Progesterone prevents epithelial-mesenchymal transition of ovine amniotic epithelial cells and enhances their immunomodulatory properties

Abstract

The in vitro expansion is detrimental to therapeutic applications of amniotic epithelial cells (AEC), an emerging source of fetal stem cells. This study provides molecular evidences of progesterone (P4) role in preventing epithelial-mesenchymal transition (EMT) in ovine AEC (oAEC). oAEC amplified under standard conditions spontaneously acquired mesenchymal properties through the up-regulation of EMT-transcription factors. P4 supplementation prevented phenotype shift by inhibiting the EMT-inducing mechanism such as the autocrine production of TGF-β and the activation of intracellular-related signaling. The effect of P4 still persisted for one passage after steroid removal from culture as well as steroid supplementation promptly reversed mesenchymal phenotype in oAEC which have experienced EMT during amplification. Furthermore, P4 promoted an acute up-regulation of pluripotent genes whereas enhanced basal and LPS-induced oAEC anti-inflammatory response with an increase in anti-inflammatory and a decrease in pro-inflammatory cytokines expression. Altogether, these results indicate that P4 supplementation is crucial to preserve epithelial phenotype and to enhance biological properties in expanded oAEC. Therefore, an innovative cultural approach is proposed in order to improve therapeutic potential of this promising source of epithelial stem cells.

Characterization of Endocannabinoid System and Interleukin Profiles in Ovine AEC: Cannabinoid Receptors Type-1 and Type-2 as Key Effectors of Pro-Inflammatory Response

“…However, it appears clear that in the presence of acute inflammatory stimuli, the AEC even reinforce their anti-inflammatory activity during the early and middle gestational stages. This seems to be a conservative mechanism that is able to increase the expression of anti-inflammatory cytokines in response to LPS stimulation, thus generating a compensatory mechanism that may limit the secretion of pro-inflammatory modulators and that could jeopardize the immune privilege during pregnancy by triggering pre-term labor [24,32].…”

“…AEC may exert a positive role in the cell-to-cell cross-talk between fetus and inflammatory system involved in pregnancy progression by supporting the high IL-10 levels, either under constitutive or LPS-activated conditions [24,32], until the late stage when AEC becomes permissive to the interleukin framework, inducing labor.…”

“…Isolated AEC were seeded at 3 × 10 3 cells/cm 2 and cultured until 80% confluence in alpha Eagle's minimum essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 20% FCS, 25 µM P 4 (Sigma-Aldrich Corp., St. Louis, MI, USA), 1% ultra glutamine (Lonza, Basel, Switzerland), 100 U/mL penicillin (Lonza Basel, Switzerland), 100 µg/mL streptomycin (Lonza Basel, Switzerland), and 2.5 µg/mL amphotericin (EuroClone Spa, Pero, MI, Italy), and incubated at 38.5 • C in 5% CO 2 according to Canciello et al [32]. This typology of cells was used in order to test CB binding.…”

“…The expression of ECS key genes such as metabolizing enzymes (NAPE-PLD, FAAH, DAGLα/β, and MAGL) and CB (CB1, CB2, and TRPV1) was assessed by using cells freshly isolated from AM epithelial layer collected at early (first month), middle (2-2.5 months), and late (3.5-4 months) stages of gestation [32]. The results showed that ECS was steady expressed in AEC, even if differently modulated during gestation ( Figure 1).…”

“…They include the secretion of cytokines and growth factors with antiapoptotic, proangiogenic, and immune-regulatory molecules [25][26][27][28][29][30][31] that overall contrast pro-inflammatory signals (cytokines and cells), thus enhancing anti-inflammatory, pro-regenerative immune pathways. Amongst them, it has recently been demonstrated in the amniotic epithelium that a highly conserved physiological mechanism controlling the expression and releasing balance of potent anti-inflammatory and pro-inflammatory cytokines such as interleukin (IL)-10, IL-4, and IL-12 under both basal and LPS-inductive conditions [24,[32][33][34].…”