HIGHLY SPECIFIC FLUORESCENT PROBE FOR MOUSE PLURIPOTENT STEM CELLS 1

HIGHLY SPECIFIC FLUORESCENT PROBE FOR MOUSE PLURIPOTENT STEM CELLS YOGESWARI CHANDRAN (Bachelors of Science, University of Melbourne) THE THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 ACKNOWLEDGEMENT It has been my great privilege and honor to thank the following people for helping me and guiding me these past two years. First and foremost, I would like to express my heartfelt gratitude to my project supervisor Professor Chang Young-tae for not only giving me an opportunity to carry out my research in his lab but also for the continual guidance, support and sharing of his immense knowledge which allowed me to better understand the project. Next I would like to express my special thanks to my internal project mentor Dr Kang Nam-Young for guiding me patiently throughout the course of the project and helping me with the numerous new techniques and the understanding of the various instruments that were required for the completion of this project. I would also like to thank Dr Park Sun Jing and Dr Yoo Jung Sun for their invaluable help despite their very busy schedule in understanding the microscopes and the Mat Lab software respectively. A very special thanks also goes to my lab mates and friends, Cheryl Leong and Dr Marc Vendrell for the laughter shared together and the short coffee breaks that we had to help in times of distress or just to chill out. Lab work and cell culturing was much more fun with them around. Not to forget Dr Marc as well for the whole bag full of compounds that he specially synthesized for my project. I would also like to thank my family and friends for their support and encouragement at all times, without whom I may not have come this far. Last but not least although many people may have been left unacknowledged in this short humble note nevertheless they are not left unappreciated. 1 TABLE OF CONTENTS Acknowledgment 1 Table of Contents 2 Summary 4 List of Tables and Figures 5 List of Abbreviations 8 CHAPTER ONE 1. Introduction 10 1.1 Summary 10 1.2 Introduction to stem cells 10 1.3 The use of fluorescent probes for in vivo tagging and imaging 13 1.4 Introduction to Diversity Oriented Fluorescent Library Approach (DOFLA) 1.5 Project aim 14 15 CHAPTER TWO 2. Materials and Methods 17 2.1 Materials 17 2.2 Methods 17 2.2.1 High Throughput screening 17 2.2.2 Flow cytometry analysis 18 2.2.3 Cell Panel Screening 19 2 2.2.4 Cytotoxicity assay of CDy9 21 2.2.5 Stability of binding 21 2.2.6 Validation of CDy9 from a mixture of MEF and mESC 21 2.2.7 CDy9 staining of miPSC and miPSC differentiation to ectoderm, endoderm and mesoderm and validation with CDy9 2.2.8 Single Cell PCR 22 23 2.2.9 Identification of potential pluripotent stem cells from mouse fats using CDy9 25 CHAPTER THREE 3. Results and Discussion 27 3.1 Hit selection and identification using mESC 27 3.2 Cell panel test to detect the most specific compound for mESC 34 3.3 Hit compound CDy9 40 3.4 CDy9 stability and survival test 41 3.5 Number of colonies observed from a mixture of mESC and MEF stained with CDy9 43 3.6 miPSC differentiation to endoderm, ectoderm and mesoderm and validation using CDy9 3.7 Single cell PCR of CDy9 treated mESC 45 47 3.8 CDy9 application in mouse abdominal fats to detect pluripotent- like stem cells 52 CHAPTER FOUR 4. Conclusion 55 Bibliography 59 Appendices 62 3 SUMMARY Stem cells are a unique population of cells that can be manipulated to be differentiated into almost all cell types of the body and thus can be used in treating many diseases. However current methods to detect stem cells have been met with limited success as these techniques usually require genetic alteration to the cells, are not specific enough or kill the cells, making them unsuitable for future applications. Therefore in this thesis, an initial high throughput screen was carried out to detect 23 potential hits which stain mESC (a type of pluripotent stem cells) specifically compared to MEF, using the compounds from DOFLA. The specificity of these compounds was then confirmed by a cell panel test to detect the most selective hit compound for mESC. It was found that the compound designation yellow 9 (CDy9) was the most selective compound. The performance of CDy9 was proved via FACS sorting and colony formation after reculturing. It was also found that CDy9 was able to stain miPSC selectively compared to differentiated miPSC. Hence in this thesis, I report the first highly selective compound for mouse pluripotent stem cells, CDy9. The potential of CDy9 to detect single stem cells was also demonstrated via single cell PCR experiment. This may then allow the purification of single stem cells from a mixture of differentiated cells. Besides this, CDy9 was also applied to mouse abdominal fats and was shown to isolate out potential pluripotent stem cells. In conclusion, CDy9 can selectively stain mouse pluripotent stem cells and therefore can be used as a useful tool for stem cell research especially since it can be applied directly to stem cells without the need of any manipulation or genetic alteration to the cells. 4 LIST OF TABLES AND FIGURES Table 1: The table summaries all the cell types that was included in the cell panel test. 6 cell lines were selected for mesoderm, 4 cell types each were selected for ectoderm and endoderm. 