184 ISOLATION AND CHARACTERIZATION OF BOVINE ENDOMETRIAL STEM CELLS

Abstract

Adult mesenchymal stem cells had been isolated from various tissues of different species, including endometrial tissue of humans, mice, and pigs, but not from cattle. The aim of our work was to identify such cells in the bovine endometrium and to establish a model system in which to test inducers of differentiation and recruiters of stem cell niches, for potential therapeutic use in this and other species, such as horses. We searched for endometrial stem cells in healthy cycling cows and in cattle with clinical (C) or subclinical (SC) endometritis. For this, the uterine tracts of slaughtered cows were collected at early (Days 2 to 5; ELF) and late luteal phases (Days 11 to 15; LLF) of the oestrus cycle of healthy cows. For endometritis-diseased cattle, uterine biopsies were taken in live animals. In all cases, markers of stemness, inflammation, uterine function, and housekeeping were studied both at mRNA and protein level, by RT-qPCR and Western blot/immunohistochemistry respectively. In addition, cell primary cultures were established in vitro from all the animals (n = 4 for ELF, n = 4 for LLF; n = 4 for C and n = 4 for SC). We found that the endometrium of the majority of studied animals expressed embryonic stem cell markers, OCT4 and SOX2, but not or little NANOG, as well as CD44, c-Kit, and STAT3, all markers of mesenchymal stem cells. The expression profile of these markers was not related to the stage of the oestrus cycle; however there was a statistically significant reduction in the expression of embryonic stem cell markers in ill animals, being the lowest in clinically ill and intermediate in subclinical endometritis, (P < 0.05 and Pearson correlation coefficient 0.92). For markers of multipotency (mesenchymal), the expression was lower in clinical endometritis (P < 0.05). In conclusion, the expression profile of stem cell markers is indicative of the presence of stem cells in the bovine endometrium. At the protein level, we verified our findings for OCT4, SOX2, and CD44 using Western blot and immunohistochemistry. In general, there was a concordance between mRNA and protein profiles. Inflammatory markers showed a pattern characteristic for each of the studied stages. In order to have an ultimate criterion of the presence of stem cells, we tested the differentiation potential of the isolated cell lines, upon induction towards chondrogenic, osteogenic, and adipogenic lineages. We found that all the cell lines tested (n = 8) displayed mesenchymal differentiation potential as demonstrated by specific staining and gene expression markers. At present, work is in progress to isolate pure stem cell populations from these primary cultures to further characterise these cells. Conclusion: we showed for the first time the presence and differentiation potential of endometrial stem cells in cattle. This can have an effect on the development of new therapeutic approaches to combat uterine diseases, such as endometritis or endometriosis (in horses).

This work was supported by grant FONDECYT REGULAR 1110642, from the Government of Chile.