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Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N -acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N -arachidonoyldopamine, N -docosahexaenoyldopamine, and N -oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

We performed a comparative study of the cytotoxic effect of endocannabinoid N-arachidonoyl dopamine (AA-DA) on cultured stromal cells of ectopic and eutopic endometrium. It was found that AA-DA in the concentration range of 1-20 μM produces more selective cytotoxic effect on the stromal cells of the ectopic endometrium due to interaction with cannabinoid type 1 receptor. In concentrations below 1 μM, AA-DA stimulated the proliferation of stromal cells of the eutopic endometrium and did not affect the division of ectopic endometrium cells. This effect was realized due to its interaction with cannabinoid type 2 receptor.

DAI-1 receptor (DNA-dependent activator of IFN-regulatory factors; DLM-1/ZBP-1) is an innate immunity cytoplasmic receptor of the DNA-recognition receptor class of antiviral immunity. DAI-1 expression reflects the severity of the inflammatory response that plays the key role in the pathogenesis of pregnancy complications. We studied DAI-1 receptor expression in the placental villi in early- and late-onset preeclampsia. In case of early-onset preeclampsia DAI-1 staining intensity was lower (p=0.01), and in case of late preeclampsia - significantly higher (p<0.005) than in the reference groups at the corresponding gestational age. There was revealed a correlation between the decrease in DAI-1 receptor expression and the severity of disease progression.

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.05). The maximum production was found for proinflammatory chemokine IP-10 and IFN-γ and anti-inflammatory cytokine IL-10. The major difference of mesenchymal stromal cells from epithelial BxPC-3 cells was 7-fold higher production of IL-10, which can explain the immunosuppressive effect of mesenchymal stromal cells.

We isolated and characterized cell cultures from eutopic endometrium and endometriotic lesions of women with malformations of the internal reproductive organs. The cells had fibroblast-like shape and intensively expressed CD90, CD73, CD105, CD44, CD146, and CD117 and were capable of induced adipogenic and osteogenic differentiation in vitro. The obtained cultures exhibited properties of multipotent mesenchymal stromal cells; at the same time, they demonstrated in vitro immunophenotypic differences from cell cultures of eutopic and ectopic endometrium of women without developmental abnormalities, which suggests their functional difference. The cells from eutopic endometrium and from ectopic endometriotic lesions can be used as the model for studying of the etiology and pathogenesis of endometriosis and for testing new drugs for this specific group of patients. Markers CD90 and CD117 were identified as promising molecules for the development of minimally invasive diagnostics of endometriosis based on cell cultures from eutopic endometrium.

Placentas from women aged 25-32 years with normal course of gestation were studied. It is essential to stick to certain methodological approaches for preparing viable multipotent mesenchymal stromal cell culture and to carry out morphological (macro and micro) evaluation of the chorionic villi, umbilical cords, and placentas. At stage I of the study, patients' histories, labor course, and examinations of the newborns should be analyzed to exclude women with genital and extragenital diseases. At stage II, it is essential to stick to special regulations and methods for collection of specimens of the cord, amnion, and placental tissue proper. Histological control of the placental structures collected for multipotent mesenchymal stromal cell culturing is obligatory.

Cell cultures isolated from endometriosis lesions by enzymatic dissociation consisted of fibroblast-like cells expressing CD90, CD73, and CD105; cell viability in these cultures was >90%, but this parameter decreased by passage 3. Zero passage cultures contained 10-25% epithelial cells expressing cytokeratin-7, but by passage 2, the cultures became more homogeneous and epithelial cells disappeared. The proportion of proliferating cells and population doubling level increased from passage 1 to passage 3. The cultures from the endometrium were induced to adipogenic and osteogenic differentiation in vitro. The cultures derived from ectopic endometrium have properties of multipotent mesenchymal stromal cells that exhibited in vitro similarities and differences from cell cultures from eutopic endometrium, which allows using this cell model for the search and testing of new drugs and technologies aimed at suppression of the growth and spread of endometriosis lesions.

Proliferative activity of mesenchymal stromal cells isolated from five sources (chorionic villi, Wharton's jelly, amnion, endometrium, and adipose tissue) was compared by flow cytometry and real-time PCR (by the content of mRNA of genes encoding of cell cycle regulators). Mesenchymal stromal cells derived from the endometrium demonstrated maximum stability and high proliferative potential.