Table 1.
Primers for the qPCR.
Figure 1.
Flow cytometric analysis and Wnt profiles of hMSCs by the induction of NTs.
(A) Bone marrow-derived hMSCs were analyzed following four cell passages. hMSCs were positive for CD44, CD73, CD105, CD166, and Stro-1, and negative for CD14 and CD34. The solid curves indicate each type of antibody, and the filled curves indicate mouse IgG as the negative control. (B) mRNA levels of MAP2 were quantified on days 7, 14, and 21 during stimulation with NTs. NTs significantly increased MAP2 levels on days 14 and 21. Untreated hMSCs served as the control. (C) mRNA levels of Wnt1, Wnt3a, Wnt5a, Wnt7a, and Wnt7b were quantified on days 7, 14, and 21 during stimulation with NTs. NTs increased the expression of Wnt1 and induced expressions of Wnt7a and Wnt7b. * p<0.05, ** p<0.01 (i.e., treated vs. control in the Wnt1, Wnt3a, and Wnt5a groups; NTs at 14 and 21 days vs. NTs at 7 days in the Wnt7a and Wnt7b groups). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. * p<0.05, ** p<0.01 (i.e., treated vs. control). ND, not determined.
Figure 2.
Neurogenic effects from different Wnts in NT-induced hMSCs.
(A) mRNA levels of MAP2, SYN1, and LEF1 were quantified after 48 h of different Wnt treatments (2 µg/ml) in hMSCs that had been treated with NTs for 1 week. All Wnts promoted MAP2 expression, and Wnt7a induced the highest SYN1 expression. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. 1 p<0.05, 1″ p<0.01 (DMEM vs. all groups); 2 p<0.05, 2″ p<0.01 (NTs vs. all groups); 3 p<0.05, 3″ p<0.01 (Wnt1 vs. all groups); 4 p<0.05, 4″ p<0.01 (Wnt3a vs. all groups); 5 p<0.05, 5″ p<0.01 (Wnt5a vs. all groups); 6 p<0.05, 6″ p<0.01 (Wnt7a vs. all groups). (B) mRNA levels of ChAT, DBH, and LEF1 were quantified after 48 h of different Wnt treatments (2 µg/ml) in hMSCs that had been treated with NTs for 1 week. Wnt1 had no effects on ChAT or DBH expressions, but Wnt7a significantly induced both genes. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. 1 p<0.05, 1″ p<0.01 (DMEM vs. all groups); 2 p<0.05, 2″ p<0.01 (NTs vs. all groups); 3 p<0.05, 3″ p<0.01 (Wnt1 vs. all groups); 4 p<0.05, 4″ p<0.01 (Wnt3a vs. all groups); 5 p<0.05, 5″ p<0.01 (Wnt5a vs. all groups); 6 p<0.05, 6″ p<0.01 (Wnt7a vs. all groups).
Figure 3.
Dose- and time-dependent effects of Wnt7a in neurogenic hMSCs.
mRNA levels of nestin, MAP2, neurotubulin, and DVL1 were quantified after 48 h of Wnt7a treatment (0.1∼1.5 µg/ml) in a dose-dependent study (A) and at various times (i.e., 6∼48 h) after Wnt7a treatment (2 µg/ml) in a time-dependent study (B). NTs combined with Wnt7a promoted expression of neuronal genes related to the canonical Wnt pathway in time- and dose-dependent manners. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. * p<0.05, ** p<0.01 (NTs and Wnt7a vs. DMEM; NTs+Wnt7a vs. NTs).
Figure 4.
Induction of synaptic markers by Wnt7a and lithium in NT-stimulated hMSCs.
(A) Wnt7a groups were treated with Wnt7a (2 µg/ml) for 2 days, and the NTs groups were treated with NTs for 9 days. After NTs treatment for 1 week, Wnt7a (2 µg/ml) was added to the NTs+Wnt7a groups for 2 days. mRNA levels of LEF1, SYN1, BSN, and SYTG were examined in hMSCs, and levels were normalized to those in the NTs control (set to 1.0). Wnt7a induced mRNA expressions of SYN1, BSN, and SYTG in NT-stimulated hMSCs, and this induction was related to upregulation of LEF1. Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. * p<0.05, ** p<0.01 (NTs and Wnt7a vs. DMEM; NTs+Wnt7a vs. NTs). (B) LiCl groups were treated with LiCl (4 mM) for 2 days, and NTs groups were treated with NTs for 9 days. After NTs treatment for 1 week, LiCl (1 or 4 mM) was added to the NTs+LiCl groups for 2 days, mRNA levels of SYN1 and LEF1 were examined in hMSCs, and their levels were normalized to those in the DMEM control (set to 1.0). * p<0.05, ** p<0.01 (NTs and LiCl vs. DMEM; NTs+LiCl vs. NTs).
Figure 5.
Immunostaining and immunoblotting of NT-stimulated hMSCs with Wnt7a or lithium.
