HIGHLY SPECIFIC FLUORESCENT PROBE FOR MOUSE PLURIPOTENT STEM CELLS 3

CHAPTER FOUR CONCLUSION Stem cells are invaluable tools for the understanding of cell based therapies and the development of important disease models to aid in drug discovery and therapy (26). Therefore it is vital to be able to control and monitor stem cell development and differentiation without any manipulations or side effects to the cells. Here we propose that a highly specific fluorescence probe is essential for the above to visualize and monitor live mESC. From all the data supported in this thesis, we report the first highly selective bioimaging fluorescent probe for mESCs (CDy9) via high throughput screening using DOFLA. Although several mESC fluorescent probes (CDy1, CDg4, CDb8) have been published previously, none of them are as specific to mESC as CDy9. The probe’s specificity to only mESC is very important especially if the probe is injected into mouse disease models. This is because; non specific binding to other cell types may result in false positive results and interfere with stem cell tracking. This may then prevent the development of drug therapies or cell based therapies that are necessary for the treatment of that disease. Furthermore, as mentioned earlier, the current methods of mESC detection via the use of immuno fluorescence, fluorescence proteins or fluorescence tags such as GFP are time consuming, expensive, require cell fixing or prior genetic modification to the cells. These are undesirable or unwanted complications to the cell behavior and thus need to be avoided. Therefore, a small molecule probe such as CDy9 will allow the visualization of mESC in their native forms. CDy9 also showed the capability to isolate out mESC successfully from a mixture of samples and it also stained other pluripotent stem cells such as miPSCs. This further illustrates the ability of CDy9 to bind to only pluripotent stem cells specifically compared to other cell types as shown in the cell panel test. It is also known that miPSC generation is a very time consuming and tedious process and so CDy9 staining can be used as an 1 additional confirmation step to the GFP signal for miPSC generation as CDy9 compound signal do not overlap with the GFP signal. The ability of CDy9 to isolate out single mESC cells also proved that the compound is able to purify undifferentiated stem cells from a mixture of differentiated cells. This way stem cell colony can be cultured from just a single cell that was isolated by CDy9 as demonstrated by the FACS analysis and reculture. Since the amount of pluripotent stem cells present in the body are very minimal, CDy9 may therefore aid in isolating just one pluripotent stem cell from the body and this cell can be cultured (given the correct culture conditions) to grow in a colony of cells which may then be used for further experiments or therapies. Besides its excellent selectivity, CDy9 also showed minimal cytotoxicity at the working concentrations and proved to bind to mESC in a stable manner. CDy9 also seemed promising in isolating potential stem cells from mouse fats and may also be employed to other organs to isolate similar pluripotent potent-like stem cells. This is especially important since fats may be voluntarily donated or removed and hence can be an easy source for pluripotent stem cells. Identification of the target to which CDy9 binds, may also be necessary to be determined in the future. This is because once the target protein is found, then its signaling pathways, its mechanisms and functions can be deduced. This will hence give a clearer picture on the compound staining and interaction with the stem cells and a better understanding of what is resulting in pluripotent stem cells to bind specifically to CDy9 as compared to the other