Wnt/β-Catenin Signaling Promotes Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells
Authors: Yu Qin , Liu Lizhen , Duan Yanping , Dong Hui , Shan Wei , Wang Biao , Yu Xiaohong , Huang He 
Published: October 2019

Abstract
Abstract 4814

Introduction
Mesenchymal stem cells (MSCs) have the potential to self-renew and differentiate into multiple cell types and have been used to replace damaged cells in the nervous system using animal models of neurological disorders or traumatic brain injury. Previous reports revealed that MSCs can differentiate into neuron-like cells in vitro. However, precise mechanisms controlling this process are unclear. Several lines of evidence suggest that Wnt signaling pathway plays pivotal roles in MSC differentiation. Here we explored the roles of Wnt/β-catenin signaling in neuronal differentiation of MSCs.

Methods
MSCs were obtained from rat bone marrow and cultured in proliferation medium containing DMEM /10%FBS. Passage 3–6 was used in this study. MSCs were induced neuronal differentiation as previously described. Twenty-four hours prior to neuronal induction, media were replaced with preinduction media consisting of DMEM/20% FBS and 1mM β-mercaptoethanol (BME). To initiate neuronal differentiation, the preinduction media were removed, and the cells were washed with PBS and transferred to neuronal induction media composed of DMEM/5mM BME and cultured for 5h. Cells after pre-induction for 6h, 12h, 24h or induction for 1h, 3h and 5h were collected. The expression of β-catenin, NSE, Nestin and Ngn1 was analyzed by Western blot, RT-PCR and immunocytochemical analysis. To further determine the roles of β-catenin, MSCs were transfected with β-catenin siRNA using Lipofectamine 2000. Cells after pre-induction for 24h and induction for 3h were collected and expression of β-catenin, NSE, Nestin and Ngn1 were detected. In addition, Wnt 3a were added to the differentiation system. Differentiation efficiency and expression of β-catenin and neuronal specific genes were analyzed.

Results
(1) MSCs could differentiate into neuron-like cells. After adding 5mM BME into MSCs culture system, cells showed neuron-like cells in morphology. Cytoplasm in the flat MSCs retracted towards the nucleus, forming a contracted multipolar. To characterize neuronal differentiation, we fixed BME-treated cultures after 5 h and stained them for the neuronal marker NSE and Nestin. The results showed that NSE+ cells were 76.00±6.50 % and Nestin+ cells were 70.52 ±5.02%. (2) Wnt/β-catenin signaling involved in the neural differentiation of MSC. β-catenin plays a central role in Wnt/β-catenin signaling pathway. Our data showed that the expression of β-catenin increased during the neural differentiation of MSCs both at mRNA and protein levels. Accordingly, the neuronal markers NSE and Nestin both increased during differentiation process. (3) β-catenin knockdown inhibited neuronal differentiation of MSCs. The western blotting and the immunocytochemical analysis revealed that the protein amount β-catenin decreased obviously after MSCs transfection with β-catenin siRNA for 72h. Subsequently, the expression of NSE, Nestin and Ngn1 decreased compared with MSCs transfected with control siRNA and non-transfected with siRNA. In addition, differentiation efficiency decreased signifufantly (P<0.05). (4) Wnt 3a promotes neuronal differentiation of MSCs. In comparison with that in control cells, Wnt3a, β-catenin protein were increased by Wnt3a (100 ng/ml)-conditioned medium, the neuronal differentiation was markedly improved in the Wnt3a treated cells in association with the increase in the protein and gene expression neural markers NSE and Nestin.

Conclusion
Our data firstly revealed that Wnt/β-catenin signaling promotes neuronal differentiation of bone marrow MSCs. β-catenin plays a pivotal role in this process and may be considered as a target to regulate in neurologic disease treatment.

Disclosures:
No relevant conflicts of interest to declare.