CHAPTER THREE RESULTS and DISCUSSION Hit selection and identification using mESC Around 10 000 compounds were screened against mESC and MEF via HTS After intensive primary, secondary and tertiary screens, a total of 23 hits were selected which showed higher staining intensity to mESC compared to MEF (see Figure 3) These 23 hits were also subjected to quantitative based analysis by flow cytometry for double confirmation (see Figure 4) In this experiment, the mESC cells (blue) were more shifted to the right compared to the MEF cells (red) indicating that they are more stained by the compound compared to MEF The optimum staining condition was observed to be uM of compound with hour of incubation for all the compounds except for BDMAC1 B9 where a higher concentration of 2uM with hour incubation was required MEF mESC Co culture Co culture MEF mESC Co culture CDg4 CDy1 MEF CDy9 mESC Hoechst 33342 Hoechst 33342 Hoechst 33342 MEF mESC Co culture Transmitted Light CDb8 MEF mESC Co culture Hoechst 33342 Hoechst 33342 BDL C2 BDL H3 MEF mESC Co culture Hoechst 33342 Hoechst 33342 BDL E5 BDD1 A2 MEF mESC Co culture Hoechst 33342 Hoechst 33342 BDD2 A6 BDD2 E6 MEF mESC Co culture MEF mESC Co culture MEF mESC Co culture MEF mESC Co culture MEF Hoechst 33342 Hoechst 33342 CLU A3 CLU G3 mESC Co culture MEF mESC Co culture MEF Hoechst 33342 Hoechst 33342 CLU B6 CLU D6 MEF mESC Co culture MEF Hoechst 33342 Hoechst 33342 CLU H7 BDNCA1 H4 MEF mESC Co culture BDNCA1 H5 Co culture MEF mESC Co culture MEF mESC Co culture BDMAC1 B9 MEF BDNCA2 D4 mESC Co culture Hoechst 33342 Hoechst 33342 Hoechst 33342 mESC mESC Co culture Hoechst 33342 BDNCA2 F3 MEF mESC Co culture MEF Hoechst 33342 mESC Hoechst 33342 BDNCA2 H7 BDNCA2 A10 MEF mESC Co culture Hoechst 33342 BDNCA2 B5 Figure 3: 23 hits selected from HTS using the high content ImageXpress machine Hits were selected after primary, secondary and tertiary screens 1uM of respective compounds were added to the cells with 1hour incubation before image acquisition except for BDMAC1 B9 where uM of compound was used instead Hoechst 33342 was used as nuclei staining Transmitted light was used to confirm the presence of cells for CDb8 compound only Scale bar: 200um Co culture Compound Flow Cytometry Data Compound Flow Cytometry Data X-axis (Fluorescence) X-axis (Fluorescence) Y-axis (Side scattering –SSC) Y-axis (Side scattering –SSC) Figure 4: Flow cytometry data of the 23 hit compounds Blue indicates mESC cells and red indicates MEF cells X-axis refers to the compound fluorescence channel and Y-axis refers to the side scattering which is the indication of the size of the cells used From all the flow cytometry data, it is noticed that mESC are more shifted compared to MEF indicating that they are more brightly stained by the compounds as compared to MEF Cell panel test to detect the most specific compound for mESC The cell panel screen was carried out to identify the most specific compound that stains mESC compared to the 23 hit compounds chosen A total of 17 different types of cells including mESC and MEF were chosen for this screen (Refer to table 1) Cell types from the germ lineages were chosen for this study as mESC can be easily differentiated into the ectoderm, endoderm and mesoderm (28 – 30) Therefore it is very important that an ideal, specific probe for mESC should not stain these other cell types that can easily arise from mESC differentiation This way undifferentiated mESC can be easily identified and isolated from differentiated or semi differentiated mESCs The ectoderm is generally defined as the outermost layer of the germ layers This layer gives rise to cells that make up the nervous system as well as the epidermis and the epidermal tissues such as skin glands, hair and tissues As such cells types were chosen from this layer, namely the NS5, NS5-D which are the NS5 differentiated astrocytes, the primary neurons and the mixed glial which consist of a mixture of microglials, astrocytes and oligodendrocytes The endoderm is the innermost layer of the embryo This layer consists of cells from the respiratory tract and the glands that are associated with the bladder, urethra and the gastrointestinal tract A total of cell types were also chosen for this lineage, namely the pancreatic cells types comprising of the alpha, beta and acinar cell types and the mouse mescenchymal stem cells The third layer, mesoderm is the middle layer and it consists of the muscles, bones, blood and connective tissues Therefore the muscle cell type C2C12, connective fibroblast such as 3T3 and 3T3-L1 and cells from the spleen were chosen as representatives