The Study On The Immunologic Function Of Two Kinds Of Amniotic Cells

Objective:Adult stem cells are one kind of cells which have capability to differentiate into multiple celltypes as well as self renew continuously. They have great therapeutic potential in tissueengineering, genetherapy and cell transplantation. In recent study, researchers have found that therewere two kinds of stem cells in human amniotic membrane: human amniotic epithelial cells(HAEC)and human amniotic mesenchymal cells(HAMC), as a new source of stem cells, these twoamniotic cells have becomed a hot spot in stem cells research due to their advantages in extensiveresource、easy to be acquired、multi-differentiation potential and negligible antigenicity. Theaims of this study were to research the immune regulatory function of amniotic epithelial cells andmesenchymal stem cells, explor the feasibility of allogeneic stem cell transplantation inimmunology point and the potential value of two amniotic cells in the prevention and treatment ofgraft versus-host disease (GVHD) and other immune diseases.Methods：1. Two kinds of human amniotic cells were cultured in vitro, they phenotypic differences wereidentified by flow cytometry and immunofluorescence.2. Induced two amniotic cells differentiation by condition and identified their multi-lineagedifferentiation potential.3. Both amniotic cells and their supernatant were mixed cultured with lymphocyte and detectedthe influence of lymphocyte proliferation by MTT assay.4. The changes of T lymphocyte subsets and intracellular factors were detected by flow cytometryafter mixed culturing.5. Established a non-contact co-culture system.by transwell system and explored the mechanismof immune suppression.Results：1. The methods of HAEC and HAMC isolation、culture and amplification were successfullyestablished.2. The results indicated that immunophenotypic characterization of both cell types showed positive expression of HLA-A,B,C and mesenchymal stem cell markers (CD29, CD73, CD44,CD59, CD90, CD105, CD166), but didn’t expressed the hematopoietic markers (CD31, CD34,CD45, HLA-DR) and weakly showed the costimulatory molecules(CD40, CD40L,CD80,CD86). The expression level of phenotypes in both cells were remained stable frompassages P3to P7.3. The immunofluorescence indicated that HAEC expressed Cytokeratin19but not Vimentin.On the contrary, HAMC expressed Vimentin but not Cytokeratin19.4. The assessment of multi-lineage differentiation potential demonstrated that HAMC showedgreater cardiomyocytic potential, while HAEC showed greater neural potential.5. Both human amniotic cells and their supernatant could inhibit lymphocyte proliferation.6. After mixed culturing with HAMC, CD4+T cell subsets were not suppressed as obviously asCD8+T cell subsets; Th1and Tc1decreased significantly contrast to control groups whichwithout HAMC, but Th2and Tc2slightly increased in all experiments. HAEC didn’t havesignificant difference.7. It was found that both human amniotic cells could secrete soluble cytokines to play immunesuppression.Conclusions：It is concluded that HAEC and HAMC can be successfully isolated from the HAM. Both cellpopulations possess similar immunophenotype. However, they differe in cell yield andmultipotential for differentiation into the major lineages, HAEC possessing a much greaterectodermal differentiation capacity while HAMC possessing a much greater mesodermaldifferentiation capacity. Both human amniotic cells could secrete soluble cytokines to play immunesuppression. HAMC can significantly suppress T-lymphocyte proliferation, especially CD8+Tcellsubsets. Otherwise it can decrease activated Th1and Tc1, increase Th2and Tc2. HAMC havepotential of alleviating acute graft-versus-host disease (aGVHD) and maintaining graft versusleukemia (GVL). HAEC also have immunosuppressive effects, due to its different mechanismwhich are more suitable for engineering seed cells in tissue repair.