A Study On Actions And Mechanisms Of Amniotic Mesenchymal Stem Cell Transplantation In Preventing Aging

To investigate actions and mechamisms of amniotic mesenchymal stem cell (AMSC) transplantation in preventing aging, the second passage AMSCs derived from the second-trimesterβ-gal transgenic mice were transplanted into Bmi-1 gene knock out (Bmi-1^-/-) mice by intraperitonal injection, the life span was recorded, body weight was measured and the phenotypes of different organs were analyzed in Bmi-1^-/- mice transplanted with AMSCs or untransplanted (control) Bmi-1^-/- mice and wild-type (WT) mice at 4 weeks of aging using techniques of histopathology, immunohistochemistry and moleculer biology, respectively.AMSCs were isolated from amnion membrane of the second-trimesterβ-gal transgenic mice and cultured. The characteristics of the second passage AMSCs were analyzed. Results showed that the second passage AMSCs derived fromβ-gal transgenic mice were positive for LacZ staining and expressed Bmi-1 gene and protein, CXCR4 and Oct4, molecular markers of pluripotent stem cells and CD44, a mesenchymal cell markers, but were not expressed CD45, a hematopoietic stem cell marker. The proliferation of AMSCs was enhanced by the treatment with bFGF, EGF or 1,25(OH)₂D₃. After the induction, AMSCs could differentiate into osteoblasts or adipocytes. These results demonstrate that AMSCs have characteristics of pluripotent stem cells.The second passage AMSCs as seed cells were transplanted into Bmi-1^-/- mice, starting at day 2 after birth, once each week for three times by intraperitonal injection. Control mice were injected with PBS. Results showed that the life span was about 4 weeks and body weight was reduced significantly in the control Bmi-1^-/- mice compared to their WT littermates. AMSC transplantation significantly prolonged the life span of Bmi-1^-/- mice and increased their body weight.The size of thymus and spleen were increased significantly in Bmi-1^-/- mice with AMSC transplantation at 4 weeks of age compared to control Bmi-1^-/- mice, although did not reach to the levels of WT mice. The ratio of lymphocytes in peripheral blood, CD4⁺CD8⁺ thymocytes, pre-B cells in bone marrow and F0 cells in spleen were all increased significantly in Bmi-1^-/- mice with AMSC transplantation at 4 weeks of age compared to control Bmi-1^-/- mice. Trabecular bone volume and osteoblast number were increased significantly, the expression of Cbfa-1 and IGF-1 protein in bone tissues was up-regulated, while the number of fat cells in bone marrow cavities was reduced significantly and the expression of PPARγprotein in bone tissues was down-regulated significantly in Bmi-1^-/- mice with AMSC transplantation at 4 weeks of age compared to control Bmi-1^-/- mice. These results indicate that ASMC transplantation prolonged the life span, partially improved the growth retardation and heamatopoietic defects and the premature osteoporosis in Bmi-1^-/- mice.To clarify the mechanism of AMSC transplantation in preventing aging, we examined the expression of Bmi-1 gene and protein and distribution of donor cells in various organs of hosts after AMSCs transplantion by PCR, Western blot and LacZ staining , respectively. Results revealed that the expression of Bmi-1 gene and protein was detected in heart, liver, spleen, lung, kidney, bone marrow and thymus from Bmi-1^-/- mice transplanted with AMSCs, but no in those from control Bmi-1^-/- mice. LacZ positive cells were detected in lung, spleen and kidney from Bmi-1^-/- mice transplanted with AMSCs, but not in those from control Bmi-1^-/- mice. These results demonstrate that AMSCs can migrate into various organs of Bmi-1^-/- mice after the transplantation.To determine whether AMSC transplantation play a role in preventing aging through reducing oxidative stress, we examined the reactive oxygen species (ROS) levels, H₂O₂ content and antioxidase activity of T-SOD and CAT in various organs from Bmi-1^-/- mice transplanted with AMSCs, control Bmi-1^-/- mice and WT mice using biochemical and flow cytometry analyses. ROS levels and H₂O₂ content were increased significantly in liver, spleen, lung, bone marrow and thymus, whereas the activities of T-SOD and CAT were reduced significantly in heart, spleen, bone marrow and thymus from control Bmi-1^-/- mice compared to their WT littermates. ROS levels and H₂O₂ content were reduced significantly in liver, spleen, lung, bone marrow and thymus, whereas the activities of T-SOD and CAT were increased significantly in heart, spleen, bone marrow and thymus from Bmi-1^-/- mice transplanted with AMSCs compared to control Bmi-1^-/- mice. These results indicate that AMSC transplantation exerts anti-aging effects through reducing oxidative stress. To determine whether the anti-aging effects of AMSC transplantation in skeleton are associated with inhibiting the expression of senescence relative genes, we examined the protein expression of Wnt16, p16, p19 and p27 in bone tissues.Results showed that the expression of Wnt16, p16, p19 and p27 was significantly up-regulated in bone tissues from control Bmi-1^-/- mice compared with those from WT mice, and was significantly down-regulated in Bmi-1^-/- mice transplanted with AMSCs compared to control Bmi-1^-/- mice. These results indicate that AMSCs may play a role in preventing aging by inhibiting the expression of senescence related genes, such as Wnt16, p16, p19 and p27.Our results demonstrate that the transplantation of AMSCs as pluripotent stem cells rescued the phenotype of premature aging caused by Bmi-1 deficiency partially either by migrating into various organs of the body and differentiating into cells of various organs, or by up-regulating the activity of antioxidant enzymes, scavenging oxygen free radicals and inhibiting the expression of senescence related genes. This study provides a theoretical and experimental basis for the AMSC transplantation to prevent aging.