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. Figure 1: A brief workflow of the hit selection and subsequent hit validation and application. Figure 2: The cell panel screen. Some representative of the cells included in the cell panel screen. Cell types were obtained from ectoderm, endoderm and mesoderm based. 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 2uM 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 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. 5 Figure 5: Cell panel screen data for the 4 hit compounds, CDy9, CDy1, CDg4 and CDb8. CDy9 appeared to be the most selective compound to mESC. 17 different cells types from ectoderm, mesoderm and endoderm including mESC and MEF were chosen to be screened in this test. Hoechst 33342 was used as nuclei staining for CDy1, CDy9 and CDg4. Transmitted light was used to show the presence of cells for CDb8. Scale Bar: 200um. 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 do not have much toxic effect on the cells. Figure 7: The stability of compound was checked by washing with different reagents after compound addition. mESC media, PBS, 100% methanol and 4% 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 4% PFA wash and slightly fainter in the case of 100% methanol wash. Scale bar 100um. 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 bright field 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 6 times more colonies than the dim. The average of the three sets of experiments were combined and plotted in a graph. Scale bar: 100um. 6 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 up regulation 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. 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. 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 2 weeks before addition of 1uM of CDy9 and image acquisition. Cells from dim population were seeded onto mitomycin treated feeder cells for 2 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. 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. 7 ABBREVIATIONS 1) ADSCs = Adipose-tissue derived stem cells 2) AP = Alkaline Phosphatase 3) BSA = Bovine Serum Albumin 4) CDb8 = Compound designation blue 8 5) CDg4 = Compound designation green 4 6) CDy9 = Compound designation yellow 9 7) CDy1 = Compound designation yellow 1 8) Cy5 = Cyanine 5 9) DAPI = 4',6-Diamidino-2-phenylindole (DAPI), 10) DOFLA = Diversity Oriented Fluorescent Library Approach 11) DOFLs = Diversity oriented florescent libraries 12) DMEM = Dulbecco’s Modified Eagle’s Medium 13) DMSO = Dimethyl Sulfoxide 14) EBs = Embryoid Bodies 15) ESc = Embryonic stem cells 16) FBS = Fetal Bovine Serum 17) FITC = Fluorescein isothiocyanate (FITC) 18) GFP = Green fluorescent protein 19) HBSS = Hank’s Buffered Saline Solution 20) hESC = human embryonic stem cells 21) HTS = High throughput screening 22) iPSC = Induced pluripotent stem cells 23) LIF = Leukemia’s Inhibitory Factor 8 24) MEF = Mouse Embryonic Fibroblast 25) mESC = Mouse embryonic stem cells 26) NEAA = Non-essential Amino Acid 27) PCA = Principle component analysis 28) PSG = Penicillin Streptomycin Glutamate 29) PFA = Para formaldehyde 30) TRITC = Tetramethylrhodamine-5- (and 6)-isothiocyanate 9 CHAPTER ONE INTRODUCTION Summary Stem cell research has gathered immense attention in the past decade due to the cell’s remarkable ability of self renewal and cell specific differentiation potential which may serve as an important basis for developmental biology research and regenerative medicine. Despite having numerous advancements in stem cell isolation and manipulation techniques, there still has not been any highly reliable or specific probe for the detection of live stem cells. This is especially important since stem cells application largely depend on the ability to isolate them from a population of mixed cells (1). This in turn has led to the need of tools and technologies which may aid in stem cells identification, isolation and characterization without any manipulations to the cells (2). Therefore the aim of my project will be to detect a highly specific fluorescent probe for mouse pluripotent stem cells to enable live imaging of the cells with very minimal or no alteration to the cell’s function or capabilities. Introduction to stem cells Stem cells are a unique population of live cells that are found in almost all multi cellular organisms. They are special due to their ability to give rise to many types of cells in the body during early development and growth. They are also referred to as immortal cells due to their ability to divide unlimited times through mitosis. During cell division, each newly formed cell can either remain being a stem cell or it can differentiate to form a cell of specialized function (1 - 3). Typically, stem cells can be broadly classified into 2 main groups, the embryonic stem cells and the somatic or adult stem cells. The latter, adult stem cells are found in various tissues in the body. These cells are undifferentiated cells residing in between a population of differentiated and specialized cell types. They are generally multi potent in nature and 10 they function primarily by replenishing and replacing worn out and damaged tissues in the body. Some examples of adult stem cells are hematopoietic stem cells which are formed in the bone marrow. These cells play a role by differentiating and replacing the blood cells in the body. Other types of adult stem cells include the mescenchymal stem cells and the gut epithelial stem cell that differentiate to line the intestinal lumen (4 -5). Embryonic stem cells (ESc) on the other end