Roles Of Wnt/β-catenin Signaling In Neural Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

Objective:To investgate whether basic fibroblast growth factor(b FGF) and epidermal growth factor(EGF) can induce the differentiation of human bone marrow mesenchymal stem cells(h BMSCs) into neural cells, and then explore the role of Wnt/β-catenin signaling in neural differentiation of h BMSCs.Methods:(1) h BMSCs at passage 2 were purchased from CHI Scientific, Inc. The marrow stromal surface markers CD44, CD90 and hematopoietic cell markers CD19, CD34 of h BMSCs at passage 4 were detected by flow cytometry.(2) To induce neural differentiation of h BMSCs, the cells from passage 4 to passage 6 were cultured in α-MEM supplemented with 1% FBS. The experiment was composed of 5 groups including control group, EGF group(h BMSCs cultured with20 ng/m L EGF), b FGF group(h BMSCs cultured with 20 ng/m L b FGF), EGF+b FGF group(h BMSCs cultured with 20 ng/m L EGF and 20 ng/m L b FGF) and EGF+b FGF+DKK-1 group(h BMSCs cultured with 20 ng/m L EGF, 20 ng/m L b FGF and 100 ng/m L DKK-1). Cellular morphologic changes were observed under inverted phase contrast microscope.(3) After 7 days of induction, the expression of neural phenotype markers including nestin, NSE, MAP-2, GFAP at m RNA levels was determined by RT-PCR assay. The expression of NSE and GFAP at protein levels was measured by Western blot assay and immunocytochemistry assay.(4) After 7 days of induction, the expression of several key components of Wnt/β-catenin signaling including GSK-3β, β-catenin, cyclin D1 at m RNA levels was determined by RT-PCR assay. The expression of total β-catenin, P-β-catenin,Cytoplasm-β-catenin, Nucleus-β-catenin at protein levels was measured by Western blot assay. The effect of β-catenin subcellular distribution of h BMSCs was observed by immunocytochemistry assay.Results:(1) h BMSCs at passage 4 predominantly consisted of homogeneous fibroblast-like and spindle-shaped cells. The expression of h BMSCs markers was highly positive for CD44(98.24%) and CD90(95.89%), but negative for CD19 and CD34(total 1.91%).(2) After 7 days of induction, some cells in b FGF group, EGF+b FGF group, and EGF+b FGF+DKK-1 group changed with shrinking and highly refractive cell bodies,and some of them extended multiple long processes. Synaptic connections were clearly visible between some cells, which suggested that h BMSCs had differentiated into neural-like cells. However, the morphological changes in EGF group and control group were not apparent.(3) The results of neural phenotype markers determined by RT-PCR showed that the cells in b FGF group and EGF+b FGF group expressed higher m RNA levels of nestin, NSE, and MAP-2 than control group(P<0.01), while the changes of that in EGF group were not obvious(P>0.05) except NSE up-regulation(P<0.05). GFAP was positive in EGF+b FGF group(P<0.01). EGF+b FGF group had higher levels of nestin, NSE, MAP-2, and GFAP compared to b FGF group and EGF+b FGF+DKK-1group(P<0.01). The results of immunocytochemistry analysis showed that NSE and GFAP proteins were positive in b FGF group, EGF+b FGF group, and EGF+b FGF+DKK-1 group(P<0.01), but negative in control group and EGF group. Moreover, the positive cell ratios in EGF+b FGF group were significantly higher than that in b FGF group and EGF+b FGF+DKK-1 group(P<0.01). The results of western blot analysis were similar to immunocytochemistry analysis to a great extent.(4) The results of several key components of Wnt/β-catenin signaling determined by RT-PCR demonstrated the down-regulation m RNA expression of GSK-3β(P<0.05) and the up-regulation m RNA expression of β-catenin and cyclin D1 in b FGF group and EGF+b FGF group(P<0.01). The results of western blot analysis suggested that the expression of P-β-catenin was decreased and the expression of totalβ-catenin, Cytoplasm-β-catenin and Nucleus-β-catenin was increased in b FGF group and EGF+b FGF group. Furthermore, the immunocytochemisry analysis of nuclearβ-catenin in EGF+b FGF group had higher intensity compared to that in control group.The changes above could be inhibited by DKK-1.Conclusion:(1) b FGF alone is able to induce the differentiation of h BMSCs into neural cells,but EGF fails to do that. However, EGF is efficient to drive neural differentiation of h BMSCs induced by b FGF。(2) Wnt/β-catenin signaling plays a postive role in neural differentiation of h BMSCs.