Amniotic Membrane-Derived Mesenchymal Cells and Their Conditioned Media: Potential Candidates for Uterine Regenerative Therapy in the Horse

<div><p>Amniotic membrane-derived mesenchymal cells (AMCs) are considered suitable candidates for a variety of cell-based applications. In view of cell therapy application in uterine pathologies, we studied AMCs in comparison to cells isolated from the endometrium of mares at diestrus (EDCs) being the endometrium during diestrus and early pregnancy similar from a hormonal standpoint. In particular, we demonstrated that amnion tissue fragments (AM) shares the same transcriptional profile with endometrial tissue fragments (ED), expressing genes involved in early pregnancy (<i>AbdB-like Hoxa</i> genes), pre-implantantion conceptus development (<i>Erα, PR</i>, <i>PGRMC1</i> and <i>mPR</i>) and their regulators (<i>Wnt7a</i>, <i>Wnt4a</i>). Soon after the isolation, only AMCs express <i>Wnt4a</i> and <i>Wnt7a</i>. Interestingly, the expression levels of prostaglandin-endoperoxide synthase 2 (<i>PTGS2</i>) were found greater in AM and AMCs than their endometrial counterparts thus confirming the role of AMCs as mediators of inflammation. The expression of nuclear progesterone receptor (PR), membrane-bound intracellular progesterone receptor component 1 (<i>PGRMC1</i>) and membrane-bound intracellular progesterone receptor (<i>mPR</i>), known to lead to improved endometrial receptivity, was maintained in AMCs over 5 passages <i>in vitro</i> when the media was supplemented with progesterone. To further explore the potential of AMCs in endometrial regeneration, their capacity to support resident cell proliferation was assessed by co-culturing them with EDCs in a transwell system or culturing in the presence of AMC-conditioned medium (AMC-CM). A significant increase in EDC proliferation rate exhibited the crucial role of soluble factors as mediators of stem cells action. The present investigation revealed that AMCs, as well as their derived conditioned media, have the potential to improve endometrial cell replenishment when low proliferation is associated to pregnancy failure. These findings make AMCs suitable candidates for the treatment of endometrosis in mares.</p></div

Molecular characterization of endometrial tissue (ED) and amnion (AM) (A), and endometrial cells at diestrus stage (EDCs) and amniotic-derived stem cells (AMCs) (B).

<p>Qualitative and quantitative RT–PCR analysis for the expression of genes associated to differentiation of uterine stromal cells during early pregnancy (<i>Hoxa9</i>), those influencing pre-implantantion conceptus development (<i>ERα</i>, <i>ERβ</i>, <i>PR, PGRMC1</i> and <i>mPR</i>) and their regulators (<i>Wnt7a</i>, <i>Wnt4a</i>), and prostaglandin E₂ synthase (<i>PTGS2</i>), <i>FOXO1</i>, <i>SGK1</i>, and <i>TP53</i>. <i>GAPDH</i> was used as reference gene.</p

Oligonucleotide sequences used for RT-PCR analysis.

<p>Oligonucleotide sequences used for RT-PCR analysis.</p

Cell morphology. Monolayer of AMCs (A) and EDCs (B); tridimensional cluster of AMCs (C).

<p>Magnification, x 20; scale bar  = 20 µm.</p

Molecular characterization of AMCs. Conventional RT-PCR analysis of evaluated genes in AMCs after culture with progesterone from passage 1 (P1) to passage (P5).

<p>Molecular characterization of AMCs. Conventional RT-PCR analysis of evaluated genes in AMCs after culture with progesterone from passage 1 (P1) to passage (P5).</p

Schematic representation of the PI3K-Akt signaling pathway model and cellular responses in ovarian follicular cells (FCs) associated with competent oocytes that were able to reach the blastocyst stage (BL) and incompetent oocytes that were unable to initiate embryo development after maturation (NC).

<p>Pentagons indicate whether a given component of the pathway was upregulated (upward arrow) or downregulated (downward arrow) in FCs from the BL and NC groups. Lines ending in arrowheads indicate stimulation, whereas lines ending in bars indicate blockage.</p

Bovine primer sequences used in the RT-PCR amplification.

<p>Bovine primer sequences used in the RT-PCR amplification.</p

Relative expression of mRNAs for genes responsible for the downstream cellular effects of PI3K-Akt signaling on non-cleaved (NC), cleaved non-blastocyst (CNB) and blastocyst (BL) groups.

<p>Bars depict means and error bars depict standard errors of the means. Different superscripts (a and b) indicate significant differences (p < 0.05).</p