cells. This may eventually aid in drug discovery function, especially if the target protein plays a role in any metabolic pathway or signaling pathway of a disease (34, 35). Knowing the target of CDy9 may also help in improving the ability of the compound so that a better version of CDy9 can be synthesized and perhaps a smaller compound concentration or incubation time is enough to detect pluripotent stem cells specifically. Furthermore, this can also be the starting point for the generation of other types of stem cell specific probes. However, current methods of target identifications using techniques such as affinity matrix have proven to be tedious and have met with limited success. This is because the above mentioned method may require the modification of compounds in order to be firmly attached to a solid-phase resin but this modification may affect the 2 biological properties of the compounds and give rise to false target identification (36). Furthermore, low abundance of target protein expression may also inhibit effective detection of the target protein. Therefore, to circumvent these issues, the DOFL compounds allow easier target identification without any modifications of the original compounds due to their intrinsic fluorescence nature. Therefore proteomics, SDS PAGE or HPLC analysis of mESC stained with CDy9 can be employed to determine the possible protein of interest that binds to the compound specifically (Figure 12). Mass spectrometry analysis may then be performed from these samples to determine the identity of the protein of interest. Western blots can also be performed to confirm the presence of the target protein that binds to the compound specifically. One success story of target identification for a DOFL compound is the fatty acid binding protein 7 for neural stem cell specific compound, CDr3 (36). As such finding the target binding partner of CDy9 may provide useful insights on the high specificity of CDy9 to mESC cells compared to the other already found mESC probes. Upon identifying the molecular binding partner of CDy9, the compound may also be translated to be a human pluripotent stem cells probe. The target protein or molecule found in the mESC can be compared with hESC using PCR or western blots. If the amount of target molecule/protein present in both stem cells is comparable, CDy9 can also be tested on hESC and be developed as a hESC specific probe. Human fats from liposuction may also be used to isolate pluripotent hESCs from human fats. This may then allow a readily available source of pluripotent human stem cells which can be utilized for the treatment of many diseases. 3 Figure 12: A general workflow of target identification for CDy9. The compounds are incubated with CDy9 and then prepared for SDS-PAGE analysis followed by target identification by mass spectrometry. This fluorescent optical probe may also be translated in the future to PET/SPECT animal imaging probes without any chemical modifications for subsequent clinical applications (33). This is because the DOFL fluorescent compounds have the ability to be altered into PET/SPECT probes via the introduction of radioisotopes into the compound structure. This will therefore allow CDy9 to be used for in vivo testing with the aim for use in animal clinical testing and to contribute as an invaluable tool for stem cell detection, disease diagnosis and treatment. 