Umbilical Cord-derived Mesenchymal Stem Cells Affected The Immune Response Of Progenitor CD34~+ Cells

Objective To investigate wheather the hypoxic/non-hypoxic umbilical cord-derived mesenchymal stem cells can inhibit the immune responses of umbilical cord blood (UCB)CD34+ progenitor cells, affecting the secretion of immunomodulattory cytokines.Methods UC-MSCs were isolated and cultured, the growth morphology were observed. Surface markers of UC-MSCs were detected by flow cytometry. The CD34 +progenitor cells in umbilical blood were isolated by immunomagnetic beads. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using Ficoll-Histopaque density gradients from heparinized samples.The different number of UC-MSCs(5×103,2×104,8×104)were added to the co-culture system of UCB CD34+ progenitor cells and peripheral blood mononuclear cells(PBMC),and cultivation of CD34+ cells and PBMC as negative group, in addition, we mixed this three kinds of cells using Transwell,UBC CD34+ cells and PBMC were cultured in the upper separated from UC-MSCs, after culturing for 96 h, supernatants were collected and detected of the IFN-y and IL-10 level by ELISA. Third passage (P3) UC-MSCs were separately cultivated in DMEM/F12 with cobalt chloride 0,50,100,150,200,250μmol/L at 37℃and 5% CO2 for 12h,1d,2d,3d,4d,5d,6d, detected the proliferation and vitality of every groups by MTT assay. The hypoxic UC-MSCs which were cultivated with CoCl2150μmol/L for 48h were added to the co-culture system of UBC CD34+ cells and PBMC, culturing for 96 h, detected cytokines using above method.Results UC-MSCs did not express CD40, CD80, CD86, HLA-DR, CD34.It can inhibit IFN-y secretion (compare with controls,P<0.05), promote IL-10 secretion(compared with controls,P<0.05) and had a dose-depended. Using Transwell,UC-MSCs have this immunomodulation too,but the effects decreased. The platform stage of UC-MSCs was in advance and decurtated after culturing with proper concentration of CoCl2(≤150μmol/L), and the proliferation decreased after 3 days,while the higher concentration of CoCl2 (200,250μmol/L) had a negative impact on the vitality of UC-MSCs.The hypoxic UC-MSCs could inhibit the secretion of IFN-y(compared with controls, P<0.05), while enhanced the secretion of IL-10(compared with controls, P<0.05), however, compared with non-hypoxia group, the secretion of IFN-y increased, IL-10 decreased. Conclusions The concentration of CoCl2(≤150μmol/L) hardly affected the vitality of UC-MSCs, so it can served as a method of hypoxia.UC-MSCs can inhibited the secretion of IFN-γand promoted the secretion of IL-10 in the immune response of UCB CD34+ progenitor cells, and partly depended on the contacts between cells. Hypoxic UC-MSCs also can inhibit the immune responses of UCB CD34+ progenitor cells, however, the effects decrease compared with normoxia. Co-transplantation of UC-MSCs and UBC CD34+ could can serve as alternative of stem cells transplantation for ischemic cardiomyopathy.