Crosstalk Between MAPK/ERK And PI3K/AKT Signal Pathways Involved In Adenovirus-mediated Cardiotrophin-1 Induced Neural Differentiation Of Human Umbilical Cord Blood Mesenchymal Stem Cells

Objective:The aim of the present study was to explore the effects of CT-1 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, and the Crosstalk Between MAPK/ERK and PI3K/AKT Signal Pathways involved.Methods:(1)Isolated, cultured and identifed h UCB-MSCs: Human UCB samples were obtained with consent from the mothers and were separated and maintained by modified density gradient centrifugation with adherent culture,The surface markers of MSCs were detected by flow cytometry.(2)transfected with Adv:h UCB-MSCs were transfected with Adv-CT-1 and Adv-EGFP with various multiplicity of infection(MOI) at 100 PFU/cell.(3)induced neural differentiation:induced by NIM(retinoic acid,glutamine,Serum-free DMEM medium with high glucose),The morphological changes and the neural markers Neu N and GFAP were observed by confocal microscopy at 6h and 4days after induction.(4)transfected with si RNA at different concentration ranging from 25,50, 100 n M respectively.The transfection efficiency was analyzed by fluorescence microscope.(5) Adv-CT-1-h UCB-MSCs were transfected with si RNA-ERK and si RNA-Akt,The expression of ERK, p-ERK, Akt, p-Akt, Bax, Bcl-2were analyzed by Western blot.Results:(1) After culture for 10 days, adherent cells showed bipolar fibroblast-like morphology,rounded by a large number of osteoclast cells. The cells reached approximately 80% confluence could be passaged. The morphology of third passage cells was uniform.we employed flow cytometry to analysis the cell-surface antigen of fibroblast-like cells showed strong expression of CD44、CD90、CD73、CD105,with weak or absent expression of the hematopoietic lineage marker CD34 and monocyte-macrophage antigens CD45.(2)h UCB-MSCs were effectively transfected with Adv-CT-1(MOI=100 PFU/cell),and Stable expression of CT-1 was confirmed by fluorescence microscope after 72 h.(3)The cells were neuron-like morphological changes at 6h and 4d after induction,in addition to the blank control group,and the cells treated with NIM and CT1 changed more prominentiy and exhibited stronger expression neural markers Neu N than those treated without Adv CT-1. The positive rate of Neu N was the highest(44.23±2.83%) at 6h after induction in CT-1 group, the differences were statistically significant(P < 0.05); The positive rate of Neu N was still highest than that of other groups(82.50±4.17%) the difference was statistically significant(P < 0.05).At 6 h after induction, the expression of GFAP in CT-1 group were 55.37±3.31%, which were higher than other groups. The positive rate of GFAP was gradually increasing to 82.04±3.23% at 4d after induction,which was higher than other groups, the difference were statistically significant(P < 0.05).(4)transfected with si RNA,when the concentration of si RNA at 100 n M, the transfection efficiency was highest(91.67±1.74%),the difference was statistically significant(P < 0.05).(5)Western Blot results show that treated with si RNA- ERK and si RNA- Akt,the expression level of p- EKR, p- Akt,Bcl-2 significantly decreased compared to CT-1 group and si RNA-control group,on the other hand, it increased the expression of Bax. the difference was statistically significant(p < 0.05).Conclusions:(1) h UCB-MSCs can be isolated and cultured by modified density gradient centrifugation with adherent culture.(2)CT1 promotes neural differentiation and survival of h UCB-MSCs.(3) It may exist crosstalk between MAPK/ERK and PI3K/Akt Signal Pathways involved in CT1-stimulated neural differentiation and survival of h UCB-MSCs.