Cell experiment:

PBMCs are cultured with pamidronate disodium for 8 days and then cells are labelled with or without 1.5 μM carboxyfluorescein succinimidyl ester (CFSE) and CFSE-labelled cells are then seeded in 6-well plates (2.5 × 106 cells/well) followed by treatment with various concentrations of TWS119 for 72 h. The total number of cultured cells is evaluated using an automated cell counter and the γδT cell proliferation is examined by flow cytometry[3].

Animal experiment:

All rats are randomly divided into four groups as follows: Sham group-rats undego the same surgical procedure, but the filament is not inserted and they receive 1 mL of dimethyl sulfoxide (1 % DMSO in saline); Vehicle group-rats undergo MCAO and receive 1 mL of DMSO; rtPA group-rats underwent MCAO and receive rtPA (10 mg/kg, Actilyse(R)) at 4 h after MCAO; and rtPA+TWS119 group-rats undergo MCAO and receive intraperitoneal TWS119 (30 mg/kg, dissolved in 1 mL 1 % DMSO) immediately after rtPA injection at 4 h after MCAO[2].

TWS119 is an inhibitor of glycogen synthase kinase-3β (GSK-3β) with IC50 value of 30nM [1].

TWS119 is screened out from a library of pyrrolopyrimidines as a molecule that selectively induces neuronal differentiation when using mouse P19 EC cells. TWS119 binds to GSK-3β tightly with a Kd value of 126nM and shows an inhibition with IC50 value of 30nM. The combination of TWS119 and GSK-3β modulates the activity of the complex and triggers downstream transcriptional events lead the neuronal induction. Besides that, TWS119 promotes neuronal differentiation of mESCs through another mechanism but not the canonical Wnt signaling pathway [1, 2].

References:
[1] Ding S, Wu T Y H, Brinker A, et al. Synthetic small molecules that control stem cell fate. Proceedings of the National Academy of Sciences, 2003, 100(13): 7632-7637.
[2] Wu T Y H, Ding S. Applying chemical tools to the discovery of novel regenerative medicine. Drug Discovery Today: Technologies, 2006, 3(3): 255-260.