Characterizing changes in the transcriptional …...Michael Schwartz Master of Science Department of Cell and Systems Biology University of Toronto 2012 Abstract Mouse embryonic stem

Characterizing changes in the transcriptional networks underlying

pluripotency in mouse embryonic stem cells upon the induction of

differentiation

by

Michael Schwartz

A thesis submitted in conformity with the requirements for the degree

of Master of Science

Department of Cell and Systems Biology

University of Toronto

© Copyright by Mike Schwartz 2012

ii

Characterizing changes in the transcriptional networks underlying

pluripotency in mouse embryonic stem cells upon the induction of

differentiation

Michael Schwartz

Master of Science

Department of Cell and Systems Biology

University of Toronto

2012

Abstract

Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating into all

three germ layers present in the adult mouse. In this thesis, I have investigated the

transcriptional changes that mESCs undergo as they are induced to differentiate towards the

mesoderm lineage by 2i/LIF withdrawal and dimethyl sulfoxide (DMSO) treatment. 5 days

of differentiation causes significant drops in expression of Sox2 and Oct4 primary transcript,

while expression of Nanog and Kit significantly drops after only 1 day. It was determined

that DMSO has no effect on the short-term changes in Nanog and Kit expression induced by

2i/LIF withdrawal. An expanded look at pluripotency-associated genes shows significant up-

regulation of Oct4 and down-regulation of Klf4 and Stat3 following only 6 hours of 2i/LIF

withdrawal. This data indicates that while some aspects of the transcriptional networks

underlying pluripotency respond quickly to mesodermal differentiation cues, others remain

unchanged for up to 5 days.

iii

Acknowledgements

I would first and foremost like to thank my family for supporting me throughout the duration

of my graduate studies. Without them, I would be nowhere (literally). I also want to thank

my friends for sticking around while I spent so much time in the lab. It is through tough

experiences where one finds who their true friends are, and I would like to formally

acknowledge my appreciation for the real friendships I have and have made over the past two

years. I would also like to express gratitude to my supervisor, Dr. Jennifer Mitchell. This

research project could not have been completed without Dr. Mitchell’s guidance and

experience. I would also like to thank my committee members Dr. David Bazett-Jones and

Dr. Ian Rogers (and his technician Mira Li). Without their help, I would not have been able

to perform the lab techniques necessary for this thesis. Finally, I would like to thank all

members of the Mitchell Lab who have helped me with my experiments and who I’ve spent

so much time with over the course of my Masters degree.

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Table of Contents

Abstract ..................................................................................................................................... ii

Acknowledgements .................................................................................................................. iii

Table of Contents ..................................................................................................................... iv

List of Figures .......................................................................................................................... vi

List of Appendices ................................................................................................................. viii

Introduction ............................................................................................................................... 1

A background on pluripotency .............................................................................................. 1

Oct4, Sox2, Nanog and transcriptional control of the pluripotent state ................................ 1

Network dynamics of Oct4, Sox2 and Nanog ....................................................................... 3

Signaling pathways underlying pluripotency in vitro ........................................................... 4

Unique features of pluripotent mESCs.................................................................................. 7

Specification of mesoderm and hematopoiesis in the murine embryo and mESCs .............. 9

Transcription factories organize the nucleus in three-dimensional space ........................... 11

Experimental Proposal ........................................................................................................ 11

Methods................................................................................................................................... 13

Cell Culture ......................................................................................................................... 13

Differentiation Protocol....................................................................................................... 13

RNA Isolation and cDNA Synthesis ................................................................................... 13

Real-Time Quantitative PCR (RT-qPCR) ........................................................................... 14

RT-qPCR Data Analysis ..................................................................................................... 15

Immunofluorescence Staining and Imaging ........................................................................ 15

Results ..................................................................................................................................... 17

Introduction ......................................................................................................................... 17

Long-term differentiation induces changes to the mESC-specific gene expression profile 17

Long-term differentiation induces changes to mESC-specific cellular morphology .......... 25

The presence of DMSO has no significant effect on 2i/Lif withdrawal after one day of

differentiation ...................................................................................................................... 43

Fine-mapping of short-term differentiation uncovers significant changes in gene expression

as early as 6 hours after treatment ....................................................................................... 51

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Discussion ............................................................................................................................... 66

Summary of Results ............................................................................................................ 66

DMSO, 2i/LIF withdrawal and changes in gene expression after 5 days of differentiation 66

