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Vertebrate reproductive science and technology
RESEARCH ARTICLE

185 Overexpression or CRISPr/Cas9-mediated inhibition of prostaglandin E2 receptors EP2 and EP4 in equine adipose mesenchymal stem cells: implications for cell migration and cellular therapies

A. C. F. Mançanares A , J. O. Manríquez A , J. Cabezas A , F. Telleria B , L. Rodriguez A and F. O. Castro A
+ Author Affiliations
- Author Affiliations

A Animal Science, University of Concepcion, Chillán, Chile;

B Ingeneria en Biotecnologia, Facultad de Ciencias Agrarias y Forestales, Universidad Catolica del Maule, Región del Maule, Talca, Chile

Reproduction, Fertility and Development 31(1) 217-217 https://doi.org/10.1071/RDv31n1Ab185
Published online: 3 December 2018

Abstract

Prostaglandin E2 (PGE2) acts through 4 cellular receptors: EP1, EP2, EP3, and EP4; only EP2 and EP4 are relevant for immunomodulation and migration in immune cells. Besides those, several cells express these receptors on their surface, including mesenchymal stem cells. Pharmacological inhibition of the EP2 receptor prevents migration of immune system cells to inflamed sites, where the concentration of PGE2 is high. Based on this, we hypothesised that overexpression of EP2 or EP4 receptors in equine mesenchymal stem cells (eMSC) will improve their migration to inflammatory sites and subsequent homing capability. Conversely, their suppression will lead to low or no migration, favouring the paracrine properties of MSC in the processes of tissue regeneration and reduction of inflammation. To test this, we manipulated the PGE2-EP2-EP4 axis and evaluated the effect of such modifications on transgenic cells in vitro. Equine MSC from adipose tissue were obtained from 5 animals. The coding sequences of both receptors were synthesised (GenScript, Hong Kong) based on the published horse genome (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/) and cloned into pcDNA3.1 overexpression vectors (Addgene). The resulting constructs were lipofected into naïve adipose eMSC. For knockouts, we PCR-amplified and sequenced horse EP2 and EP4 receptors, and gRNAs were created based on the obtained sequences and ligated into LentiCRISPRV2 plasmid (Addgene, Cambridge, MA, USA). The lentiviral vector plus helping packaging plasmids were co-transfected into HEK293FT cells and the produced viral particles were harvested and transduced into adipose eMSC. After 48 h of transfection (for overexpression) or transduction (for knockout, KO), cells were probed for the presence/absence of EP2 and EP4 receptors by immunohistochemistry and/or quantitative (q)PCR. Mitomycin-C-treated cells of both phenotypes and naïve, were subjected to migration in scratch assay, towards 3 mM PGE2. Fetal calf serum (10 or 0%) was used as positive or negative control, respectively, in migration experiments. Receptors EP2 and EP4 were clearly overexpressed after transfection as determined by immunocytochemistry or qPCR assays (phenotype MSC-EP2+/EP4+), whereas in the cells that underwent KO, little or no expression of EP2 and EP4 was detected (phenotype MSC-EP2ko/EP4ko) compared with unmanipulated cells (naïve MSC-Ctr). In the migration experiments towards 3 mM of PGE2, MSC-EP2+/EP4+ cells at 24 h filled the scratch faster (P < 0.05) than MSC-EP2ko/EP4ko or MSC-Ctr. These results showed that manipulation of PGE2-EP2/EP4 axis receptors led to changes in cell surface receptor availability and increased the migration pattern in overexpressed cells compared with KO and unmanipulated cells. These factors may affect the design of cellular therapeutic tools for inflammatory diseases in the equine species.

This research was supported by FONDECYT 3170390 to ACFM, Ministry of Education, Chile.