BIO (6-bromoindirubin-3'-oxime) GSK3 inhibitor induces dopaminergic differentiation of human immortalized RenVm cells

Abstract

Parkinson's disease (PD) is one of the most neurodegenerative disorders which can lead to severe neural disability and neurological defects. Cell-based therapy using fully differentiated cells is a new method for the treatment of this abnormal condition. In the present study, we investigated the effects of 6-bromoindirubin-3'-oxime (BIO) on dopaminergic differentiation of human immortalized RenVm cells in order to obtain a set of fully differentiated cells for transplantation in Parkinson's disease. To this end, the immortalized RenVm cells were induced to dopaminergic differentiation using a neuro basal medium supplemented with N2 and different concentrations (75, 150, 300, 600, and 1200 nM) of BIO for 4, 8, and 12 days. The efficiency of dopaminergic differentiation was determined using immunocytochemistry for tyrosine hydroxylase expressions. In addition, the expression of a β-catenin marker was measured using the western blot technique. The results of immunocytochemistry revealed that the mean percentage of Tuj1- and TH-positive sells in 150- and 300-nM-BIO-treated groups was significantly increased compared to that of other groups (p ≤ 0.01). In addition, the expression of the β-catenin marker was higher in these groups as compared with that of other groups. Overall, BIO through its effect on the Wnt-Frizzled signaling pathway can promote dopaminergic differentiation of RenVm cells in a dose-dependent manner.

Keywords: 6-Bromoindirubin-3′-oxime; Beta catenin; Tyrosine hydroxylase; Wnt signaling pathway.

Fig. 1

Generation of dopaminergic neuron from induced RenVm cells by different concentrations of BIO. RenVm cells at the beginning of the neural differentiation (a). RenVm cells which expressed Tuj1 (b) and tyrosine hydroxylase (TH) (c) markers after differentiation. Scale bars represent 200 μm in a and 100 μm in b and c

Fig. 2

Immunocytochemistry images of differentiated cells which expressed the Tuj1 marker in different BIO concentrations. The scale bar represents 100 μm

Fig. 3

The mean percentage of differentiated cells which expressed the Tuj1 markers. In the 150 and 300 nM concentrations of BIO in the fourth and eighth days, the mean percentage of Tuj1-positive cells was significantly increased compared to that of the other groups (**p ≤ 0.01)

Fig. 4

Immunocytochemistry images of differentiated cells which expressed the tyrosine hydroxylase (TH) marker in different BIO concentrations. The scale bar represents 100 μm

Fig. 5

The mean percentage of differentiated cells which expressed the tyrosine hydroxylase (TH) marker. In the 150 and 300 nM concentrations of BIO in the fourth and eighth days, the mean percentage of TH-positive cells was significantly increased compared to that of the other groups (**p ≤ 0.01)

Fig. 6

Western analysis of β-catenin in differentiated and undifferentiated cells in different concentrations of BIO at 4, 8, and 12 days. β-Actin was used as a control marker