Bovine endometrial MSC: mesenchymal to epithelial transition during luteolysis and tropism to implantation niche for immunomodulation

Abstract

Background: The uterus is a histologically dynamic organ, and the mechanisms coordinating its regeneration during the oestrous cycle and implantation are poorly understood. The aim of this study was to isolate, immortalize and characterize bovine endometrial mesenchymal stem cell (eMSC) lines from different oestrous cycle stages (embryo in the oviduct, embryo in the uterus or absence of embryo) and examine their migratory and immunomodulatory properties in an inflammatory or implantation-like environment, as well as possible changes in cell transdifferentiation.

Methods: eMSCs were isolated and analysed in terms of morphological features, expression of cell surface and intracellular markers of pluripotency, inmunocytochemical analyses, alkaline phosphatase activity, proliferation and osteogenic or chondrogenic differentiation capacities, as well as their ability to migrate in response to inflammatory (TNF-α or IL-1β) or implantation (IFN-τ) cytokines and their immunomodulatory effect in the proliferation of T cells.

Results: All eMSCs showed MSC properties such as adherence to plastic, high proliferative capacity, expression of CD44 and vimentin, undetectable expression of CD34 or MHCII, positivity for Pou5F1 and alkaline phosphatase activity. In the absence of an embryo, eMSC showed an apparent mesenchymal to epithelial transition state. eMSC during the entire oestrous cycle differentiated to osteogenic or chondrogenic lineages, showed the ability to suppress T cell proliferation and showed migratory capacity towards pro-inflammatory signal, while responded with a block in their migration to the embryo-derived pregnancy signal.

Conclusion: This study describes for the first time the isolation, immortalization and characterization of bovine mesenchymal stem cell lines from different oestrous cycle stages, with a clear mesenchymal pattern and immunomodulatory properties. Our study also reports that the migratory capacity of the eMSC was increased towards an inflammatory niche but was reduced in response to the expression of implantation cytokine by the embryo. The combination of both signals (pro-inflammatory and implantation) would ensure the retention of eMSC in case of pregnancy, to ensure the immunomodulation necessary in the mother for embryo survival. In addition, in the absence of an embryo, eMSC showed an apparent mesenchymal to epithelial transition state.

Keywords: Cell migration; Embryo implantation; Endometrial mesenchymal stem cells; Inflammation.

Fig. 1

Morphology of MSCs. Pre-immortalized mesenchymal stem cell cultures at passage 0 (upper panels) and immortalized mesenchymal stem cell lines at passages 10–15 (lower panels). Phase-contrast images were acquired with × 100 magnification

Fig. 2

Expression of cell surface, intracellular and pluripotent specific markers and alkaline phosphatase activity. a–c Analysis by flow cytometry of the expression levels of cell surface markers CD34, CD44 and MHCII and intracellular markers cytokeratin, vimentin and POU5F1 in eMSC. Data correspond to the mean fluorescence intensity (folds of negative control) for each sample. d Analysis of AP activity: bright field images were obtained at × 50 magnifications, showing some red-stained cell groups after the action of alkaline phosphatase on Fast Red in the presence of Napthol AS-mx phosphate. Bars = 150 μm

Fig. 3

In vitro proliferation of mesenchymal stem cells. a Absolute number of cells/dish (mean ± SD) at indicated points. b Doubling time in days of each MSC line (mean ± SD). Different letters indicate significant differences (p < 0.05)

Fig. 4

In vitro differentiation of eMSC to different lineages. Images show Alizarin Red S staining of calcium deposits in cells cultured in basal medium (control) or in osteogenic differentiation medium (top panel); Alcian blue staining of acidic proteoglycan in cells cultured in basal medium (control) or in chondrogenic differentiation medium (bottom panel). Bright field images were acquired with a × 3 magnification (bars = 150 μm)

Fig. 5

Migration analysis in an agarose spot assay. A Representative images of eMSC-4C migration assay into a PBS-, TNF-α-, IL-1β- or IFN-τ-containing agarose spot at the indicated times. Images were obtained in a light stereomicroscope at × 20 magnification. b and c Migratory response of MSC to inflammatory and implantational stimuli. b The distance migrated from the border of the PBS-agarose spot was measured in two independent experiments for all eMSC lines at the indicated times (mean ± SD). Different letters indicate significant differences: p < 0.005 for eMSC migration mediated by PBS at day 7 (a, b, c, d, e); C p < 0.05 for eMSC migration mediated by PBS (a, b, c), p < 0.005 for eMSC migration mediated by TNF-α (e, f, g) and p < 0.05 for eMSC migration mediated by IL-1β (i, j, k, l) and IFN-τ (p, q). *p < 0.05; ***p < 0.0005; ****p < 0.0001

Fig. 6

Measurement of T cell proliferation suppression capacity by eMSC. a, b Representative plots of non-stimulated and stimulated (anti-CD2/CD3/CD28-coated microbeads) human T cell control conditions (in the absence of MSCs). c Percentage of proliferating stimulated T cells in the presence of eMSC-1A or eMSC-4H (X:X T:eMSC ratio ± 0.54 and ± 0.63 respectively); proliferative T cell were analysed by Violet CellTrace fluorescence loss after a 4.5-day culture. Results are reported as percentage of proliferating T cells relative to the proliferation of activated T cells in the absence of eMSCs (positive control). ****p < 0.0001

Fig. 7

Model for bovine endometrial MSC: mesenchymal to epithelial transition during luteolysis and tropism to inflammation niche for immunomodulation