are isolated from very young embryos (3.5 days old) (3) and they are found in the inner cell mass of the blastocyst (an embryo of around 100 cells) of a fertilized egg (3). These cells are pluripotent in nature as they are able to proliferate and remain undifferentiated in vitro and can be manipulated into all the cells types comprising the three germ layers when given the correct cues (6). As such these stem cells assure a bottomless reserve of specialized cell types, not only for basic research such as understanding cell differentiation but also for transplantation therapies for a whole range of diseases such as leukemia’s and Parkinson’s disease. But the isolation of ES from embryos has raised many ethical concerns over the past few decades and despite its numerous advantages this has limited its use especially in the case of human embryonic stem cells (hESC). Until recently, another type of stem cells known as induced pluripotent stem cells (iPSC) have been made available to overcome these ethical concerns (7). These cells are made by inducing non stem cells such as mouse fibroblast cells with four important factors that are needed to maintain pluripotency (Oct4, Sox2, c-Myc and Klf4). Similar to embryonic stem cells, these cells have shown great promise in differentiating into other cell types and can be used in treating a range of diseases such as diabetes, spinal cord injuries, Gaucher disease and Parkinson’s diseases (8). However, there are still some disadvantages associated with the use of iPSC such as the complexity and time taken for their generation. There is also the danger of tumor formation from the presence of residual iPS cells during transplantation and other therapies (9). Nevertheless these cells are still an amazing breakthrough in the field of stem cell research and are the target stem cells of choice for many researchers. Therefore the primary cell type of choice for my thesis will be mainly mESC followed by miPSC. 11 Despite these numerous promising applications of stem cells for the treatment of complex diseases, there are still no effective ways to detect them in vivo or ex vivo. This is mainly contributed to their heterogenetic nature and their unpredictable pattern of proliferation and differentiation in culture, ex vivo (2). Usually stem cells are isolated and characterized based on their morphology in culture such as their distinctive colony and sphere formation. Enzymatic reactions such as alkaline phosphatase (AP) activity can also be used to detect the presence of stem cells. This is possible since undifferentiated pluripotent stem cells express a high level of alkaline phosphatase (10 - 11). The AP levels in stem cells can be easily detected by currently available alkaline phosphatase assay detection or characteristics kits. These kits are sensitive and specific and are carried out by adding a mixture of fast red violet solution and naphthol AS-BI phosphate solution to the stem cells. They then allow the phenotypic measurement of pluripotent stem cell differentiation which can be observed with a microscope. Immunofluorescene detection using specific protein markers are also used for the detection of these cells. Stem cell specific markers such as SSEA-1, Nanog, Sox2 and Oct4 can be coupled with a fluorescent secondary antibody to visualize these stem cells (12). However the above mentioned techniques have some disadvantages as they are not only tedious due to the multiple washing steps that are required which may result in the detaching or lost of some cells which may be critical for the experiment. But the methods also require fixing of the cells with 4% Para formaldehyde (PFA) which eventually kills the cells. As such, the cells can no longer be used for subsequent tracking and monitoring purposes. One way to overcome the above mentioned problems is to label the stem cells specifically with probes for easier visualization and tracking. The probes can be easily incorporated to the cells and can allow tracking of live cells with no or very minimal damage to the cell behavior. However, it is essential that this probe not only allows live staining and visualization of stem cell specifically but it must also be robust and be expressed in a stable manner in the cells without degrading easily. The probe must also be sensitive so that a small amount of probe will be enough to detect the cells. It is also vital that the probes do not alter the cells functions or have any side effects on the cells. 12 The use of fluorescent probes for in vivo tagging and imaging Almost all organisms are comprised of diverse tissues which are made up of many different cell types. The interactions between cells and within cells are highly complex and controlled and therefore the deciphering and understanding of this complex biological system is the primary aim of biology (13). Special techniques and strategies have been employed till now to detect and monitor these cells as it is almost impossible to accurately distinguish them by just mere observations. In comparison with other techniques such as MRI and radioisotope labeling, the use of fluorescent probes coupled with a suitable imaging system to monitor and track biological targets such as cellular organelles, polymeric biomolecules or stem cells will allow better understanding of the functions and the characteristics of the complex biological systems (14). Fluorescent imaging has many advantages for such labeling as it is highly specific