of the mesoderm layer The embryoid bodies (EBs) were also included in this screen as they were made from mESCs and contain a mixture of differentiated cells and undifferentiated stem cells As mentioned earlier, compounds namely CDy1, CDy8 and CDg4 have been previously reported as different colored mESC specific probes by our group (14, 22, and 23) But when subjected to the cell panel test, it was noticed that these compounds showed positive staining for at least one other cell type from the other lineages CDg4, despite showing negative staining for the cells from the mesoderm and the ectoderm lineages showed staining for almost all the other cell types from the endoderm lineage CDy1 showed a very strong staining pattern to the acinar cells and appeared faintly in several other cell types from the lineages CDb8 also stained beta and acinar cells as well as fibroblast such as 3T3 and 3T3 – L1 very strongly (see Figure 5) These cell panel results clearly show that despite being previously identified as mESC specific compounds and functioning well in identifying and isolating mESC from a mixture of mESC and MEF and identifying miPSC (in the case of CDy1), these compounds cannot be considered as the most specific compound for mESC But, it was found that one compound CDy9 ( ex/ em= 510 nm / 570 nm) seemed promising as it did not stain all the other cell types except for mESC CDy9 staining may not be as bright as the other yellow compound CDy1, but it noticeably stained mESC more brightly than all the other cell types after one hour of incubation, resulting in being the most specific compound for mESC (see Figure 5) Mesoderm C2C12 B cell T cell Splenocytes 3T3 3T3-L1 Ectoderm NS5 NS5-D Primary Neurons Mixed glial Endoderm Alpha Beta Acinar mMSC Others Embryoid Bodies Table 1: The table summaries all the cell types that was included in the cell panel test cell lines were selected for mesoderm, cell types each were selected for ectoderm and endoderm mESC MEF Acinar Alpha CDy9 Hoechst 33342 Beta mMSC NS5 NS5-D Mixed glial 3T3 3T3 –L1 CDy9 Hoechst 33342 Pri Neurons CDy9 Hoechst 33342 C2C12 CDy9 Hoechst 33342 Splenocytes B cell T cell EBs CDg4 Transmitted light was used to show the presence of cells for CDb8 Scale Bar: 200 um Hit compound CDy9 The hit compound CDy9 was synthesized by our group by solid phase synthesis approach (31) A BODIPY core scaffold with a free amine was first made This was followed by chloroacetylation to form the final compound that is seen below (see Figure 6a) The CellTiter 96® AQueous Non-Radioactive Cell Proliferation assay was also carried out to assess the cytotoxicity of CDy9 The viability of mESC cells was determined by using an increasing concentration of CDy9 treated mESC compared to non CDy9 treated mESC The absorbances measured were then used to plot a graph to determine the cytotoxicity The readings from the non CDy9 treated mESC were normalized to 100 % under the assumption that all cells in these wells have a survival rate of 100 % It was noticed that CDy9 did not show any toxic effect on the cells at the working concentration of 1uM as they show an absorbance reading similar to that from the non compound treated samples (see Figure 6b) This was indicated by the formation of formazan which arises due to the enzymatic reactions of the components in the assay, which directly correlates to the number of living cells in that well a b CDy9 - Day0 1hr and 2hr 120 Absorbance 490nm 100 80 hour 60 hour 40 20 No Compound 100nM 300nM 500nM 1uM Com pound concentration Figure 6: (a) Structure of hit compound CDy9 (b) The cytotoxicity test for CDy9 It was found that at the working concentration of 1uM, CDy9 not have much toxic effect on the cells CDy9 stability and survival tests One of the most important criteria needed for a probe is to be able to bind to its target cells in a stable manner and not be washed away easily by other reagents Therefore to further characterize CDy9’s stability and survival, the staining of the compound to mESC was checked by washing with different reagents after compound incubation PBS, mESC media, % PFA and 100 % methanol were used as reagents for this experiment PBS and media were chosen as of the reagents for this experiment as normal cell culturing techniques require the cells to be washed by either PBS or media frequently especially during trpsinization of the cells or during daily maintenance of the cells Therefore it was important to ensure that these two reagents not wash out the compounds easily Furthermore % PFA is required to fix the cells during immunocytochemistry and hence the CDy9 