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PNAS. 10214–10217 vol 109. no. 26 7 APPENDICES Cell Panel results for other hit compounds mESC MEF Beta mMSC Acinar Alpha BDL C2 Hoechst 33342 NS5 NS5-D BDL C2 Hoechst 33342 Pri Neurons Mixed glial 3T3 –L1 3T3 BDL C2 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDL C2 Hoechst 33342 8 mESC MEF Alpha Acinar BDL H3 Hoechst 33342 Beta mMSC NS5 NS5-D 3T3 3T3 –L1 BDL H3 Hoechst 33342 Pri Neurons Mixed glial BDL H3 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDL H3 Hoechst 33342 9 mESC MEF Beta mMSC Alpha Acinar NS5 NS5-D BDL E5 Hoechst 33342 BDL E5 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDL E5 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDL E5 Hoechst 33342 10 mESC MEF Alpha Acinar BDD1 A2 Hoechst 33342 Beta mMSC NS5 Mixed glial 3T3 NS5-D BDD1 A2 Hoechst 33342 Pri Neurons 3T3 –L1 BDD1 A2 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDD1 A2 Hoechst 33342 11 mESC MEF Alpha Acinar BDD1 A6 Hoechst 33342 Beta mMSC NS5 Mixed glial 3T3 NS5-D BDD1 A6 Hoechst 33342 Pri Neurons 3T3 –L1 BDD1 A6 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDD1 A6 Hoechst 33342 12 mESC MEF Alpha Acinar NS5 NS5-D BDD1 E6 Hoechst 33342 Beta mMSC BDD1 E6 Hoechst 33342 Pri Neurons 3T3 Mixed glial 3T3 –L1 BDD1 E6 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDD1 E6 Hoechst 33342 13 mESC MEF Alpha Acinar mMSC NS5 NS5-D Mixed glial 3T3 3T3 –L1 BDNCA1 H4 Hoechst 33342 Beta BDNCA1 H4 Hoechst 33342 Pri Neurons BDNCA1 H4 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDNCA1 H4 Hoechst 33342 14 mESC MEF Alpha Beta mMSC NS5 Acinar BDNCA1 H5 Hoechst 33342 NS5-D BDNCA1 H5 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 H5 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDNCA1 H5 Hoechst 33342 15 mESC MEF Beta mMSC Alpha Acinar NS5 NS5-D BDNCA1 F3 Hoechst 33342 BDNCA1 F3 Hoechst 33342 Pri Neurons 3T3 Mixed glial 3T3 –L1 BDNCA1 F3 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDNCA1 F3 Hoechst 33342 16 mESC MEF Alpha Beta mMSC NS5 Acinar BDNCA1 D4 Hoechst 33342 NS5-D BDNCA1 D4 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 D4 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDNCA1 D4 Hoechst 33342 17 mESC MEF Alpha Beta mMSC NS5 Acinar BDNCA1 B5 Hoechst 33342 NS5-D BDNCA1 B5 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 B5 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDNCA1 B5 Hoechst 33342 18 mESC MEF Alpha Acinar BDNCA1 H7 Hoechst 33342 mMSC NS5 NS5-D Pri Neurons Mixed glial 3T3 3T3 –L1 C2C12 Splenocytes Beta BDNCA1 H7 Hoechst 33342 BDNCA1 H7 Hoechst 33342 B cell T cell EBs BDNCA1 H7 Hoechst 33342 19 mESC MEF Beta mMSC Alpha Acinar NS5 NS5-D BDNCA1 A10 Hoechst 33342 BDNCA1 A10 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 A10 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDNCA1 A10 Hoechst 33342 20 mESC MEF Beta mMSC Alpha Acinar BDMAC1 B9 Hoechst 33342 NS5 NS5-D BDMAC1 B9 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDMAC1 B9 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs BDMAC1 B9 Hoechst 33342 21 mESC MEF Alpha Acinar Beta mMSC NS5 NS5-D CLU A3 Hoechst 33342 CLU A3 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 CLU A3 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs CLU A3 Hoechst 33342 22 mESC MEF Alpha Acinar NS5 NS5-D CLU G3 Hoechst 33342 Beta mMSC CLU G3 Hoechst 33342 Pri Neurons Mixed glial 3T3 3T3 –L1 CLU G3 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs CLU G3 Hoechst 33342 23 mESC MEF Alpha Acinar mMSC NS5 NS5-D CLU B6 Hoechst 33342 Beta CLU B6 Hoechst 33342 Pri Neurons Mixed glial 3T3 –L1 3T3 CLU B6 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs CLU B6 Hoechst 33342 24 mESC MEF Beta mMSC Alpha Acinar CLU D6 Hoechst 33342 NS5 NS5-D 3T3 3T3 –L1 CLU D6 Hoechst 33342 Pri Neurons Mixed glial CLU D6 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs CLU D6 Hoechst 33342 25 mESC MEF Alpha Beta mMSC NS5 Acinar CLU H7 Hoechst 33342 NS5-D CLU H7 Hoechst 33342 Pri Neurons Mixed glial 3T3 –L1 3T3 CLU H7 Hoechst 33342 C2C12 Splenocytes B cell T cell EBs CLU H7 Hoechst 33342 26 [...]