and sensitive and can be easily coupled with different instruments for safe detection (13). Over the past few years, much effort and time have been invested in this field for the development and improvement of numerous molecular imaging techniques (15-16). A very important advancement in this field was the discovery of the Green fluorescent protein (GFP) isolated from the Aequorea jellyfish which as been used as a marker for gene expression and to understand protein targeting, interactions and structures (17). Despite being able to serve as an outstanding fluorescent tag for cellular imaging, there are some disadvantages associated with its use. Firstly the protein is made up of 238 amino acids which may be too bulky to be incorporated as a tag to disrupt regular protein function and folding (17). Secondly, prior genetic modifications are required when tagging the desired proteins with GFP. This may alter the cellular function of the cells and thus may inhibit monitoring or tracking of these cells over a period of time. Therefore, although much effort have been taken to give rise to a whole range of molecular imaging techniques using many different modalities such as optical and nuclear imaging and magnetic imaging, there is still an limitation in the number of reporter molecules that are available to be used in these applications. 13 Introduction to Diversity Oriented Fluorescent Library Approach (DOFLA) Another alternative way to overcome these limitations is the use of small fluorescent molecules or bioprobes to label cells in vivo (18 -19). One way to achieve this is via the Diversity Oriented Fluorescence Library Approach (DOFLA) which was pioneered by our research group to discover and develop many different fluorescent probes. Traditionally, small fluorescent molecules are developed via the hypothesis-drive approach where information on the fluorphore, linker region and target recognition motif are required from the beginning itself. Thus this may, narrow and limit the scope for the generation of a vast number of small fluorescent molecules based on the existing target information. Therefore the alternative approach will be the use of DOFLA. DOFLA is a diversity-driven approach that incorporates forward chemical genetics. As such no prior information of the target is known initially. This technique uses combinatorial chemistry to synthesize many different fluorescent scaffolds with different moieties and functional groups. By doing this, whole arrays of structurally and spectrally diverse libraries of fluorescent molecules are created for unbiased screening (20). These diversity oriented fluorescent libraries (DOFLs) are then coupled with high throughput imaging techniques to maximize the chances of finding a hit compound (20). Till today, many sensors and probes have been discovered by the DOFLA compounds. Some examples of DOFLA success compounds are sensors and small molecules for polymers such as DNA and heparin or GTP and glutathione respectively (21). Three pluripotent stem cells markers specifically for mESC probes have been previously identified by DOFLA. They were named compound designation yellow 1 (CDy1), compound designation green 4 (CDg4) and compound designation blue 8 (CDb8) based on their fluorescent color (14, 22 – 23). CDy1 ( ex/ em = 535/570 nm), was the first mESC probe to be detected by our group via the DOFLA. It was found to be able to detect miPSC prior to the appearance of the GFP signal which confirms the presence of iPSC formation, therefore allowing early detection of miPSC (14). CDb8 ( ex/ em = 369/487nm) and CDg4 ( ex/ em = 430/560nm) were the next 2 mESC compounds to be discovered which are blue and green colored mESC probes respectively. However, these 14 2 probes despite being able to stain mESC did not show the potential to stain other pluripotent stem cells such as hESC and iPSC. Furthermore not many applications for these 2 compounds were proven yet (22). It was also noted that even though these 3 compounds preferentially stained mESC compared to the negative control, mouse embryonic fibroblast (MEF), the compounds were also noted to stain many other cell types from other lineages such as the ectoderm, endoderm and mesoderm (data shown later). As such it was important to identify a compound that does not only stain mESC preferentially stain but it must also be highly specific and not stain the other cell types from the different lineages. Project aim Therefore, the primary aim of my thesis will be the discovery of a highly specific fluorescent probe for mouse pluripotent stem cells especially the mESC and miPSC. This is aimed to be achieved via an initial high throughput screening (HTS) of the DOFLs compounds. Hence here, I report the first highly specific mouse pluripotent stem cell fluorescent probe named compound designation yellow 9 (CDy9). After an initial screen to detect potential hit compounds that stains mESC more selectively than MEF, the specificity of CDy9 to mESC was validated and confirmed via the cell panel screen which included many different cell types from the three germ lineages. The cell panel test was also necessary to prove that CDy9 was the most specific compared to the previously identified mESC probes CDy1, CDg4 and CDb8. A mixture of MEF and mESC were also treated with CDy9 and FACS sorting was carried out to validate CDy9’s ability to isolate mESC. mIPSC were also semi-differentiation to confirm CDy9’s ability to stain other