Separation, Cultivation And Immune-suppressive Effects Of Mesenchymal Stem Cells Derived From Human Amniotic Fluid

Researchers are trying to find out new resources of stem cells because of the ethical arguments of embryonic stem cells (ESs). Recent studies demonstrateded that a few cells displaying ES's characteristics exist in amniotic fluid, which was named human amniotic fluid stem cell (hAFS cell). hAFS cells that express embryonic and adult stem cell markers can differentiate into cell types of three embryonic germ layer and display specialized functions. All these confirmed that the hAFS cells have value of application. On the other hand, hAFS cells can be obtained easily and do not damage the mother body and the embryo. So the hAFS cells may offer an alternative approach for the cell therapy and tissue engineering.Mesenchymal stem cells (MSCs) resemble a multipotent adult stem cell population capable of differentiating into different mesodermal cell lineages including osteoblasts, chondroblasts and adipocytes. Research on the MSCs mainly concentrated in two areas: the multi-differentiation potential and the immune-suppression. MSCs have generated a great deal of excitement as a potential source of cells for regenerative medicine and tissue engineering owing to their dramatic potential of proliferation and differentiation, and raise high hopes in clinical application. On the other side, this is an opportunity for the cell therapy providing the low side effects.In this study, in the first part, we isolated and purified the mesenchymal stem cells from human amniotic fluid. First, we isolated and cultured the cells in human amniotic fluid, and choosed the fibroblastoid-like cells to purify and expand. Then, we identified the source and molecular markers of cell clones by Chromosome Giemsa staining, RT-PCR and flow cytometer, and they were identified as the mesenchymal-like cells. Mesenchymal-like cells could be induced to differentiate into adipocytes and osteoblasts in the special medium, and they were confirmed as MSCs because of the differentiation ability. Additionally, we found that AF-MSCs, which can double in 33h on average, have strong proliferation ability and they hold the expression of Oct-4 that is the maker of pluripotency and the normal chromosome ploidy in the different passages.In the second part, we discovered that the AF-MSCs have inhibitory effects on the proliferation of peripheral blood mononuclear cells (PBMC). And this inhibition showed a linear correlation with the AF-MSCs'quantity. We further explored that the AF-MSCs could secrete the TGF-beta1, which mediated the AF-MSCs'suppressive effects on the PBMC. Adding anti-TGF-beta1 in co-culture system found that TGF-beta1 does not stimulate cells'apoptosis but induce the CD4+CD25- T cells into CD4+CD25+Foxp3+ T cells, which have the suppressive function to inhibit other cells'proliferation. So AF-MSCs suppress PBMC's proliferation via secreting TGF-beta1 to induce the regulatory T cells.