A fluorescent rosamine compound selectively stains pluripotent stem cells.

Stem cells, which are capable of self-renewing and differentiating into various types of cells, have captured great interest as a valuable resource for regenerative medicine and developmental biology research. Technical progress during the last decade has enabled the isolation of stem cells from a wide range of tissues, their differentiation into specific types of cells, and the generation of induced pluripotent stem cells (iPSC) from somatic cells. The recent success of patientspecific iPSC generation and their differentiation into functional cells exemplifies how stem cells can be used for drug discovery and treatment of specific patients having complex diseases. However, despite the general enthusiasm for the multiple applications of stem cells, their practical use both in research and disease therapy has been hampered by the heterogeneity of stem cells and their unpredictable proliferation and differentiation. The current methods of isolation and characterization of stem cells mostly depend on their morphology in the culture, such as colony or sphere formation, and immunodetection of marker proteins. These methods, however, require extended times and antibody reactions which may make the cells unsuitable for further usage. Therefore, the development of tools and technologies that may facilitate the isolation, identification, and characterization of stem cells is one of the most demanding requisites in the field of stem-cell research and applications. Fluorescent small molecules have been widely used for the visualization of polymeric biomolecules or cellular organelles. We have employed combinatorial chemistry to develop several diversity-oriented fluorescence libraries (DOFL) and successfully applied them to the discovery of imaging probes for a number of biological targets. Among our libraries is a rosamine library synthesized using solidphase chemistry to provide more flexibility within the rhodamine scaffold, which has excellent photophysical properties. By screening this library against a muscle-formation cell culture, we previously discovered a compound that controls muscle differentiation. To additionally evaluate the application of rosamine derivatives as stem-cell-selective probes, we have screened the library against embryonic stem cells (ESC) in this study and discovered a novel fluorescent compound, called the compound of designation yellow 1 (CDy1, lex/lem= 535/570 nm), that selectively stains ESC and iPSC as well. For high-throughput screening, we incubated mouse ESC (mESC) and mouse embryonic fibroblast (MEF) feeder cells with 280 rosamine compounds at a concentration of 500 nm in 384-well microplates. After 0.5, 24, and 48 hours, tetramethylrhodamine isothiocyanate (TRITC) fluorescence and bright-field images were taken using an ImageXpress imaging system. From this primary screening, 20 compounds that stained mESC consistently with stronger intensity than MEF were manually selected. As a secondary screening, we incubated mESC and MEF separately with each of the hit compounds and analyzed them using flow cytometry and [*] Dr. C.-N. Im, Dr. H.-H. Ha, Dr. S. Y. Lee, Dr. Y. K. Kim, Dr. J.-S. Lee, Prof. Dr. Y.-T. Chang Department of Chemistry, National University of Singapore 3 Science Drive 3, Singapore 117543 (Republic of Singapore) Fax: (+65)6779-1691 E-mail: [email protected] Homepage: http://ytchang.science.nus.edu.sg Dr. N.-Y. Kang, Dr. X. Bi, Dr. J. J. Lee, Dr. S.-J. Park, Dr. M. Vendrell, Dr. J. Li, Dr. S.-W. Yun, Prof. Dr. Y.-T. Chang Laboratory of Bioimaging Probe Development Singapore Bioimaging Consortium, Agency for Science Technology and Research (A*STAR), 11 Biopolis Way # 02-02 Helios, Singapore 138667 (Republic of Singapore) E-mail: [email protected]