treated cells were incubated with % PFA and the staining pattern was observed again after 15 minutes of incubation 100 % methanol was necessary to ensure that CDy9 staining survives when subjected to organic solvent treatment Washing with these reagents helped in removing the background signal and it was noticed that after washing with PBS and % PFA, the mESCs appeared brighter compared to just incubating with CDy9 for hour In the case of 100 % methanol, the staining pattern appears slightly fainter than before washing But from the data, it can be assumed that CDy9 survives and exhibit a stable binding pattern to mESC (see Figure 7) Media Wash Bright field Before Wash After Wash Before Wash After Wash Before Wash After Wash Before Wash After Wash PBS Wash Bright field 100% Methanol Wash Bright field 4% PFA Bright field Figure 7: The stability of compound was checked by washing with different reagents after compound addition mESC media, PBS, 100 % methanol and % PFA were used as reagents for this experiment It was noticed that CDy9 survives washing with these reagents and the background signal was also eliminated in all cases The degree of CDy9 staining remains the same in the case of media wash, appears slightly brighter in the case of PBS and % PFA wash and slightly fainter in the case of 100 % methanol wash Scale bar 100 um Number of colonies observed from sorting a mixture of mESC and MEF stained with CDy9 Mixture of MEF and mESC were also stained with 1uM of CDy9 to test if there were any co culture effect that indirectly affects the staining pattern This experimental setup is important since stem cells require a layer of feeder cells (usually MEF) for growth Since CDy9’s capability to distinguish between mESC and MEF when they are cultured separately has already been proven previously, it is now necessary to mimic this same behavior of CDy9 for a mixture of samples Visually it was noticed that CDy9 stains mESC more brightly than MEF cells in mixed samples (see Figure 8a,b) but to confirm this observation quantitatively, these cells were subjected to FACS analysis After gating to exclude debris and dead cells, the most brightly shifted cells and the population appearing before this group of cells were sorted under the assumption that they were mESC and MEF respectively (see Figure 8c) After days of culturing on gelatin coated plates, it was noticed that at least times more colonies were observed in the bright wells as compared to the dim wells ((see Figure 8d,e) This data clearly indicate that CDy9 shows an affinity to stain mESC specifically despite being cultured together with MEF a b c d !" No of colonies counted Bright Dim Set 392 54 Set 495 79 Set 490 65 e e Co Culture Sorting with CDy9 600 No of colonies 500 400 300 200 100 Bright Dim Bright VS Dim Figure 8: The specificity of CDy9 staining towards mESC was validated by treating a mixture of mESC and MEF with CDy9 (a,b) Shows the brightfield and fluorescence images of CDy9 treated co culture before sample preparation for FACS analysis (c) The side scattering and fluorescence profile of CDy9 treated co culture sample The brightest cells were gated under the assumption that they were mESC and the population before that was gated under the assumption that they were MEF for sorting purposes (d, e) The sorted cells were seeded onto gelatin coated plates and the numbers of colonies formed on each well were counted Dim represents MEF cells and the bright represents mESCs It was noted that in all three sets, the bright wells formed at least times more colonies than the dim The average of the three sets of experiments were combined and plotted in a graph Scale bar: 100 um mIPSC differentiation to endoderm, ectoderm and mesoderm and validation using CDy9 It was tested that CDy9 also showed specific staining for miPSC Thus, miPSC fused with a GFP tag was cultured and allowed to differentiate so that there will be a mixture of stem cells and differentiated cells CDy9 was added to this semi-differentiated cells and it was observed that CDy9 only stained the miPSC specifically This staining was confirmed by the GFP signal which corresponds with the presence of stem cells Immunocytochemistry was also carried out to confirm differentiation to ectoderm, endoderm and mesoderm It was also noticed that there was always a very strong antibody signal surrounding the colonies indicating that the circumference of the colony is more prone to differentiation than the inner core of the colony Both 10X and 40X images were taken and the nuclei staining (dapi channel), antibody signal (Cy5 channel), CDy9 staining (TRITC channel) and GFP signal (FITC) were merged together as well a Mesoderm Ectoderm Endoderm Bright field 10X CDy9 GFP Antibody Merge b Bright field 40X CDy9 GFP Antibody Merge Mesoderm Ectoderm Endoderm Figure 10: CDy9 and immunocytochemistry staining of a mixture of differentiated cells and GFP-miPSCs (a) and (b) are 10X and 40 X images respectively CDy9 signal is only noticed for the stem cells which are confirmed by the GFP signal The differentiated cells are confirmed by the lineage specific antibody staining The nuclei staining (dapi), antibody (Cy5), CDy9 (TRITC) and miPSC-GFP (FITC) staining patterns were merged as well Scale bar: a) 100um and b) 20um Single cell PCR of CDy9 treated mESC It has been proven via the earlier experiments that CDy9 stains mESC more specifically than the other compounds and thus this compound was applied to an emerging new technique called single cell PCR to validate CDy9’s ability to distinguish between mESC and other cells by analyzing the gene expression profiles of individual cells Single cell PCR was chosen for the validation of CDy9 as it allows high throughput analysis of a series of individual cells at a relatively short period of time Even though mESC cell lines are used for this experiment, this technique is especially important in real live situations when analyzing stem cells that are usually very limited in number (32) For this experiment, single cell PCR was performed on individual CDy9 treated cells that were previously isolated from a mixture of cells (mESC and MEF) via FACS The gene expression profile of 48 individual cells from the brightly stained mixtures of cells and 48 individual dimly stained mixtures of cells was determined by an array of primers from the TaqMan® Gene Expression Assay A total of 33 primers were selected to determine the pluripotent and differentiated states of the cells (refer to Table 2) 21 primers were chosen that define the pluripotent states of the cells These genes are upregulated in ES cells and are involved in stem cell initiation, maturation and stabilization 12 primers were selected that were needed to define the differentiated states of the cells These genes are upregulated in differentiated cells and are EMT, mescenchymal and differentiation markers GAPDH primers were used as the housekeeping genes After single cell PCR, the Ct values obtained were normalized using the GAPDH Ct values Then the inverse Ct values were calculated and used to plot the heatmap (see Figure 9) The heatmap was plotted by using the bioinformatics toolbox of the Matlab It can be observed from the heatmap that there is an upregulation of pluripotent genes for the cells from the CDy9 brightly stained population and an up regulation of differentiated genes for the CDy9 dimly stained population of cells Grey color indicates no or very low gene expressions, green color indicates median gene expression, black color indicates high gene expression and red color indicates very high gene expression (see Figure 9a) The blue box indicates the 21 genes that are known to be upregulated in stem cells and the red box indicates the 12 genes that are upregulated in differentiated cells It can also be seen that some genes such as Oct4, Esg1, Ccnd2 and FN1 show expression in both cells types but it can be noted that a much higher gene expression (red color) is observed only for the cells that express them This non specific amplification of genes could be due to the presence of genes in both cell types but at different levels or because some of the single mESC cells were already priming towards differentiation and hence they were starting to show the expression of differentiation genes as well Principle component analysis (PCA) was also carried out for these cells The R software followed by the minor 3D program was used to create this plot Similar result as the heatmap was also reflected in the PCA From the PCA, a clear discrimination between the brightly stained and the dimly stained population of cells can be noted (see Figure 9b) These separate clusters of cells represent mESC and MEF respectively The green triangles each represent a single cell from the bright population and the red triangles each represent a cell from the dim population There are a few cells that are present in the other cluster and this observation may be due to the reasons described above Therefore both the heatmap and the PCA analysis reveal that CDy9 is able to selectively detect mESC from a mixture of cells It is also important to note that CDy9 does not only stain a colony of stem cells and allow easy identification and isolation