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Pancreatic Langerhans islets are mainly composed of insulinsecreting beta cells and glucagon-secreting alpha cells, along with other minor cell types, and play a central role in the regulation of blood glucose levels. Because of this, imaging of viable pancreatic islets is an important component in research on diabetes both in clinical and experimental medicine. The conventional imaging technique for pancreatic islets is antibody-based immunostaining directly on pancreatic sections, or using transgenic mice with luminescent reporter genes linked to islet-specific promoters. Among small molecule probes, Newport Green and dithiazone (DTZ) have been used for ex vivo fluorescent staining of pancreatic islets, based on their Zn ion binding affinity, which are abundant in beta cells in complex with insulin. For in situ application, fluorescently labeled exendin-4 (a GLP1R binding peptide: M.W. is about 5 kDa) has been recently introduced for the measurement of the mass of pancreatic islet beta cells. However, small molecule probes for selective staining of beta cells in pancreatic islets of live animals have not yet been reported. We predicted that a diversity-oriented fluorescence library approach (DOFLA), an expedited bioimaging probe discovery method using high throughput synthesis and high contents screening, would be a powerful method to achieve this goal. Using a similar approach, we have previously elucidated probes for pluripotent stem cells (CDy1), muscle cells (CDy2), neuronal stem cells (CDr3), and pancreatic alpha cells (GY= glucagon yellow). Although the glucagon-targeting probe GY selectively stains alpha cells in isolated cell culture, it did not clearly mark mouse pancreatic islets in tissue, partially owing to the small population of alpha cells (around 15–20% in mouse islets). We expected that a fluorescent probe for pancreatic beta cells (with a larger population of 75–80% in mouse islets) would be more effective for visualizing pancreatic islets. As a first step, we synthesized fluorescent smallmolecule libraries composed of 1200 compounds, and screened them against beta TC-6 cells in comparison to alpha TC-1 cells and acinar cells (exocrine cells in the pancreas) as controls. The three cell types were compared in 384-well plates and incubated with the library compounds (1 mm) at incubation times ranging from one to 48 hours. The fluorescence live-cell images were acquired by an automated imaging microscope system, ImageXpress Micro. One compound from the BDNCA series (Scheme 1), BDNCA-325 (labs/lem= 558/585 nm, extinction cooefficient e= 58,000m 1 cm , quantum yield F= 0.06) was chosen as the most selective for the beta TC-6 cells (Figure 1a) in comparison to the two control cell types in terms of relative fluorescence intensity. The BDNCA library was prepared from a BODIPY-aniline (BDN) series by chloroacetylation. While BDN has very low fluorescence emission (less than 1% quantum yield) owing to photoinduced electron transfer (PET), by converting the amine to an amide, the fluorescence of BDNCA was moderately increased (F= 5–10%). Therefore, this amide motif is a modulator of the fluorescence intensity of the BDNCA series through interaction with the surrounding environment or binding partner. However, when we injected BDNCA-325 intravenously into a mouse, the pancreatic islets were not selectively stained at various incubation times and concentrations (data not shown). Because BDNCA-325 contains a chemically reactive chloroacetyl group, we hypothesized that the compound might have reacted with other tissues in the animal before reaching the pancreatic islets. Thus, BDNCA-325 was modified by removing the reactive alpha-chloride and also by [*] Dr. N.-Y. Kang, Dr. S.-C. Lee, Dr. S.-J. Park, Dr. S.-W. Yun, Dr. E. Kostromina, Dr. N. Gustavsson, Dr. Y. Ali, Y. Chandran, Dr. W. Han, Dr. G. K. Radda, Prof. Y.-T. Chang Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR) 138667, Singapore (Singapore) E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

Current strategies to monitor reprogramming into induced pluripotent stem cells (iPSCs) are limited in that they rely on the recognition of advanced stage biomarkers or they involve the transduction of genetically-modified cells. These limitations are particularly problematic in high-throughput screenings where cell availability, low cost and a rapid experimental protocol are critical issues. Herein we report the application of a pluripotent stem cell fluorescent probe (i.e. CDy1) as a reporter for the rapid screening of chemicals in reprogramming iPSCs. CDy1 stains early-stage iPSCs at 7dpi as well as matured iPSCs; hence it can partially overcome the slow kinetics of the reprogramming process. As a proof of concept, we employed a CDy1-based screening in 384 well-plates to examine the effect of newly synthesized hydroxamic acid derivatives in reprogramming mouse fibroblasts transduced with Oct4, Sox2 and Klf-4 without c-Myc. One compound (1–26) was identified as a reprogramming enhancer by 2.5-fold and we confirmed that 1–26 behaves as a histone deacetylase (HDAC) inhibitor. The successful identification of novel small molecules enhancing the generation of iPSCs by means of a rapid and simple protocol demonstrates the suitability of this CDy1-based screening platform for the large scale and high-throughput evaluation of iPSC modulators.

A better understanding of the cellular and molecular mechanisms involved in the reprogramming of somatic cells is essential for further improvement of induced pluripotent stem (iPS) cell technology. In this study, we enriched for cells actively undergoing reprogramming at different time points by sorting the cells stained with a stem cell-selective fluorescent chemical probe CDy1 for their global gene expression analysis. Day-to-day comparison of differentially expressed genes showed highly dynamic and transient gene expressions during reprogramming, which were largely distinct from those of fully-reprogrammed cells. An unbiased analysis of functional regulation indicated robust modulation of concurrent programs at critical junctures. Globally, transcriptional programs involved in cell proliferation, morphology and signal transduction were instantly triggered as early as 3days-post-infection to prepare the cell for reprogramming but became somewhat muted in the final iPS cells. On the other hand, the highly coordinated metabolic reprogramming process was more gradually activated. Subsequent network analysis of differentially expressed genes indicated PDGF-BB as a core player in reprogramming which was verified by our gain- and loss-of-function experiments. As such, our study has revealed previously-unknown insights into the mechanisms of cellular reprogramming.

We report the first fluorescent diamino-chalcone library and its application in the discovery of a mouse embryonic stem cell (mESC) probe. CDg4, a novel green fluorescent mESC probe was discovered through a high-content image based screening of 160 members of the chalcone library. Interestingly, the molecular binding target of CDg4 was identified as the glycogen of the stem cell colony surface, rather than a conventional protein target from an intracellular source.

We report the first solid phase synthesis of a xanthone library CX and its application to embryonic stem cell probe development. The CX library was further derivatised with an activated ester resin to provide an acetylated CX (CXAC) library. Screening of these libraries led to the discovery of a novel fluorescent mESC probe, CDb8.