Objective: The aim of the study was to investigate the effects of CT1 on the stimulation of human umbilical cord blood mesenchymal stem cells(h UCB-MSCs) in terms of their potential to differentiate into neuron-like cells, their survival characteristics, and the PI3K/Akt pathway involved. Methods:(1) Mononuclear cells(MNCs) were isolated by density gradient centrifugation from human umbilical cord blood. The cell surface markers were detected by flow cytometry.(2) h UCB-MSCs were transfected with Adv-CT1-EGFP at various multiplicity of infection(MOI) ranging from 50 to 200 for 3 days. The transfection efficiency was determined by fluorescence microscope.(3) h UCB-MSCs were transfected with Adv-CT1-EGFP(MOI=100PFU/cell) and then induced by NIM(retinoic acid treatment without serum). The neural markers nestin and βⅢ-tubulin were analyzed by immunofluorescences at 6h and 4days after induction.(4) The cell viability and apoptosis of h UCB-MSCs was determined by CCK8 and TUNEL after 6h and 4days of incubation in NIM with or without CT1.(5) The expression of Akt, p-Akt and apoptotic factors(Bcl-2, Bax, Bak, caspase 3, cleaved-caspase 3) were compared between the different groups by Western blot. Results:(1) It was found that human cord blood-derived MSCs expressed CD29, CD44, CD105, but did not express CD34.(2) h UCB-MSCs were effectively transfected by Adv-CT1-EGFP(MOI=100PFU/cell).(3) The treatment of cells with NIM and CT1 generated more cells that were neuron-like and produced stronger expression of neural markers than those cells treated with NIM without CT1(4)h UCB-MSCs after 6h or 4days of incubation in NIM with CT1 compared with those without CT1, the viability of cells were higher, and the percentage of apoptotic cells was lower;(5)CT1 significantly increased the level of p-Akt in NIM+CT1 group compared to NIM group; on the other hand, it significantly decreased the expression of Bax, Bak and cleved-caspase3 expression. In addition, the effect of CT1 was blocked by PI3K/Akt inhibitors LY294002. Conclusions:(1) Retinoic acid treatment without serum was the reliable one in terms of stability of differentiation and neural protein expression. CT-1 promotes neuronal differentiation in h UCB-MSCs.(2) The survival of h UCB-MSCs after incubation in NIM could be improved by CT1.(3) PI3K/Akt signals contribute to CT1-stimulated neural differentiation and to the survival of differentiated cells. |