... Acinar NS5 NS5-D CLU G3 Hoechst 33 342 Beta mMSC CLU G3 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 CLU G3 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU G3 Hoechst 33 342 23 mESC MEF Alpha Acinar mMSC NS5 NS5-D CLU B6 Hoechst 33 342 Beta CLU B6 Hoechst 33 342 Pri Neurons Mixed glial 3T3 –L1 3T3 CLU B6 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU B6 Hoechst 33 342 24 mESC MEF Beta mMSC... Acinar BDMAC1 B9 Hoechst 33 342 NS5 NS5-D BDMAC1 B9 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDMAC1 B9 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDMAC1 B9 Hoechst 33 342 21 mESC MEF Alpha Acinar Beta mMSC NS5 NS5-D CLU A3 Hoechst 33 342 CLU A3 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 CLU A3 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU A3 Hoechst 33 342 22 mESC MEF Alpha... BDNCA1 H5 Hoechst 33 342 NS5-D BDNCA1 H5 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 H5 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDNCA1 H5 Hoechst 33 342 15 mESC MEF Beta mMSC Alpha Acinar NS5 NS5-D BDNCA1 F3 Hoechst 33 342 BDNCA1 F3 Hoechst 33 342 Pri Neurons 3T3 Mixed glial 3T3 –L1 BDNCA1 F3 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDNCA1 F3 Hoechst 33 342 16 mESC MEF Alpha... NS5-D BDD1 E6 Hoechst 33 342 Beta mMSC BDD1 E6 Hoechst 33 342 Pri Neurons 3T3 Mixed glial 3T3 –L1 BDD1 E6 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDD1 E6 Hoechst 33 342 13 mESC MEF Alpha Acinar mMSC NS5 NS5-D Mixed glial 3T3 3T3 –L1 BDNCA1 H4 Hoechst 33 342 Beta BDNCA1 H4 Hoechst 33 342 Pri Neurons BDNCA1 H4 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDNCA1 H4 Hoechst 33 342 14 mESC MEF Alpha... H7 Hoechst 33 342 mMSC NS5 NS5-D Pri Neurons Mixed glial 3T3 3T3 –L1 C2C12 Splenocytes Beta BDNCA1 H7 Hoechst 33 342 BDNCA1 H7 Hoechst 33 342 B cell T cell EBs BDNCA1 H7 Hoechst 33 342 19 mESC MEF Beta mMSC Alpha Acinar NS5 NS5-D BDNCA1 A10 Hoechst 33 342 BDNCA1 A10 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 A10 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDNCA1 A10 Hoechst 33 342 20 mESC... Acinar BDD1 A2 Hoechst 33 342 Beta mMSC NS5 Mixed glial 3T3 NS5-D BDD1 A2 Hoechst 33 342 Pri Neurons 3T3 –L1 BDD1 A2 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDD1 A2 Hoechst 33 342 11 mESC MEF Alpha Acinar BDD1 A6 Hoechst 33 342 Beta mMSC NS5 Mixed glial 3T3 NS5-D BDD1 A6 Hoechst 33 342 Pri Neurons 3T3 –L1 BDD1 A6 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDD1 A6 Hoechst 33 342 12 mESC MEF Alpha... BDNCA1 D4 Hoechst 33 342 NS5-D BDNCA1 D4 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 D4 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDNCA1 D4 Hoechst 33 342 17 mESC MEF Alpha Beta mMSC NS5 Acinar BDNCA1 B5 Hoechst 33 342 NS5-D BDNCA1 B5 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 BDNCA1 B5 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDNCA1 B5 Hoechst 33 342 18 mESC MEF Alpha... Alpha Acinar CLU D6 Hoechst 33 342 NS5 NS5-D 3T3 3T3 –L1 CLU D6 Hoechst 33 342 Pri Neurons Mixed glial CLU D6 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU D6 Hoechst 33 342 25 mESC MEF Alpha Beta mMSC NS5 Acinar CLU H7 Hoechst 33 342 NS5-D CLU H7 Hoechst 33 342 Pri Neurons Mixed glial 3T3 –L1 3T3 CLU H7 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU H7 Hoechst 33 342 26 ... NS5 NS5-D CLU G3 Hoechst 33 342 Beta mMSC CLU G3 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 CLU G3 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU G3 Hoechst 33 342 23 mESC MEF Alpha... Beta mMSC NS5 NS5-D CLU A3 Hoechst 33 342 CLU A3 Hoechst 33 342 Pri Neurons Mixed glial 3T3 3T3 –L1 CLU A3 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs CLU A3 Hoechst 33 342 22 mESC MEF Alpha... 33 342 NS5 NS5-D BDL C2 Hoechst 33 342 Pri Neurons Mixed glial 3T3 –L1 3T3 BDL C2 Hoechst 33 342 C2C12 Splenocytes B cell T cell EBs BDL C2 Hoechst 33 342 mESC MEF Alpha Acinar BDL H3 Hoechst 33 342