Robust differentiation of human pluripotent stem cells into Schwann cells.

Research into Schwann cell (SC)-related diseases has been hampered by the difficulty of obtaining human-derived SCs, which have limited proliferative capacity. This has resulted in a delay in progress in drug discovery and cell therapy targeting SCs. To overcome these limitations, we developed a robust method for inducing the differentiation of human induced pluripotent stem cells (hiPSCs) into SCs. We established hiPSC lines and successfully generated high-purity Schwann cell precursors (SCPs) from size-controlled hiPSC aggregates by precisely timed treatment with our proprietary enzyme solution. Such SCPs were successfully expanded and further differentiated into myelin basic protein (MBP) expressing SC populations when treated with an appropriate medium containing dibutyryl-cAMP (db-cAMP). These differentiated cells secreted factors that induced neurite outgrowth in vitro. Our method allows for the efficient and stable production of SCPs and SCs from hiPSCs. This robust induction and maturation method has the potential to be a valuable tool in drug discovery and cell therapy targeting SC-related diseases.

Assessing the activation/inhibition of tyrosine kinase-related pathways with a newly developed platform.

The phosphorylation of cellular proteins plays a crucial role in the transduction of various signals from outside the cell into the nucleus. The signals are transduced by phosphorylation chain reactions within multiple pathways; however, determining which pathways are responsible for each defined signal has proven challenging. To estimate the activity of each pathway, we developed a phosphorylation array platform comprising a protein array with 1200 proteins belonging to 376 signalling pathways and an analytical method to estimate pathway activity based on the phosphorylation levels of proteins. The performance of our system was assessed by reconstructing kinase-substrate relationships, as well as by estimating pathway activity upon epidermal growth factor (EGF) stimulation and the pharmacological inhibition of epidermal growth factor receptor (EGFR). As a result, kinase-substrate relationships were reliably reconstructed based on the precise measurement of phosphorylation levels of constituent proteins on the array. Furthermore, the pathway activities associated with EGF stimulation and EGFR inhibition were successfully traced through the related pathways from the outer membrane to the nucleus along a time course. Thus, our phosphorylation array system can effectively assess the activity of specific signalling pathways that are perturbed by extracellular stimuli, such as various drugs.

Nonmuscle myosin II folds into a 10S form via two portions of tail for dynamic subcellular localization.

Nonmuscle myosin II forms a folded conformation (10S form) in the inactivated state; however, the physiological importance of the 10S form is still unclear. To investigate the role of 10S form, we generated a chimeric mutant of nonmuscle myosin IIB (IIB-SK1·2), in which S1462-R1490 and L1551-E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB-SK1·2 mutant did not fold into a 10S form under physiological condition in vitro. IIB-SK1·2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB-SK1·2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB-SK1·2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB-SK1·2, which may underlie the change of the cellular migratory properties. These results suggest that the 10S form is necessary for maintaining nonmuscle myosin II in an unassembled state and for recruitment of nonmuscle myosin II to a specific region of the cell.

Synthesis and Bioevaluation of 3-(Arylmethylene)indole Derivatives: Discovery of a Novel ALK Modulator with Antiglioblastoma Activities.

Glioblastoma is the most common brain tumor, with high recurrence and low survival rates. An integrative bioinformatics analysis demonstrated that anaplastic lymphoma kinase (ALK) is a promising therapeutic target for glioblastoma. We designed and synthesized a series of 3-(arylmethylene)indole derivatives, which were further evaluated for antiproliferative activity using glioma cell lines. Among them, compound 4a significantly inhibited the viability of glioblastoma cells. With favorable pharmacokinetic characteristics and blood-brain barrier permeability, 4a improved the survival rate and inhibited the growth of orthotopic glioblastoma. The Phospho-Totum system revealed that ALK was a potential target for the antiglioblastoma activity of 4a. Further experiments indicated that 4a might be a novel ALK modulator, which interacted with the extracellular ligand-binding domain of ALK, thus selectively induced ERK-mediated autophagy and apoptosis. Our findings provide an alternative ALK-based targeting strategy and a new drug candidate for glioblastoma therapy.