Comparison of cell morphology after 5 days of differentiation indicates that 2i/LIF

withdrawal and addition of DMSO enables BMP4-mediated non-neural differentiation .. 69

Immunofluorescence uncovers changes in Oct4, Sox2 and Nanog localization following

2i/LIF withdrawal and DMSO treatment ............................................................................ 70

The presence of DMSO has no significant effects on the changes of gene expression

induced by 1 day of 2i/LIF withdrawal ............................................................................... 71

Changes in the expression of Kit, Oct4, Zic3, Klf4 and Stat3 are detected as early as 6

hours after withdrawal of 2i/LIF ......................................................................................... 72

Changes in the expression of Nanog and Lefty2 are detected as early as 12 hours after

withdrawal of 2i/LIF, while Tdgf1 changes are detected after 24 hours ............................. 74

Future Direction .................................................................................................................. 76

Conclusions ......................................................................................................................... 76

References ............................................................................................................................... 78

Appendices .............................................................................................................................. 87

vi

List of Figures

Figure 1. Nanog expression significantly drops after 1 day of differentiation ............................. 19

Figure 2. Kit expression significantly drops after 1 day of differentiation ................................... 20

Figure 3. Oct4 primary transcript expression significantly drops after 5 days of differentiation . 21

Figure 4. Sox2 expression significantly drops after 5 days of differentiation .............................. 22

Figure 5. Brachyury expression significantly increases after 5 days of differentiation................ 23

Figure 6. Flk1 transcription significantly drops after 1 day of differentiation ............................. 24

Figure 7. Brightfield microscopy images of pluripotent and differentiated mESCs .................... 26

Figure 8. Differentiation causes a loss of OCT4 staining ............................................................. 28

Figure 9. OCT4 immunofluorescence in pluripotent mESCs ....................................................... 29

Figure 10. OCT4 immunofluorescence in mESCs subject to 2i/LIF withdrawal and DMSO

treatment for 1 day ........................................................................................................................ 30


treatment for 2 days ...................................................................................................................... 31

Figure 12. Oct4 immunofluorescence in mESCs subject to 2i/LIF withdrawal and DMSO


Figure 13. Differentiation causes a loss of SOX2 staining ........................................................... 33

Figure 14. SOX2 immunofluorescence in pluripotent mESCs ..................................................... 34

Figure 15. SOX2 immunofluorescence in mESCs subject to 2i/LIF withdrawal and DMSO




Figure 18. Differentiation causes a loss of NANOG staining ...................................................... 38

Figure 19. NANOG immunofluorescence in pluripotent mESCs ................................................ 39

Figure 20. NANOG immunofluorescence in mESCs subject to 2i/LIF withdrawal and DMSO






Figure 23. Oct4 expression is not significantly affected by 1 day of differentiation .................... 45

Figure 24. Sox2 expression is not significantly affected by 1 day of differentiation .................... 46

vii

Figure 25. Nanog expression significantly drops after 1 day of differentiation, but the

magnitude of drop is not affected by the presence of DMSO ....................................................... 47

Figure 26. Kit expression significantly drops after 1 day of differentiation, but the magnitude

of drop is not affected by the presence of DMSO ........................................................................ 48

Figure 27. Brachyury processed transcript expression significantly drops after 1 day of

differentiation, but the magnitude of drop is not affected by the presence of DMSO .................. 49

Figure 28. Flk1 processed transcript expression significantly drops after 1 day of

differentiation, but the magnitude of drop is not affected by the presence of DMSO .................. 50

Figure 29. Kit expression significantly drops after 6 hours of 2i/LIF withdrawal ....................... 53

Figure 30. Klf4 expression significantly drops after 6 hours of 2i/LIF withdrawal ..................... 54

Figure 31. Stat3 processed transcript expression significantly drops after 6 hours of 2i/LIF

withdrawal..................................................................................................................................... 55

Figure 32. Zic3 primary transcript expression is significantly up-regulated after 6 hours of

2i/LIF withdrawal ......................................................................................................................... 56

Figure 33. Oct4 expression is significantly up-regulated after 6 hours of 2i/LIF withdrawal...... 57

Figure 34. Nanog expression significantly drops after 12 hours of 2i/LIF withdrawal ................ 58

Figure 35. Lefty2 expression significantly drops after 12 hours of 2i/LIF withdrawal ................ 59

