The Hematopoietic Supportive Effect Of Amniotic Mesenchymal Stem Cells Conditioned Culture With Amniotic Epithelial Cells In NID/SCID Mice And Long-term Cultures Of AMSC Without Spontaneous Tumorigenic Transformation

Objective:Mesenchymal stem cells (MSC) received much attention because of multiple differentiation potential, hematopoietic support, immune regulation self-renewal. Many studies have showed that MSC could promote the homing of hematopoietic stem cells (HSC) and the hematopoietic reconstruction. And SDF-1/ CXCR4 axis has been shown to be critical for the migration of MSC to the bone marrow. Previously we identified amniotic mesenchymal stem cells (AMSC) had the immune phenotype, multi-directional differentiation potential and hematopoietic support function similar to the bone marrow mesenchymal stem cells (BMSC). And they can secrete more hematopoietic factors than BMSC. It’s also be shown that the co-culture with amniotic epithelial cells (AEC) resulted in up-regulation of cell surface CXCR4 of AMSC. In this study we investigated whether AMSC could promote the hemopoietic reconstruction in NOD/SCID mice, and observed the role of SDF-1/CXCR4 axis in the hematopoietic supportive effect of AMSC. In the second part, we tried to explore the possibility of long-term culture of AMSC in vitro and detecte the spontaneous tumorigenic transformation during culture expansion..Methods:First, We explored the hematopoietic supportive ability of AMSC condition cultured with AEC 24 hours after intravenous infusion into irradiated NOD/SCID mice. The peripheral blood was analyzed and the bone marrows were evaluated after 14 days transplantation. In the second part, we analyzed the characters of AMSC during long-term expansion such as:1.the alterations of morphology; 2. the proliferation capability; 3. the immunophenotype of AMSC:4. the karyotyp; 5. the expressions of the cytokines, such as IL1β,IL-6, IL-8, IL-10, IL-12, TNF-aand IL-3; 6. the osteogenic and adipogenic differentiation potential of AMSC; 7.the colony-forming ability in soft agar;8.the tumorigenicity of AMSC in immunodeficient(BLAB/C) mice.Results:1. AMSC alleviated the bone marrow damage after irradiation, promoted hematopoietic recovery, and increased the survival rate of mice.2. Transplantation of AMSC conditioned cultur with AEC resulted in faster hematological recovery than non-treated cells while the number of AMSC homing to the bone marrow increased. Neutralization of CXCR4 significantly reduced the hematopoietic supportive effect of AMSC.3. AMSC maintained their normal morphology, immunophenotype, karyotype and preserved multilineage differentiation potential without spontaneous tumorigenic transformation in long-term culture in vitro. However the proliferation activity decreased after P25.Conclusions:AMSC could promote hematopoietic reconstruction in irradiated NOD/SCID mice. SDF-1/CXCR4 axis played an important role in the migration and homing of AMSCand accelerated the hematopoietic recovery. AMSC could be long-term cultured in vitrowithout spontaneous tumorigenic transformation while maintaining the biological characteristics unchanged.