pluripotent stem cells. In addition to this, the low toxicity of CDy9 within its working concentration was demonstrated. Next single cell PCR was carried out to determine the ability of CDy9 to detect single stem cells from a mixture of cells. An array of different genes that define pluripotency and differentiation were used in this experiment to illustrate the potential of CDy9 to stain and detect single mESC specifically. The binding stability of CDy9 was also proved via treating CDy9 stained 15 mESC to different reagents. CDy9 was also applied in real situations to isolate out potential pluripotent –like stem cells from mouse abdominal fats (refer to Figure 1). Plating of cells into 3 8 4 p lates and com p ou nd ad d ition 1 H igh th rou gh p u t screening • C om p ou nd stab ility and cy totox icty ch eck • C D y 9 stained M E F and m E S C co cu ltu re sorting u sing F A C S 2 D ata analy sis and h it selection and confirm ation u sing flow cy tom etry 3 B est h it selection u sing cell p anel test – C D y 9 • S ingle C ell PC R • D etection of p lu rip otent-lik e stem cells in m ou se fats u sing C D y9 • m IPS C d ifferentiation and stainng w ith C D y 9 Figure 1: A brief workflow of the hit selection and subsequent hit validation and application. 16 CHAPTER TWO MATERIALS AND METHODS Materials Initial mESC and MEF cell lines were provided by Sai Kiang Lim (Institute of Medical Biology, Singapore). The cells were passaged and stocks were made for subsequent uses. All mice used in the experiment were provided by Biological Resource Centre (BRC), A*STAR with approval from Institutional Animal Care and Use Committee (IACUC). Methods High throughput screening MEF was used as the negative control for the identification of probe for mESC / miPSC. This is because most stem cells require a layer of feeder cells to provide the structural support for their proliferation and undifferentiated growth. Therefore an ideal probe will be one which stains mouse pluripotent stem cells but not MEF cells. Specialized media were prepared for the two cell types. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose (Gibco) , 20 % Fetal Bovine Serum (FBS) (Gibco) , 1 % Non Essential Amino Acid (NEAA) (Gibco), 0.1 % beta-mercaptoethanol (Gibco) and 1 % Penicillin Streptomycin Glutamate (PSG) (Gibco) were added together to prepare the media for MEF cells. The same media was used for mESC with the addition of Leukemia Inhibitory factor (LIF) to prevent differentiation of the cells. 384 well plates (Grenier) were used for the high throughput screens. 0.1 % Gelatin in sterile water (Merck- ES-006-B) was used to coat the plates before seeding of mESC to enable adherence of the cells to the wells. The plates were incubated with 50 ul of 0.1 % Gelatin for 30 minutes before removal and seeding of the cells into the appropriate well. The cells were plated in the following order, MEF, mESC and a mixture of mESC and MEF cells. 17 Based on the size of the cells, 1X105 cells/mL mESC and 2.5X104 cells/mL MEF were seeded per well. The cell viability was checked via Trypan blue staining and counted using haemocytometer before seeding into the wells. The cells were allowed 1 day to settle before addition of the DOFL compounds the next day. Around 10 000 DOFL compounds were screened before hit identification. 1 uM of compounds previously dissolved in dimethyl sulfoxide (DMSO) were added to the wells with 1 hour incubation at 37 °C before image acquisition using a high content automated imaging microscope known as ImageXpress (Molecular Device). The Cyanine-5 (Cy5) - ( 646/666nm), 4',6-Diamidino-2-phenylindole Fluorescein isothiocyanate (FITC) - ( (and 6)-isothiocyanate (TRITC) - ( ex/ em= ex/ em= (DAPI) - ( ex/ em= ex/ em= 350/470nm), 490/525nm) and Tetramethylrhodamine-5- 557/576nm) filters were used. 30 minutes prior to image acquisition, 1ug/mL Hoechst 33342 was added per well for nuclei staining. The transmitted light was used to confirm the presence of cells for compounds that showed a signal in the DAPI channel. After image acquisition, the images were manually selected using the MetaXpress High Content Image Acquisition & Analysis Software. The selected pool of hit compounds was further narrowed via subsequent secondary and tertiary screening. Flow cytometry analysis Next the image-based selected hit compounds were subjected to intensity based selection via flow cytometry analysis. Flow cytometry scrutinizes individual cells as they are suspended in a sheath of PBS which passes through a laser light. It utilizes light emission, excitation and scattering properties of fluorescent molecules to yield multi-parameter information from individual cells specifically (24). 1X105cells/mL mESC and 2.5X104 cells/mL MEF cells were cultured in 6 well plates until they reach confluency. 1 uM of compounds were added into each well with 1hour incubation at 37 ºC. After incubation, the wells were washed with Phosphate Buffered Saline (PBS) for 3 times. The staining pattern was checked using the Nikon ECLIPSE Ti Microscope. The cells were then treated with Trypsin (Invitrogen) for 5 minutes before neutralizing with the appropriate culture media and centrifuging at 1500 rpm for 3 minutes in 5 ml tubes (Becton 18 Dickinson). PBS washing was carried out 3 more times with centrifugation. Finally 500 uL of PBS was added per tube. Unstained MEF and mESC were prepared to serve as controls. The BD LSR II Bioanalyzer (BD Bioscience) was used to analyze the samples. 