Senescence And IDO-1/COX-2Improved The Immunoregulation Capability Of Human Umbilical Cord Mesenchymal Stem Cells

Objective:To investigate the immunomodulatory ability of human umbilical cord-derived MSCs (UC-MSCs) along with culture time and generations in vitro. To improve the immunomodulation capability of MSCs by overexpressing indoleamine-pyrrole2,3-dioxygenase (IDO-1) or cyclooxygenase2(COX-2).Methods:1. MSCs were isolated from human umbilical cord with the tissue explants adherent method and cultured under normoxic conditions in vitro. The morphological characterization and nucleocytoplasmic ratio of MSCs were observed using Giemsa staining.2. The fifth generation MSCs were selected as control group and the thirteenth generation cells as senile group. Cellular senescence was detected by aging cell in situ test kit. And Cell Counting Kit-WST-8was used to determine the proliferation of lymphocytes in lymphocyte coculture system with different generations’MSCs.3. The expression levels of immunomodulation related genes in senile MSCs were investigated by real-time quantitative PCR.4. Recombinant plasmids, pEGFP-N3/IDO-1and pEGFP-N3/COX-2, were constructed and transferred into MSCs by electroporation. Western Blot was used to verify the expression levels of IDO-1and COX-2proteins. 5. Four experiment groups were established, in which lymphocytes were co-cultured separately with MSCs transferred with pEGFP-N3, pEGFP-N3/IDO-1, pEGFP-N3/COX-2, and both recombinant plasmids. They separetely served as the control group, IDO-1+MSCs group, COX-2+MSCs group, and IDO-1+COX-2+MSCs group. The proliferation of lymphocytes was determined by Cell Counting Kit-WST-8.6. The expression levels of immune response-related genes were detected by real-time quantitative reverse transcriptase (qRT-PCR) in control MSCs, IDO-1+MSCs, COX-2+MSCs, and IDO-1+COX-2+MSCs.Results:1. Length breadth ratio of MSCs increased with persistent culture and nucleocytoplasmic ratio decreased. The aging cells’volume was bigger, cell-edge dim, and intracellular particles increased.2. The senium extent of the thirteenth passage cells was higher than the fifth passage cells. The capacity of suppressing lymphocytes proliferation of the thirteenth passage MSCs enhanced compared to the fifth passage ones.3. The expression of immunosuppression related genes of senile MSCs increased when compared with young MSCs by real time PCR, such as HO-1, IL-10and iNOS. And the expression of most inflammatory associated genes declined, such as IL-1α, IL-1β and IFN-γ.4. The MSCs with recombinant plasmids could significantly upregulate the expression levels of the inserted genes.5.The IDO-1+MSCs, COX-2+MSCs, and IDO-1+COX-2+MSCs could suppress lymphocyte proliferation more effectively than the control group. The enhancement effect was not obvious in IDO-1+COX-2+MSCs.6. The expression levels of inflammatory cytokines were downregulated in pEGFP-N3/IDO-1positive MSCs, while those of immunosuppression factors were increased in pEGFP-N3/COX-2positive MSCs.Conclusion:The immunosuppressive property of human umbilical cord-derived mesenchymal stem cells was strengthened with continuous culture and increasing generations under normoxic conditions in vitro. The expression levels of IDO-1and COX-2were upregulated obviously in IDO-1+MSCs and COX-2+MSCs. The overexpression of IDO-1and COX-2improved the immunoregulation capability of UC-MSCs dramatically.