The Effect On Biological Characteristics Of Amniotic Mesenchymal Stem Cells Co-culture With Amnotic Epithelial Cells

Objective:Adult stem cells are one kind of cells, which have capability to differentiate into multiple cell types as well as self-renew continuously. They have great therapeutic potential in tissue engineering, gene therapy and cell transplantation. Recently human amniotic mesenchymal stem cells (hAMSC) have been showed the similar biological characteristics and supportive effect on hematopoiesis with bone marrow mesenchymal stem cells (BMSC) in vitro. Because of their easily obtained, abundant source and other advantages, AMSC has been expected to be a more desirable new source of MSC. Amniotic epithelial cells (AEC) can secrete a variety of cytokines. In this paper we focus on the changes in the biological characteristics of AMSC, which were co-cultureed with AEC. SDF-1/CXCR4axis is the significant biological axis, which play an important role in the homing of MSC and implantation in vivo. This study compared the expression of CXCR4in AMSC before and after co-culture with AEC, and investigated the role SDF-1/CXCR4axis in homing and migration process of AMSC.Methods:AMSC and AEC were isolated from human amnion and cultured primarily by the tissue pieces culture method and enzymatic digestion method respectively. BMSCs wre isolated from the specimens of healthy human bone marrow by density gradient centrifugation. The levels of IL-6in the culture supernatant of P3cells of AMSC and BMSC were detected, and the expressiones of IL-6R (CD126) also were compared. The indirect co-culture systems were established with millicell chamber. Three groups were established:AMSC co-cultured with AEC group (in serum-free), AMSC serum-free cultured group, AMSC serum cultured group(conventional culture group). The effects on cell vitality after co-culture24h,48h,72h were detected by CCK-8assay and trypan blue stain. The expressing of CXCR4in the AMSC were analysied with flow immunoassay, real-time RT-PCR etc.to detect. The migration ability of AMSC were detected by Millicell chamber method.Reault:1. AMSC separated from human amniotic membrane have the similar phenotypes and morphology of MSC derived from other tissues. The phenotype of these progenitor cells was similar to BMSC, i.e., CD29, CD44and CD105positive; CDlla、CD11b、 CD31、CD34、CD45and HLA-DR were negative, and they were negative for marker of epithelial cells.2. AMSC and BMSC had similar biological characteristics and phenotypes.3. AMSC synthesized and secreted IL-6more abundant than BMSC, while the expression of CD126on AMSC was also higher than on BMSC.4.Although there was no significant differences in cell proliferation activity among the three groups at24h, but at the48h,72h, the cell proliferation activity of co-culture group and serum culture group showing a significant higher than serum-free culture group.5. Trypan blue staining further confirmed, no matter on which time sites, the survival rate of co-culture group and serum culture group were higher than serum-free culture group.6. After24h, higher expression of intracellular CXCR4was showed in co-culture group than in serum-free culture group by flow cytometry, but no significant difference was founded in co-culture group and in the serum culture group. In the next48h and72h, there were no significant differences in the expression of intracellular CXCR4in the three groups. At all the three time sites, serum culture group showed inferior on the expression of the surface CXCR4, while co-culture group and serum-free culture group had no difference.7. The expression of CXCR4in the three groups were detected at the mRNA level, the results displayed that co-culture group and serum-free culture group were higher expression than serum culture group in all the three time sites. And there was no significant difference between co-culture group and serum-free culture group.8. AMSCs are able to migrate chemotactically along a concentration gradient of SDF-1in vitro. The migration ability of co-culture group and serum-free culture group were higher than the serum culture group, while there was no statistical difference between co-culture group and serum-free culture group.Conclusion:AMSC and AEC can be isolated, cultured and amplified in vitro. AMSC was similar with BMSC on the biological characteristics. Both of them can synthesis and secrete IL-6. And the expression of IL-6receptor (CD126) on AMSC was more abundant. Prompted that in the fact of the restoration of damaged tissues and the regulation of hematopoiesis, AMSC has more broad application prospects than BMSC probably. AMSC after co-culture with AEC exhibited a better growth activity, higher expression of CXCR4, greater ability of migration.