Synthetic small molecules that control stem cell fate

Abstract

In an attempt to better understand and control the processes that regulate stem cell fate, we have set out to identify small molecules that induce neuronal differentiation in embryonic stem cells (ESCs). A high-throughput phenotypic cell-based screen of kinase-directed combinatorial libraries led to the discovery of TWS119, a 4,6-disubstituted pyrrolopyrimidine that can induce neurogenesis in murine ESCs. The target of TWS119 was shown to be glycogen synthase kinase-3beta (GSK-3beta) by both affinity-based and biochemical methods. This study provides evidence that GSK-3beta is involved in the induction of mammalian neurogenesis in ESCs. This and such other molecules are likely to provide insights into the molecular mechanisms that control stem cell fate, and may ultimately be useful to in vivo stem cell biology and therapy.

Fig. 1.

Structures of 4,6-disubstituted pyrrolopyrimidines and affinity matrices.

Fig. 2.

Induction of neurogenesis by TWS119. P19 cells were treated with 1–5 μM TWS119 for 2 days and cultured in compound-free MEMα supplemented with 2% FBS for an additional 2–14 days (B27-supplemented neurobasal medium was used for culturing longer than 10 days) before fixing and immunostaining with various neuronal specific antibodies. (A–F) Immunofluorescence staining for nestin, neurofilament-M/TuJ1, Map2(a+b)/TuJ1, NeuN/TuJ1, glutamate, and synapsin 1. (G–H) Under similar conditions, TWS119 can also induce murine ESCs (D3 cell line) to differentiate into neurons as indicated by immunofluorescence staining for neurofilament-M/TuJ1 and Map2(a+b)/TuJ1.

Fig. 3.

Silver staining and Western blot analysis of proteins retained by affinity supports and SPR-based binding assay. (A) Total protein extracts from P19 cells lysed with PY buffer plus 0.4% Nonidet P-40 were loaded onto affinity supports AS·101, AS·102, AS·119, AS·113, and AS·121. After three washes with bead buffer, bound proteins were resolved by SDS/PAGE and revealed by silver staining. (B) Proteins retained by AS·101, AS·102, and AS·119 affinity supports were confirmed to be GSK-3β by Western blotting with anti-GSK-3β antibody. (C) SPR-based interaction analysis of TWS119 with recombinant GSK-3β. A GST-GSK-3β fusion protein was immobilized on an S series CM5 biosensor chip (≈17,000 relative light units) via EDC coupling reactions. Ligand binding with increasing concentrations of TWS119 (2–1,000 nM) were analyzed on a BIAcore S51 instrument. Analysis of the equilibrium binding responses was performed assuming a 1:1 binding model. The average affinity for TWS119 binding to GSK-3β was determined at K_D = 126 ± 11 nM in four independent experiments.

Fig. 4.

Effects on β-catenin-dependent signaling on treatment of P19 cells with TWS119. (A) β-catenin levels were analyzed by Western blot with anti-β-catenin antibody in P19 cells treated with TWS121 (24 h, 10 μM), TWS101 (24 h, 5 μM), and TWS119 (24 h, 3 μM). (B) pTOPFLASH luciferase reporter assay in a P19 monolayer treated with TWS analogs and P19 aggregates treated with RA: P19 cells were cotransfected with pTOPFLASH (containing four consensus LEF-1/TCF-1 binding sites, a minimal promoter, and a firefly luciferase reporter) and Renilla luciferase control reporter (Promega). After 24 h, cells were trypsinized and replated into a 96-well tissue culture plate and treated with TWS analogs, or plated into Petri dishes and treated with RA in MEMα with 5% FBS 12 h postplating. Thirty-six hours later, cells were lysed, and protein extracts were assayed for luciferase activity. The increase in firefly luciferase activity was normalized to Renilla luciferase activity and represents the average of three experiments.