10 000 cells were analyzed per tube and subsequent data processing was carried out using the Flowjo (Tree Star, Inc). Cell Panel Screening Almost all organisms consist of cells derived from the 3 germ layers, the ectoderm, the endoderm and the mesoderm (25). Since the hit compound is expected to stain only mESC and not other differentiated cells, the potential hit compounds selected were subjected to cell panel test. The cell panel test as its name suggest, contains a panel of different cells from all 3 lineages. Figure 2 summarizes some of the cell types that were used in the screen. A total of 17 cell types including mESC and MEF were selected for the screen. Four cell types, namely the alpha cells, beta cells, acinar cells from the pancreas and mouse mescenchymal stem cells were chosen as representatives for the endoderm lineage. Four cell types, NS5, NS5 derived astrocytes (NS5-D), primary neurons and mixed glial were chosen for the ectoderm lineage. A total of 6 cell types, 3T3, 3T3-L1, C2C12, splenocytes, B cell and T cells were chosen as representatives of mesoderm. mESCs were also converted to embryoid bodies (EBs) which are aggregates of pluripotent stem cells. These EBs were allowed to differentiate for 5 days so that they consist of both pluripotent cells and differentiated cells. These cells were seeded in 384 well plates and 1uM of compounds were added to the cells. 1 ug/mL Hoechst 33342 was added 15 minutes prior to image acquisition for nuclei staining. The images were taken using the ImageXpress machine and the images obtained were analyzed using the MetaXpress High Content Image Acquisition & Analysis Software. 19 mESC !" # NS5 Beta cell Alpha cell $ NS5-D Primary neuron Mixed-glial 3T3 $ C2C12 3T3L1 Figure 2: The cell panel screen. Some representative of the cells included in the cell panel screen. Cell types were obtained from ectoderm, endoderm and mesoderm based. 20 Cytotoxicity assay of CDy9 The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) – Promega was used to perform the cytotoxicity assay. This assay is a colorimetric technique for analyzing the total quantity of live cells in chemosensitivity or proliferation assays. 1X105 cells/ mL of mESC cells were seeded onto gelatin coated 96 well black clear bottom plates and allowed one day to settle. The next day, 100 uL of fresh medium were added into the wells. Empty wells without any cells but only media were also prepared to serve as a negative control. No compound, 100 nM, 300 nM, 500 nM and 1 uM concentration of CDy9 was tested. After 1 hour incubation, 20 uL of the assay solution was added per well and further incubated for another hour. The absorbance was then measured at 490 nm using a plate reader. Stability of binding In order to determine the stability of binding of CDy9 to mESC cells, the cells were washed with media, PBS, 100 % methonal and 4 % PFA respectively. mESC (1 X 105 per ml) were seeded into gelatin coated 6 well plates. Then 1uM of CDy9 was added into every well with 1 hour incubation at 37 ºC. After incubation, the images were observed and acquired using the Nikon ECLIPSE Ti Microscope using the TRITC filter. The cells were then washed with mESC media, PBS, 100 % methanol respectively and imaged again. As for 4 % PFA, the cells were incubated with 4 % PFA for 15 minutes at room temperature before imaging. Validation of CDy9 from a mixture of MEF and mESC A mixture of MEF (2.5X104 per ml) and mESC (1X105 per ml) were prepared in each well of a gelatin-coated 6 well plate. 1uM CDy9 was added with 1 hour incubation at 37 ºC. After 1 hour, the cells were washed 3 times with PBS and treated with Trypsin for 5 21 minutes at 37 ºC. The cells were neutralized with culture media before centrifugation at 1500 rpm for 3 minutes. The cells were washed 3 more times with PBS before adding a final volume of 500 uL of PBS in 5mL FACS tubes. Stained and unstained individually grown MEF and mESC were prepared as controls. The FACSAria IIu SORP Cell sorter was used for sorting purposes. The bright and the dim populations observed from CDy9 staining were gated and a total of 100 000 cells were collected from the dim and bright cells respectively. These cells were then seeded equally into 3 wells of gelatin coated 6 well plates each and grown for 5 days with media change everyday. The total number of colonies formed in each well was counted after 5 days. CDy9 staining of miPSC and miPSC differentiation to ectoderm, endoderm and mesoderm and validation with CDy9 As mentioned previously, miPSCs are similar to mESC and are pluripotent in nature. As such, CDy9 was tested on miPSC as well. For this experiment, miPSCs were prepared accordingly as mentioned in Yamaka’s paper (7). Oct4 tagged MEF cells were used for the iPSC production so that the presence of iPSC will be supported by the GFP signal. 1 uM of CDy9 was added to the cells with 1hour incubation at 37 ºC followed by image acquisition using the Nikon ECLIPSE Ti Microscope. Embryoid bodies were then prepared from these miPSCs by seeding them on low adherent plates with MEF media for 4 days. After which around 5-10 colonies were transferred into 6 well plates and the cells were allowed to grow until they attach and differentiate so that there will be a combination of stem cells and differentiated cells. Then 1uM of CDy9 was added to confirm that it stains only the stem cells and not the differentiated cells. Immunocytochemistry was carried out for the differentiated cells to identify mesoderm, ectoderm and endoderm differentiation. 