Figure 36. Tdgf1 expression is significantly up-regulated after 1 day of differentiation ............. 60

Figure 37. c-Myc processed transcription is up-regulated after 1 day of differentiation .............. 61

Figure 38. Dppa2 processed transcription is up-regulated after 1 day of differentiation ............. 62

Figure 39. Rex1 primary transcript expression significantly drops after 1 day of

differentiation ................................................................................................................................ 63

Figure 40. Tet1, Fgf4 and Sall4 expression does not significantly change after 1 day of


Figure 41. Smad1, Tcfcp2 and Sox2 expression does not significantly change after 1 day of


viii

List of Appendices

Appendix 1. Chart of primers used for RT-qPCR ........................................................................ 87

Appendix 2. Gel electrophoresis of RT-qPCR primer products ................................................... 90

Appendix 3. Antibodies used for immunofluorescence ................................................................ 91

1

Introduction

A background on pluripotency

While the phenomenon of pluripotency is currently associated with embryonic stem cells

(ESCs), this was not always the case. Science’s first experiences with pluripotency came via

research into malignant teratocarcinomas. While rare in occurrence, they attracted lots of

attention due to the fact that these tumours commonly contained mixtures of cells in various

stages of development from all three embryonic lineages (reviewed by (Solter, 2006)). In

1964, intraperitoneal injection of a single tumour cell yielded new tumours containing all cell

types from the donor teratocarcinoma (Kleinsmith and Pierce, 1964), and in 1970, it was

discovered that pre- and post-implantation mouse embryos grafted onto adult mouse testes

yielded transplantable teratocarcinomas (Stevens, 1970). This introduced the concept of self-

renewal and the notion that pluripotency arises from the embryo. The first pluripotent mouse

ESC (mESC) lines were established in 1981 by culturing the inner cell mass (ICM) of

delayed blastocysts (Evans and Kaufman, 1981). By definition, a pluripotent cell is capable

of forming all cell types present in the adult form of an organism, as well as possessing the

ability to proliferate in an undifferentiated state (Jaenisch and Young, 2008). On top of this,

when injected into host blastocysts, mESCs are also able to contribute to chimera formation

(Nagy et al., 1993). In their initial publication, Evans and Kaufman discussed the possibilities

of using ESCs to introduce mutant alleles into the mouse genome. Thus, ESCs have been

linked with genome manipulation and field-changing innovation from their inception.

Currently, great efforts are being made to understand the biological underpinnings of

pluripotency (Chen et al., 2008) and to therapeutically harness the vast developmental

potential that pluripotent cells possess (Warren et al., 2010).

Oct4, Sox2, Nanog and transcriptional control of the pluripotent state

While there are many pathways and biological processes involved in the development and

maintenance of pluripotency, three transcription factors, Pou5f1 (commonly termed Oct4),

Sox2 and Nanog, comprise the core regulatory circuit underlying this phenotype (Young,

2011). Each of these genes when studied in isolation have been found to control different

aspects of differentiation and pluripotency. The collective interaction of Oct4, Sox2 and

2

Nanog serves to co-ordinate multiple networks which allows for both self-renewal and

differentiation in response to intrinsic and extrinsic signals.

Oct4 is a member of the POU transcription factor (TF) family and it is only expressed in

blastomeres, pluripotent early embryo cells and the germ cell lineage. Embryos lacking Oct4

will develop to the blastocyst stage, but their ICM is not pluripotent and therefore

differentiates only to the extra-embryonic trophectoderm lineage (Nichols et al., 1998). Oct4

over-expression causes differentiation to mesoderm and primitive endoderm lineages, while

Oct4 inactivation in mESCs causes differentiation to the trophectoderm lineage (Niwa et al.,

2000). Inhibition of Oct4 in mESCs results in their inability to differentiate to the mesoderm

lineage, and Oct4 siRNA injection into the ICM impairs cardiogenesis (Zeineddine et al.,

2006). Deviations in Oct4 expression by 50% (in either direction) from levels in mESCs are

sufficient for the induction of differentiation. Notably, in the absence of Oct4, the presence of

LIF (leukemia inhibitory factor) does not maintain pluripotency (Niwa et al., 2000).

Sox2 is a part of the SRY-related high-mobility-group box family of TFs that has only 1 exon

(Lefebvre et al., 2007). Unlike Oct4 and Nanog, the expression of Sox2 is widespread,

including tissues such as extraembryonic ectoderm (Avilion et al., 2003) and a variety of

endodermal and ectodermal tissue progenitor cells in both the developing and adult mouse

(Arnold et al., 2011). Sox2-null embryos die shortly after implantation (Avilion et al., 2003).

