HIGHLY SPECIFIC FLUORESCENT PROBE FOR MOUSE PLURIPOTENT STEM CELLS 1

2.2.9 Identification of potential pluripotent stem cells from CHAPTER THREE 3.1 Hit selection and identification using mESC 27 3.2 Cell panel test to detect the most specific compound f

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

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

2.2.9 Identification of potential pluripotent stem cells from

CHAPTER THREE

3.1 Hit selection and identification using mESC 27 3.2 Cell panel test to detect the most specific compound for mESC 34

3.5 Number of colonies observed from a mixture of mESC

3.6 miPSC differentiation to endoderm, ectoderm and mesoderm

3.8 CDy9 application in mouse abdominal fats to

CHAPTER FOUR

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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

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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

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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

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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

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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

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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

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

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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

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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