Progesterone Prolongs Viability and Anti-inflammatory Functions of Explanted Preterm Ovine Amniotic Membrane

Abstract

Amniotic membrane (AM) is considered an important medical device with many applications in regenerative medicine. The therapeutic properties of AM are due to its resistant extracellular matrix and to the large number of bioactive molecules released by its cells. An important goal that still remains to be achieved is the identification of cultural and preservation protocols able to maintain in time the membrane morphology and the biological properties of its cells. Recently, our research group demonstrated that progesterone (P₄) is crucial in preventing the loss of the epithelial phenotype of amniotic epithelial cells in vitro. Followed by this premise, it has been evaluated whether P₄ may also affect AM properties in a short-term culture. Results confirm that P₄ preserves AM integrity and architecture with respect to untreated AM, which showed alterations in morphology. Transmission electron microscopy (TEM) analyses demonstrate that P₄ also maintains unaltered cell-cell junctions, nuclear status, and intracellular organelles. On the contrary, an untreated AM experienced an extensive cell death and a strong reduction of immunomodulatory properties, measured in terms of anti-inflammatory cytokine expression and secretion. Overall, these results could open to new strategies to ameliorate the protocols for cryopreservation and tissue culture, which represent preliminary stages of AM application in regenerative medicine.

Keywords: amniotic epithelial stem cells; amniotic membrane; immunomodulation; progesterone; regenerative medicine; tissue culture.

Figure 1

Amniotic membrane isolation and culture. (A) Sheep fetus isolated from the uterus according to Canciello et al. (2018) (see references). (B) Isolated AM pieces contain chorioallantois. (C) Amnion pieces appearance after mechanical removal of chorioallantois. (D) AM pieces during the in vitro culture.

Figure 2

Fresh AM histology, viability, and ultrastructure. (A) Fresh AM pieces were subjected to hematoxylin–eosin staining to evaluate their membrane architecture. Magnification 40×. (B) Fresh AMs were subjected to staining by using Calcein AM (green), DAPI (blue), and propidium iodide (red) to assess cell viability and mortality. Magnification 40×. (C–F) Ultrastructure of fresh AM shows cell morphology (C), nuclear details (n) (C,F), apical specializations and basal lamina (bl) (D), cell–cell junctions (cj) (E), and mitochondria (m) and RER (rer) (F).

Figure 3

AM histology, morphology, and viability. (A) AM pieces were cultured up to 3 days in the presence (P4) or in the absence (CTR) of P₄ supplementation and then were subjected to hematoxylin–eosin staining to evaluate their membrane architecture. (B) CTR and P4-treated AM were in vitro cultured up to 3 days and then were subjected to staining by using Calcein AM (green), DAPI (blue), and propidium iodide (red) to assess cell viability and mortality. Representative 40× magnification is shown for CTR and P4 day 1 acquisition; 60× magnification or days 2 and 3. Scale bar: 50 μm. (C) Quantification of Calcein AM, propidium iodide, and DAPI fluorescence was performed by using image-processing software ImageJ, version 1.40g (http://rsbweb.nih.gov/ij). The viability of freshly isolated AM was taken as 100%. Each experimental group was analyzed in triplicate, and the relative levels of fluorescence were normalized by the mean level of fluorescence obtained from three replicates of the medium alone.

Figure 4

AM ultrastructural analysis. Ultrastructure of AM pieces after 1 (A) and 3 (B) days of culture in the presence (P4) or in the absence (CTR) of P₄ supplementation was evaluated by using TEM. (A) From the top to the bottom of the panel, cell morphology, nuclear details (n), apical and basal specializations, cell–cell junctions (cj), and mitochondria (m) are shown. (B) From the top to the bottom of the panel, cell morphology, cell–cell junction (cj), nuclear details (n) and basal lamina (bl), RER (rer), Golgi (g), and mitochondria (m) are shown.

Figure 5

Anti-inflammatory cytokine mRNA expression and culture medium content. (A) Comparison between basal (blue bars) and LPS-induced (red bars) IL-10, IL-4, and TGF-β mRNA expression in CTR and P4-treated AM, after 1 (1 d) and 3 (3 d) days of in vitro culture. (B) Comparison between basal (black bars) and LPS-induced (gray bars) IL-10, IL-4, and TGF-β content in CM collected from CTR and P4-treated AM, after 1 (1 d) and 3 (3 d) days of in vitro culture. Data are the mean ± SEM, from n = 4 independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.