The Effect On Homing Efficiency Of Amniotic Mesenchymal Stem Cells Conditioned Culture With Amniotic Epithelial Cells

Objective:Previously we have demonstrated the capacity of hematopoitic support of amniotic mesenchymal stem cells(AMSC) was similar to the bone marrow mesenchymal stem cells(BMSC). Interestingly AMSC could even express the hematopoietic cytokines that were not expressed in BMSC.And the co-culture with amniotic epithelial cells (AEC) resulted in up-regulation of cell surface CXCR4 of AMSC. We speculate that may be related to co-culture of AMSC with AEC produces a variety of cytokines. However, for clinical use, AMSC co-cultured with AEC may be limited due to the high cost,and the operation is relatively complex and it is difficult to meet the large number of cells required for clinical treatment.The conditioned medium of AEC have many advantages,such as low cost, simple method,and which can be frozen.In this study we will investigate the feasibility of AEC condition culture medium used to culture AMSC. First,we detected the changes in biological characteristics of AMSC before and after cultured with AEC condition culture medium.And compared with co-culture with AEC.Second.we detected cytokines in the culture medium and to explore the role of cytokines in the culture medium.and to explore the role of cytokines in expression of CXCR4.Finally,we investigated the surface expression of CXCR4 on AMSC with condition cultured and explored their homing ability after intravenous infusion into sublethally irradiated NOD/SCID mice. Some valuable in formations may be provided to modulate AMSC for the clinical using.Methods:AMSC and AEC were isolated from human amnion and cultured primarily by the tissue pieces culture method and enzymatic digestion method respectively. The condition culture systems were established.Three groups were established.AMSC condition cultured with AEC,(conventional culture group),serum cultured AMSC,and serum-free cultured group.The effects on cell vitality after condition cultured 48h were detected by MTT assay and trypan blue stain. The expressing of CXCR4 and IL-6R (CD 126) in the AMSC,were analyzed with flow immunoassay.The secretion of cytokine(IL-8、IL-1β、IL-6、IL-10、TNF-α、IL-12P70)in culture were detected. The migration ability of AMSC were detected by Millicell chamber method.NOD/SCID mice were sublethally irradiated prior to transplantation with AMSC stained with PKH26. Control mice received the same volume of saline. At 24 hours after transplantation, the presences of donor cells were analyzed by flow cytometry.Results:1. The survival rate of AMSC in condition culture group and serum culture group were higher than serum-free culture group.2.The proliferation activity of AMSC in condition culture group and serum culture group showing a significant higher than in serum-free culture group.3.Higher expression of CXCR4 and CD 126 were detected in condition culture group and serum-free culture than in serum culture group by flow cytometry,4.After blocking IL-6, the expressions of CXCR4 and CD 126 in condition culture group were decreased.5.Compared with in the co-culture system,condition culture on the biological characteristics of AMSC in conventional culture group showed no significant difference.6.The levels of IL-8, IL-6, IL-1β were high in AMSC culture system.7.AMSC could migrate chemotactically along a concentration gradient of SDF-1 in vitro. The migration ability in condition culture group were higher than in serum culture group.8. Transplantation of condition cultured AMSC resulted in higher levels of donor chimerism in bone marrow.Neutralization of CXCR4 significantly reduced homing and engraftment of AMSC in murine bone marrow.Conclusions:1.AMSC condition cultured with AEC maintained good proliferation, and higher expression of CXCR4, greater ability of migration.Compared with the previous co-culture system with AEC,the biological characteristics of AMSC showed no significant difference in condition culture group.It confirmed the feasibility of condition culture with AEC.2.The cytokines and their receptors such as IL-6 and CD 126 may play important roles in the up-regulation of CXCR4 on the surface of AMSC in condition culture group.3.Stromal cell-derived factor-1/CXCR4 axis plays an important role in the regulation of motility of AMSC. Increasing CXCR4 expression might be a potential strategy to improve homing of AMSC in bone marrow and accelerate hematopoiesis recovery.