(A) p4 NT-treated hMSCs were stained with β-catenin (green). The NTs+Wnt7a groups were treated with NTs+Wnt7a for 24 h, and the NTs+lithium groups were treated with NTs+lithium for 24 h. 4,6-Diamidino-2-phenylindole (DAPI) (blue) was used as a counterstain. (B) p4 NT-treated hMSCs were immunoblotted with MAP2, SYN1, and GAPDH. The NT group was treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then with NTs+Wnt7a or lithium for 7 days. DMEM groups served as controls. (C) p4 NT-treated hMSCs were stained with MAP2 (red), SYN1 (red), and β-catenin (green). NTs groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then NTs+Wnt7a or lithium for 7 days. DAPI (blue) was used as a counterstain. DMEM groups served as the control. The white bar represents 50 µm. (D) Percentages of MAP2-positive cells, SYN1-positive cells, and neurite-positive cells among all DAPI-positive cells. All data are presented as the mean ± SD. * p<0.05, ** p<0.01 (all vs. NTs). (E) Cell areas were calculated by β-catenin-positive cells from (B).
Figure 6.
Inhibitory effects of Wnt inhibitors and blocking antibodies in Wnt7a-induced synapsin expression.
(A) As described in "Materials and Methods", mRNA levels of SYN1 and LEF1 were examined by a qPCR. sFRP4 showed significant inhibition of SYN1 expression, and Frz5 blocking antibodies greatly inhibited gene expressions. Levels were normalized to those in NTs groups (set to 1.0). * p<0.05, ** p<0.01 (NTs+Wnt7a vs. all groups). (B) Percentages of inhibition calculated from (A). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments.
Figure 7.
Neuronal specification by the non-canonical Wnt7a pathway in NT-induced hMSCs.
(A) mRNA levels of ChAT and DBH were examined in NT-induced hMSCs, and SP600125 (15 µM) and Wnt7a (2 µg/ml) or LiCl (4 mM) were added to NT-induced hMSCs at the same time. Levels were normalized to those in the NTs control (set to 1.0). Wnt7a, but not lithium, stimulated mRNA levels in NT-induced hMSCs, and SP600125 totally inhibited Wnt7a-induced ChAT and DBH expressions. Data are presented as the mean ± SD of one triplicate experiment that was representative of the three independent experiments. * p<0.05, ** p<0.01 (all vs. NTs). # p<0.05, ## p<0.01 (all vs. NTs+Wnt7a+SP600125). + p<0.05, ++ p<0.01 (all vs. NTs+LiCl+SP600125). (B) p4 NT-treated hMSCs were immunoblotted with ChAT, DBH, and GAPDH. The NT groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then with NTs+Wnt7a or lithium for 7 days. DMEM groups served as controls. (C) Expression levels of MAP2 and SYN1 in NT-induced hMSCs with SP600125/Wnt7a or SP600125/LiCl are shown. SP600125 had no effect in MAP2 or SYN1 expression. Levels were normalized to those in NTs groups (set to 1.0). * p<0.05, ** p<0.01 (NTs vs. all groups). # p<0.05, ## p<0.01 (all vs. NTs+Wnt7a+SP600125). + p<0.05, ++ p<0.01 (all vs. NTs+LiCl+SP600125). (D) p4 NT-treated hMSCs were stained with ChAT (green) and DBH (red). NTs groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for the first 7 days and then with NTs+Wnt7a or lithium for the next 7 days. In inhibitory groups, SP600125 was added with Wnt7a or lithium in NT-induced hMSCs at the same time. DAPI (blue) was used as a counterstain. DMEM groups were used as controls. The white bar represents 50 µm. (E) Percentages of ChAT-positive cells and DBH-positive cells among all DAPI-positive cells calculated from (D). All data are presented as the mean ± SD. * p<0.05, ** p<0.01 (all vs. NTs). # p<0.05, ## p<0.01 (all vs. NTs+Wnt7a+SP600125). + p<0.05, ++ p<0.01 (all vs. NTs+LiCl+SP600125). (F) As described in "Materials and Methods", mRNA levels of ChAT and DBH were examined by a qPCR. Levels were normalized to those in NTs groups (set to 1.0). * p<0.05, ** p<0.01 (NTs+Wnt7a vs. all groups). (G) Percentages of inhibition calculated from (E). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments.
Figure 8.
Summary of neuronal transdifferentiation regulation by Wnt7a in human bone marrow-derived MSCs.
Accompanying NGF, BDNF, and RA, Wnt7a activated the canonical/β-catenin pathway via receptor Frz5 to promote neurogenesis and trigger synaptic marker (SYN1) expression. Furthermore, Wnt7a also triggered differentiations of cholinergic and dopaminergic neurons, but this effect was induced by another non-canonical/JNK pathway through Frz9 receptors. In this study, we showed that Wnt7a utilized two pathways to promote hMSC neurogenesis.