Wnt/β-catenin signaling promotes midbrain specification of neural precursor cells derived from human embryonic stem cells

Abstract

Effective differentiation of midbrain dopaminergic (mDA) neurons derived from human embryonic stem (ES) cells is crucial for cell replacement therapy to treat Parkinson’s disease (PD). Therefore, in order to differentiate human ES cells into bona fide mDA neurons in vitro, mechanisms which control development of mDA neurons needs to be fully understood.In this study, I show the involvement of Wnt/β-catenin signaling in the anterior-posterior patterning during human ES cell-derived DA neuron development and the activation of Wnt signaling by small molecule glycogen synthase kinase-3 (GSK-3) inhibitor to obtain the midbrain specific characteristics. I introduced the small molecule GSK-3 inhibitor, 6-bromoindirubin-3''-oxime (BIO) to neural precursor cells (NPCs) derived from human ES cells by the simultaneous inhibition of BMP and Activin/Nodal signals. Once presented to NPCs, BIO exclusively promoted the expression of midbrain marker engrailed 1 (En1) while reducing the expression of forebrain marker brain factor 1 (Bf1) and hindbrain marker gastrulation brain homeo box 2 (Gbx2). Other known specific GSK-3 inhibitors such as 1-Azakenpaullone and lithium chloride (LiCl) also showed compatible results to BIO. However, NPCs treated with Wnt antagonist Dickkopf homolog 1 (DKK-1) and Frizzled-5 (Frz-5) revealed the significantly reduced expression of En1. Beta-catenin specific shRNA transduction also decreased β-catenin and En1 protein expression. Furthermore, a combined treatment of NPCs with BIO and fibroblast growth factor 8 (FGF8), a potent factor that promotes midbrain specification of NPCs, increased the level of En1 expression additively compared with BIO-only group while the decreased expression was found when SU5402, a FGF receptor inhibitor, was added to the combined treatment. This suggests that the endogenous FGF signal effects in En1 expression along with Wnt signal.Followed by midbrain specification using GSK-3 inhibitors, NPCs then differentiated into mDA neurons by a serial treatment of Sonic Hedgehog (SHH), FGF8, and ascorbic acid. Among the differentiated TH-positive neurons, an increased number of cells coexpressed En1 while having a significantly low number of TH-positive cells coexpressed GABA. These results show that the activation of the canonical Wnt signaling pathway of NPCs increases the number of midbrain specific neurons within TH-positive neurons.