but it also allows single stem cells to be detected from a mixture of cells Hence from this observation, it can be noted that CDy9 can be used to purify a single mESC cell from a mixture of differentiated cells and this single cell can be cultured to give rise to a whole colony Primers to define pluripotent states of cells Oct-04 Esg1 Myst4 Dnm3tl Nanog Lin28 Utf1 Cbx7 Alp1 Nodal Fgf4 E-cadherin Tcl1 Gdr3 Pecam1 Dax1 Zfp295 Sall4 Stella Crb3 Suv39h2 Primers to define differentiated states of cells Casp8 Cbx4 Twist2 Twist1 Snail Slug Tgfbr2 N-cadherin Jak2 Camk2d Ccnd2 FN1 Table 2: The table summarizes all the primers that were used in the Single cell PCR experiment A total of 33 primers were selected in which 21 were required to define pluripotency and 12 primers were required to define differentiation a No or very low expression Median expression High expression Very high expression mESC MEF b Figure 9: (a) The heat map obtained from single cell PCR analysis of a co culture of CDy9 treated mESC and MEF The blue box indicates pluripotent genes and the red box indicates differentiation genes It can be noted that there is an upregualtion of pluripotent genes for mESC and an upregulation of differentiated genes for the MEF cells (b) The PCA plot shows that the populations of cells are clearly separated from one another The red color indicates the cells from the dim population and the green color indicates the cells from the bright population and hence this suggests that they are MEF and mESC respectively CDy9 application in mouse abdominal fats to detect pluripotent-like stem cells From all the previous experiments and observation, CDy9 can now be safely classified as a compound that selectively stains mouse pluripotent stem cells only As such it was applied in mouse abdominal fats to isolate out any potential pluripotent-like stem cells It was noticed that round stem cell-like colonies started forming in the wells that were containing CDy9-bright cells after several days of culture These colonies also showed positive staining for CDy9 (see Figure 11b) The surrounding feeder cells were negative for CDy9 Next immunocytochemistry was carried out for the colonies using pluripotentstem cells specific antibody, anti-oct4 (ab19857) and it was also noticed that these cells stained positive for the antibody (See Figure 10d) Ever though these observations suggest that the colonies formed may be potential pluripotent-like stem cells, more experiments such as the ability to re culture these colonies numerous times without them dying is also necessary to indicate their real pluripotent stem cell identity But, this has proved to be difficult, as the numbers of colonies formed were little and it was also crucial to provide them with the same environment so that their niche is preserved and they can exhibit continued survival Also the presence of stem cells in these mice differs from one mouse to another and as such there is no guaranteed measure that every mouse used for this experiment will give rise to the same result a Count Dim Bright CDy9 Control PE texas red b Transmitted Light Bright Population CDy9 CDy9 Transmitted Light Transmitted Light Dim Population CDy9 I Transmitted Light II CDy9 c )" *+ III #$%& '"( ) IV & & Figure 11: FACS analysis of mFats stained with 1uM of CDy9 (a) FACS analysis data 5% of the most bright and dim populations were gated respectively and 10 000 cells were collected from each population (b) Cells from bright population were seeded onto mitomycin treated feeder cells for weeks before addition of 1uM of CDy9 and image acquisition Cells from dim population were seeded onto mitomycin treated feeder cells for weeks before addition of 1uM of CDy9 and image acquisition (c) Immunocytochemistry was carried out for the colonies using anti-Oct4 primary antibody and cy5 goat anti-rabbit IgG (H+L) secondary antibody The CDy9 (pseudo color), antibody (red), nuclei staining (blue) and merge images (of CDy9 and antibody) are shown Scale bar: 100um ... culture Hoechst 333 42 Hoechst 333 42 BDL C2 BDL H3 MEF mESC Co culture Hoechst 333 42 Hoechst 333 42 BDL E5 BDD1 A2 MEF mESC Co culture Hoechst 333 42 Hoechst 333 42 BDD2 A6 BDD2 E6 MEF mESC Co culture... BDNCA2 D4 mESC Co culture Hoechst 333 42 Hoechst 333 42 Hoechst 333 42 mESC mESC Co culture Hoechst 333 42 BDNCA2 F3 MEF mESC Co culture MEF Hoechst 333 42 mESC Hoechst 333 42 BDNCA2 H7 BDNCA2 A10... pluripotent stem cells only As such it was applied in mouse abdominal fats to isolate out any potential pluripotent- like stem cells It was noticed that round stem cell-like colonies started forming