22 Mesoderm: Primary antibody: Mouse mAb [0.N.5] to alpha smooth muscle Actin (AB18147 – Abcam) Secondary antibody: Cy5 goat anti-mouse IgG (H+L) (A10524 – Invitrogen) Ectoderm: Primary antibody: Anti-Nestin, clone rat-401 (MAB353 – Millipore) Secondary antibody: Cy5 goat anti-mouse IgG (H+L) (A10524 – Invitrogen) Endoderm: Primary antibody: Anti-hSOX17 (AF1924- R&D systems) Secondary antibody: Alexa Fluor 647 donkey anti-goat IgG (H+L) (A21447 – Invitrogen) Single cell PCR Single cell PCR has now allowed the analysis of an array of gene expression profiles for a number of cells at a relatively short period of time (26). This new technique is also useful in eliminating issues that arise due to small sample size and heterogeneity property of the sample (26). This is extremely important for stem cell analysis especially due to the limiting number of stem cells available. In this experiment, single cell PCR was carried out to determine the expression of genes in different population of cells sorted from a mixture of mESC and MEF stained with CDy9. As such, a mixture of MEF (5 X 104 per ml) and mESC (1 X 105 per ml) were prepared and 1uM CDy9 was added with 1 hour incubation at 37ºC. After incubation, the cells were prepared for FACS sorting. 96 well plates (Sorenson- 28440) with 10 uL of master mix per well were prepared for single cell sorting. 23 For 1 sample/ cell For 100 samples/ cells CellsDirect 2X Reaction Mix 5ul 500ul 0.2x Assay pool 2.5ul 250ul RT/ Taq enzyme 0.5ul 50ul TE buffer/ ultrapure water 2ul 200ul Total 10ul 1000ul - 0.2X assay pool (contains primers and ultrapure water) The bright and the dim populations were gated and 1 cell/ well were sorted into the 96 well plates. The plates were then stored at -80 ºC freezer for at least 1 hour and thawed to promote cell lysis. Next reverse transcription and cDNA amplification was carried out using the following settings. (a) (b) (c) (d) (e) 50deg, 20min (reverse transcribe RNA to cDNA) 95deg, 2 min (Inactivate RT enzyme and start Taq) 95deg, 15 sec 60deg, 4 min Repeat c and d for 17x. (preamplify cDNA by denaturing for 18 cycles (13 cycles for single spheres) at 95deg for 15 sec each, and annealing at 60deg for 4 mins). (f) 8deg, forever 40 uL of nuclease free water was added per well and the amplified cDNAs were stored at -80 ºC. On the day of real-time PCR, 10uL of 2X DA assay loading reagent (Fluidigm, PN 85000736) was added into each of 48 PCR tubes, containing 10 uL of the respective Taqman probe. The pre sample mix was then prepared containing, 182 uL of Taqman® Universal PCR Master Mix (Applied Biosystems, PN 4304437) and 18.2ul DA sample loading reagent (Fluidigm, PN 85000735). Lastly, 3.8 uL of pre-sample mix was added to 3.2 uL of respective diluted cDNA before priming the 48 sample*48 assay integrated fluidic circuit (IFC) with tuberculin. Next 5 uL of the real –time assay mix and 5 uL of the respective final sample mix are loaded into the chambers and the sample was loaded into the BioMark™ HD System. 24 Identification of potential pluripotent stem cells from mouse fats using CDy9 It is well known that only embryonic stem cells have the potential to differentiate and form all cell types of the body. Furthermore there has been evidence that specific proteins that are expressed in embryonic stem cells are also found to be expressed in adiposetissue derived stem cells (ADSCs) (27). This is particularly useful as adipose tissues or fats are readily available via procedures such as liposuction and therefore can be a good source of embryonic-like stem cell supply. In this experiment, adipose tissues were obtained from mouse instead of human since CDy9 have been selected specifically for mESC. Abdominal mouse fat pads or the epididymal fat pads were obtained from 2 male BL6 mice aged 3 months old. Male mouse are preferred compared to female due to the larger amount of fat pads available. These fat pads were washed 3 times with PBS before mincing. Dissociation solution consisting of 30mL of Hank’s Buffered Saline Solution (HBSS) (Invitrogen) , 2 mg / mL collagenase Type 1 (Invitrogen), 1% Bovine Serum Albumin (BSA) (Sigma) and 50mg / mL of D-glucose (Sigma) was prepared. 20mL of this solution was added to the minced tissues and placed in a 37 ºC water bath for 30 minutes with mixing every 10 minutes. After dissociation, the minced tissue was centrifuged for 10minutes at 1500rpm and the supernatant was discarded. The pellets was washed 3 times with PBS and passed through a 100 micron cell strainer. The cells obtained were divided equally in 2 FACs tubes and 1uM of CDy9 diluted in MEF media was added to one of the tubes with 1 hour incubation at a 37 ºC incubator. After incubation, the cells were washed 3 times with PBS and prepared for FACs. MEF and mESC with and without CDy9 were also prepared to serve as positive and negative controls. The MoFlo XDP Cell sorter was used to sort out 10 000 cells from the CDy9 bright and CDy9 dim populations respectively. These cells were then seeded separately onto 1 well of a 6 well plate containing mitomycin c treated feeder cells. Fresh mESC media was added each day and the cells were grown until colonies start forming. To make feeder plates, BL6 MEF were grown to confluency on 10 cm culture dishes. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose (Gibco) , 10% Fetal Bovine Serum (FBS) (Gibco) , 1 % Anti-Anti and 1 % Penicilin Streptomycin 25 Glutamate (PSG) (Gibco) was prepared as culture media for the growth of these cells. Then 300uL of mitomycin C (Sigma) was added to the cells with 2hours and 20 minutes of incubation. After incubation, the cells were washed 3 times with PBS. Then the cells were dissociated using Trypsin and 100 000 cells were added into each well of a 6 well plate. Immunocytochemistry was carried out on the newly formed colonies from mouse fat pads. The wells were treated with 4 % PFA for 15 minutes. The 0.1 % Triton X was added with 15 minutes incubation. 1 % BSA diluted in PBS was using as the blocking solution. Primary antibody used was Anti-Oct4 antibody (ab19857) and secondary antibody used was Cy5 goat anti-rabbit IgG (H+L). 26 [...]... be to detect a highly specific fluorescent probe for mouse pluripotent stem cells to enable live imaging of the cells with very minimal or no alteration to the cell’s function or capabilities Introduction to stem cells Stem cells are a unique population of live cells that are found in almost all multi cellular organisms They are special due to their ability to give rise to many types of cells in the... embryonic stem cells (hESC) Until recently, another type of stem cells known as induced pluripotent stem cells (iPSC) have been made available to overcome these ethical concerns (7) These cells are made by inducing non stem cells such as mouse fibroblast cells with four important factors that are needed to maintain pluripotency (Oct4, Sox2, c-Myc and Klf4) Similar to embryonic stem cells, these cells have... MEF was used as the negative control for the identification of probe for mESC / miPSC This is because most stem cells require a layer of feeder cells to provide the structural support for their proliferation and undifferentiated growth Therefore an ideal probe will be one which stains mouse pluripotent stem cells but not MEF cells Specialized media were prepared for the two cell types Dulbecco’s Modified... to coat the plates before seeding of mESC to enable adherence of the cells to the wells The plates were incubated with 50 ul of 0 .1 % Gelatin for 30 minutes before removal and seeding of the cells into the appropriate well The cells were plated in the following order, MEF, mESC and a mixture of mESC and MEF cells 17 Based on the size of the cells, 1X105 cells/ mL mESC and 2.5X104 cells/ mL MEF were seeded... also be highly specific and not stain the other cell types from the different lineages Project aim Therefore, the primary aim of my thesis will be the discovery of a highly specific fluorescent probe for mouse pluripotent stem cells especially the mESC and miPSC This is aimed to be achieved via an initial high throughput screening (HTS) of the DOFLs compounds Hence here, I report the first highly specific. .. highly reliable or specific probe for the detection of live stem cells This is especially important since stem cells application largely depend on the ability to isolate them from a population of mixed cells (1) This in turn has led to the need of tools and technologies which may aid in stem cells identification, isolation and characterization without any manipulations to the cells (2) Therefore the aim... to as immortal cells due to their ability to divide unlimited times through mitosis During cell division, each newly formed cell can either remain being a stem cell or it can differentiate to form a cell of specialized function (1 - 3) Typically, stem cells can be broadly classified into 2 main groups, the embryonic stem cells and the somatic or adult stem cells The latter, adult stem cells are found... the BioMark™ HD System 24 Identification of potential pluripotent stem cells from mouse fats using CDy9 It is well known that only embryonic stem cells have the potential to differentiate and form all cell types of the body Furthermore there has been evidence that specific proteins that are expressed in embryonic stem cells are also found to be expressed in adiposetissue derived stem cells (ADSCs) (27)... this probe not only allows live staining and visualization of stem cell specifically but it must also be robust and be expressed in a stable manner in the cells without degrading easily The probe must also be sensitive so that a small amount of probe will be enough to detect the cells It is also vital that the probes do not alter the cells functions or have any side effects on the cells 12 The use of fluorescent. .. of the complex biological systems (14 ) Fluorescent imaging has many advantages for such labeling as it is highly specific and sensitive and can be easily coupled with different instruments for safe detection (13 ) Over the past few years, much effort and time have been invested in this field for the development and improvement of numerous molecular imaging techniques (15 -16 ) A very important advancement ... ONE Introduction 10 1. 1 Summary 10 1. 2 Introduction to stem cells 10 1. 3 The use of fluorescent probes for in vivo tagging and imaging 13 1. 4 Introduction to Diversity Oriented Fluorescent Library... (DOFLA) 1. 5 Project aim 14 15 CHAPTER TWO Materials and Methods 17 2 .1 Materials 17 2.2 Methods 17 2.2 .1 High Throughput screening 17 2.2.2 Flow cytometry analysis 18 2.2.3 Cell Panel Screening 19 ... manipulations to the cells (2) Therefore the aim of my project will be to detect a highly specific fluorescent probe for mouse pluripotent stem cells to enable live imaging of the cells with very minimal