate the effect of different times of culture on quality of secretome. An enrichment of miRNAs, that have an important role in the immunity and anti-inflammatory response, is found both in cells and MVs at 4 nights of culture compared to 1 night. Simultaneously, an increase of MVs size due to the presence of apoptotic bodies is discovered. It will be interesting to isolate these apoptotic bodies in order to understand how their miRNA content is able to affect the miRNA library. In this way, it would be possible to give a more precise answer to the question if is better the secretoma of 1 night or 4 nights.