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Stem cells isolated from amniotic epithelium (AECs) have shown great potential in cell-based regenerative therapies. Because of their fetal origin, these cells exhibit elevated proliferation rates and plasticity, as well as, immune tolerance and anti-inflammatory properties. These inherent attitudes make AECs well-suited for both allogenic and xenogenic cellular transplants in animal models. Since in human only at term amnion is easily obtainable after childbirth, limited information are so far available concerning the phenotypic and functional difference between AECs isolated from early and late amnia. To this regard, the sheep animal model offers an undoubted advantage in allowing the easy collection of both types of AECs in large quantity. The aim of this study was to determine the effect of gestational age on ovine AECs (oAECs) phenotype, immunomodulatory properties, global DNA methylation status and pluripotent differentiation ability towards mesodermic and ectodermic lineages. The immunomodulatory property of oAECs in inhibiting lymphocyte proliferation was mainly unaffected by gestational age. Conversely, gestation considerably affected the expression of surface markers, as well the expression and localization of pluripotency markers. In detail, with progression of gestation the mRNA expression of NANOG and SOX2 markers was reduced, while the ones of TERT and OCT4A was unaltered; but at the end of gestation NANOG, SOX2 and TERT proteins mainly localized outside the nuclear compartment. Regarding the differentiation ability, LPL (adipogenic-specific gene) mRNA content significantly increased in oAECs isolated from early amnia, while OCN (osteogenic-specific gene) and NEFM (neurogenic-specific gene) mRNA content significantly increased in oAECs isolated from late amnia, suggesting that gestational stage affected cell plasticity. Finally, the degree of global DNA methylation increased with gestational age. All these results indicate that gestational age is a key factor capable of influencing morphological and functional properties of oAECs, and thus probably affecting the outcome of cell transplantation therapies.

Gestational stage affects amniotic epithelial cells phenotype, methylation status, immunomodulatory and stemness properties