Different culture times affect MicroRNA cargo in equine amniotic mesenchymal cells and their microvesicles

Conditioned medium (CM) and microvesicles (MVs) are produced using different protocols: CM is collected following 12-96 h of cell culture without renewal of tissue culture medium, while MVs are collected after overnight cell culture. For future comparative studies in regenerative medicine looking at the efficacy of CM and MVs, it is important to understand how the quality of cell secretions is affected by culture. The aim of this study was to evaluate whether the duration of culturing influences the micro-RNAs (miRNAs) cargo of equine amniotic mesenchymal cells (AMCs) and their MVs. The analysis identified 990 miRNAs. After one night, there were 347 differently expressed (DE)-miRNAs between MVs and cells, whereas after four nights there were 359. About 58.3% of the DE-miRNAs were shared between samples produced under the two conditions. The comparison between miRNA content in AMC cells cultured for one night versus four nights showed eight DE-Equus caballus (eca)-miRNAs, which target genes were involved in immune response to external stimulus, inflammatory response, and production of reactive oxygen species. Comparing MVs isolated from one or four nights, four DE-miRNAs that target genes regulating cell cycle progression and production of reactive oxygen species were found, but only eca-miR-214 was enriched in the MVs after four nights. In conclusion, after 4 days of cell culture, the profile of AMC miRNAs was altered, indicating a probable phenotypic transition versus a new cell culture environment and aging. After this time, MVs accumulated eca-miR-214, which may help cells survive or adapt to new culture conditions.