WNT-C59, a Small-Molecule WNT Inhibitor, Efficiently Induces Anterior Cortex That Includes Cortical Motor Neurons From Human Pluripotent Stem Cells

Abstract

The recapitulation of human neural development in a controlled, defined manner from pluripotent stem cells (PSCs) has considerable potential for studies of human neural development, circuit formation and function, and the construction of in vitro models of neurological diseases. The inhibition of Wnt signaling, often by the recombinant protein DKK1, is important for the induction of cortical neurons. Here, we report a novel differentiation method using a small-molecule WNT inhibitor, WNT-C59 (C59), to efficiently induce human anterior cortex. We compared two types of small molecules, C59 and XAV939 (XAV), as substitutes for DKK1 to induce cortical neurons from PSCs in serum-free embryoid body-like aggregate culture. DKK1 and XAV inhibited only the canonical pathway of Wnt signaling, whereas C59 inhibited both the canonical and noncanonical pathways. C59 efficiently induced CTIP2 + /COUP-TF1 2 cells, which are characteristic of the cells found in the anterior cortex. In addition, when grafted into the cortex of adult mice, the C59-induced cells showed abundant axonal fiber extension toward the spinal cord. These results raise the possibility of C59 contributing to cell replacement therapy for motor neuron diseases or insults. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:552-560 SIGNIFICANCEFor a cell therapy against damaged corticospinal tract caused by neurodegenerative diseases or insults, cortical motor neurons are needed. Currently, their induction from pluripotent stem cells is considered very promising; however, an efficient protocol to induce motor neurons is not available. For efficient induction of anterior cortex, where motor neurons are located, various WNT inhibitors were investigated. It was found that one of them could induce anterior cortical cells efficiently. In addition, when grafted into the cortex of adult mice, the induced cells showed more abundant axonal fiber extension toward spinal cord. These results raise the possibility that this inhibitor contributes to a cell-replacement therapy for motor neuron diseases or insults.

Transplantation of feeder‐free human induced pluripotent stem cell–derived cortical neuron progenitors in adult male Wistar rats with focal brain ischemia

“…We obtained a comparable cortical neuron induction with previous feeder‐maintained hiPSC neural induction (Gomi et al, ; Motono et al, ). The previous study showed that xeno‐free neural induction could be performed with insulin, transferrin, and sodium selenite and taurine cocktail (Lukovic et al, ), but there was no cortical neuron formation.…”

“…In this study, we maintained on laminin E8 fragment (iMatrix 511, Nippi, Ibaraki, Japan) with feeder‐free culture medium (Stemfit [AKO3], Ajinomoto, Tokyo, Japan). To adjust the differentiation in the feeder‐free condition, we modified a previously described protocol on SNL feeder condition (Motono et al, ). Confluent hiPSC colonies were pretreated with Y‐27632 15 µM (WAKO, Tokyo, Japan) prior to differentiation.…”

“…The colonies were dissociated with Accumax (Innovative Cell Tech., San Diego, CA, USA), and 12,000 live cells per well were then allowed to form embryoid body–like aggregates in U‐bottom 96‐well plates (Sumitomo Bakelite). To adjust the induction in the feeder‐free condition, we modified a previously described protocol on the SNL feeder condition (Motono, Ioroi, Ogura, & Takahashi, ). The colonies were dissociated with Accumax (Innovative Cell Tech., San Diego, CA, USA), and 12,000 live cells per well were then allowed to form embryoid body–like aggregates in U‐bottom 96‐well plates (Sumitomo Bakelite, Kobe, Japan).…”

Rostrocaudal Areal Patterning of Human PSC-Derived Cortical Neurons by FGF8 Signaling

“…A previous study showed that PSCs can be specified to differentiate into hippocampal neurons by activating Wnt and BMP signaling ( Sakaguchi et al, 2015 ). In another study, inhibition of Wnt signaling induced rostral marker expression ( Motono et al, 2016 ). The modulation of these signaling pathways, along with that of FGF8, will enable finer control of areal identity in PSC cultures.…”

L1CAM Is a Marker for Enriching Corticospinal Motor Neurons in the Developing Brain

“…To investigate the characteristics of CTIP2 + cells, we generated CTIP2:GFP knock-in (KI) mESCs and differentiated them into neural lineage by inhibiting WNT and TGF/Activin/Nodal signaling in a floating culture of cell aggregates (Figure 2A; Motono et al, 2016). The sphere size gradually increased, and GFP expression became detectable by around day 12 ( Figure 2B).…”