Menstrual Blood-Derived Stromal Stem Cells Augment CD4+ T Cells Proliferation

Abstract

Background: It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells (MenSCs), as surrogate for endometrial stem cells, on proliferative capacity of CD4+ T cells was tested.

Methods: MenSCs and Bone marrow Mesenchymal Stem Cells (BMSCs) were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFNγ pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4+ T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFNγ by a colorimetric assay.

Results: MenSCs exhibited dual mesenchymal and embryonic markers and multi-lineage differentiation capacity. MenSCs significantly increased proliferation of CD4+ cells at ratios 1:2, 1:4 and 1:8. IFNγ pre-treated BMSCs but not MenSCs significantly suppressed CD4+ T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFNγ treatment.

Conclusion: Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy.

Keywords: Endometrium; Immunological tolerance; Menstrual blood stem cells; Pregnancy; Proliferation; T lymphocytes.

Figure 1.

Immunophenotyping of MenSCs and BMSCs. MenSCs and BMSCs were evaluated for the expression of MSCs markers, CD9, CD10, CD29, CD44, CD73 and CD105, hematopoietic makers, CD34, CD38, CD45 and CD133, and pluripotency makers, Nanog, Oct-4, SSEA-4 and Stro-1. The grey and empty histograms represent unstained sample and test samples, respectively. Results are representative of three individual experiments.

Figure 2.

Multi-lineage differentiation potential of MenSCs and BMSCs. The left and right pictures of each panel represent differentiated (Dif) and undifferentiated (Undif) stem cells, respectively. Differentiation of stem cells toward osteocytes, chondrocytes and adipocytes were assessed by Alizarin red, Alcian blue and Oil red staining, respectively. Results are representative of three individual experiments.

Figure 3.

Effect of MenSCs on proliferation of CD4+ T cells. A) MenSCs were co-cultured at different ratios with anti-CD3/CD28-activated purified CD4+ T cells for 5 days and the percent of proliferation was assessed by CFSE flow cytometry. B) Representative histogram plots are shown. The grey and empty histograms represent test samples (co-culture) and biological controls (BC) (CD4+ T cells cultured alone), respectively. Results are representative of nine individual experiments. ***: p<0.001.

Figure 4.

Effect of IFNγ stimulation of MenSCs on proliferation of CD4+ T cells: A) MenSCs were co-cultured with CD4+ T cells at 1:4 and 1:8 (MenSCs:CD4+ T cells) ratios with or without IFNγ pre-stimulation for five days and the percent of proliferation was assessed by CFSE flow cytometry. B) IFNγ pre-stimulated BMSCs were used as positive control in CD4+ T cells proliferation assay. Figures on the right in each panel represent histogram plots of corresponding proliferation assays. The empty histograms represent biological controls (BC) (CD4+ T cells cultured alone) and grey histograms represent test samples (co-culture). Results are representative of ten individual experiments *: p<0.05 and ****: p<0.0001.

Figure 5.

Assessment of IDO activity in MenSC and BMSC supernatants after stimulation with IFNγ. IDO activity was measured using kynurenine colorimetric assay. The results are median and rage of four BMSCs and six MenSCs samples ****: p<0.0001.