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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.

Cell-derived microvesicles (MVs) are a recently discovered mechanism of cell-to-cell communication. Our previous data show that MVs secreted by equine amniotic mesenchymal-derived cells (AMCs) are involved in downregulation of proinflammatory genes in lipopolysaccharide-stressed equine tendon and endometrial cells. The aim of the present study was to evaluate whether AMC-MVs contain selected microRNAs (miRNAs) involved in inflammation. Two pools of cells, derived from 3 amniotic membranes each, and their respective MVs were collected. Small RNAs were extracted and deep sequenced, followed by miRNA in silicodetection. The analysis identified 1,285 miRNAs, which were quantified both in AMCs and MVs. Among these miRNAs, 401 were classified as Equus caballusmiRNAs, 257 were predicted by homology with other species (cow, sheep, and goat), and 627 were novel candidate miRNAs. Moreover, 146 miRNAs differentially expressed (DE) in AMCs and MVs were identified, 36 of which were known and the remaining were novel. Among the known DE miRNAs, 17 showed higher expression in MVs. Three of these were validated by real time polymerase chain reaction: eca-miR-26, eca-miR-146a, and eca-miR-223. Gene ontology analysis of validated targets showed that the DE miRNAs in cells and MVs could be involved both in immune system regulation by modulating interleukin signaling and in the inflammatory process. In conclusion, this study suggests a significant role of AMCs in modulating immune response through cell–cell communication via MV-shuttling miRNAs.

Purpose Adult bone marrow mesenchymal stem cells (BM-MSCs) are a potential cell source for tendon repair in direct cell therapy and tissue engineering investigations. The purpose of this study was to evaluate the tenogenic induction of undifferentiated BM-MSCs under indirect co-culture technique with trimmed native tendon tissue. Since the horse represents a preferred species to study tendon regenerative strategies, this work was conducted on equine BM-MSCs.Methods Equine BM-MSCs were co-cultured in a transwell system with tendon tissue fragments. The BM-MSC tenogenic differentiation was evaluated by cytochemical staining and real time PCR for gene expression. Cell viability in tendon fragments and cultured cells was analyzed.Results Our results indicate that under indirect co-culture with native and healthy tendon tissue the BM-MSCs expressed tendon-specific markers such as decorin, tenomodulin, tenascin-C, and collagen type I. They also retained a tenocyte-like phenotype during monolayer culture.Conclusions Data are very encouraging for future in vitro investigations into committing cells to the tenogenic lineage without adding growth factors or serum to the culture medium for both cell therapy and tissue engineering.

To examine the paternal genome's role in reprogramming metabolic activity in one-cell embryos, we investigated metabolic aspects of bovine oocytes after in vitro maturation and in vitro fertilization and after in vitro parthenogenetic activation with a Ca²⁺ ionophore and 6-dimethylaminopurine. We assayed succinate dehydrogenase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities by microspectrophotometry in immature oocytes and oocytes after maturation, in vitro fertilization and parthenogenetic activation. Succinate dehydrogenase activity significantly increased after in vitro maturation, significantly decreased after Ca²⁺ ionophore activation and further decreased after 6-dimethylaminopurine treatment. Lactate dehydrogenase activity showed a significant decrease in bovine oocytes after in vitro maturation, remained unchanged in Ca²⁺ ionophore-treated oocytes and rose significantly after 6-dimethylaminopurine treatment. This activity was dramatically reduced after in vitro fertilization, reaching absorbance levels that were not different from those in mature and Ca²⁺ ionophore-treated oocytes. Glucose-6-phosphate dehydrogenase activity was significantly lower in matured oocytes as compared to immature oocytes, was significantly higher after artificial activation with Ca²⁺ ionophore and remained constant after 6-dimethylaminopurine treatment or after in vitro fertilization. We suggest that metabolic changes involved in parthenogenetic activation are similar to those occurring after fertilization.

A technology for encapsulation of swine semen in barium alginate and protamine alginate has recently been proposed for the controlled release of the spermatozoa, thus reducing the number of instrumental inseminations required. Controlled-release capsules containing swine spermatozoa were prepared by adding saturated BaCl2 solution to ejaculate and dropping the resulting suspension into a sodium alginate solution, leading to the formation of barium alginate capsules. A second type of capsule was obtained by cross-linking the barium alginate with protamine sulfate. Two types of membrane were thus obtained: barium alginate gel and a protamine cross-linked alginate membrane. Morphological (scanning electron microscopy and transmission electron microscopy), functional (motility, membrane integrity and in vitro fertilization test) and technological (capsule structure and weight) approaches were used to characterize the encapsulated spermatozoa and the controlled-delivery system. No differences in terms of morphological and functional characteristics (acrosome integrity and spermatozoa motility) between free and encapsulated semen were found. The technological process did not compromise in vitro fertilization potency of the spermatazoa, although seasonal variability was found. The capsule weight was related to either the pH of the semen or the season. This study represents the starting point for the development of further investigations into the storage and release kinetics of cells from the capsules and for the development of an in vivo fertilization protocol.