Sox2 is necessary for pluripotency in mESCs as inducible Sox2-null cell lines differentiate

mainly into trophectoderm-like cells. This elimination of Sox2 also causes the expression of

Oct4 and Nanog to drop by 50% 72 hours after induction (Masui et al., 2007). During the

same timeframe, over-expression of Sox2 causes an increase in expression of genes

associated with ectodermal and mesodermal lineages, even in the presence of LIF. At this

timepoint, while Nanog expression drops, Oct4 expression is not modulated by the increase

in Sox2 (Kopp et al., 2008).

Nanog is a highly divergent homeodomain-containing TF which was identified by a screen

using mESCs with the LIF receptor deleted (Chambers et al., 2003). mESC cDNAs were

transfected into these modified cells and cultured without LIF. In contrast to Oct4 and Sox2,

3

the forced expression of the Nanog cDNA was able to maintain pluripotential self-renewal of

cells without any LIF signaling (Chambers et al., 2003). Nanog is expressed only in the ICM,

mESCs (derived from the ICM) and in the proximal epiblast at embryonic day 6 (Hart et al.,

2004). While Nanog null embryos do develop an ICM by embryonic day 3.5, the ICM does

not proliferate when cultured on gelatinized plastic, instead differentiating into parietal

endoderm-like cells (Mitsui et al., 2003). Interestingly, Nanog is dispensable for the

maintenance of pluripotency. While conditionally-deleted Nanog mESCs are more likely to

differentiate, undifferentiated cells remained after continuous passaging (Chambers et al.,

2007).

Network dynamics of Oct4, Sox2 and Nanog

Numerous results indicate that Oct4, Sox2 and Nanog are extensively interconnected and

function together in a network-like manner. OCT4, SOX2 and NANOG TFs (the proteins are

collectively referred to as OSN) have been found, as a group, to bind to the promoters of the

Oct4, Sox2 and Nanog genes, pointing to the existence of autoregulatory feedback loops

(Kim et al., 2008). Chromatin immunoprecipitation followed by paired-end tag sequencing

(ChIP-PET) has shown that around 70% of OCT4 clusters also contain an adjacent SOX2

binding site, termed sox-oct composite elements (Loh et al., 2006). In fact, motif

identification of this OCT4 ChIP-PET data, revealed a perfect match to previously validated

OCT4-SOX2 elements, highlighting the frequent co-localization of these transcription factor

binding sites (TFBS) (Loh et al., 2006). By way of these composite elements, SOX2 has been

found to regulate expression of multiple TFs which influence Oct4 expression (Masui et al.,

2007), suggesting that the OCT4/SOX2 heterodimer is the functional binding unit (Chen et

al., 2008). ChIP-seq (ChIP followed by deep sequencing) analysis has shown that OSN TFBS

extensively co-localize, with over 600 sites in the mouse genome being bound by OSN and at

least one other TF. SMAD1 and STAT3 (other TFs integrated into the signaling of

pluripotency) also frequently co-bind to regions occupied by OSN (Chen et al., 2008).

Multiple TF –binding loci (MTLs, sites containing more than three bound TFs) containing

OSN binding sites have been shown to be bound by p300 and have robust, mESC-specific

enhancer activity (Chen et al., 2008). In addition, only 2% of all genes bound by OSN are not

bound by any other pluripotency-associated TFs, suggesting extended co-ordination of the

4

Oct4, Sox2 and Nanog circuit with other pathways implicated in pluripotency (Young, 2011).

OSN has also been shown to bind to and repress genes involved in regulation of lineage

specification (Young, 2011). Interestingly, many Polycomb Group proteins, which are known

to epigenetically silence genes, are bound to the same repressed lineage-specific genes as

OSN, indicating a distinct linkage between chromatin remodeling and transcriptional control

of pluripotency (Boyer et al., 2006). These results highlight the widespread influence on gene

expression and network organization that OSN collectively exerts on pluripotent mESCs.

Signaling pathways underlying pluripotency in vitro

Another important development which came from embryonal carcinoma research was the use

of mitotically-inactivated mouse embryonic fibroblasts (MEFs) in co-cultures to maintain

pluripotency (Smith, 2001). MEF co-culture was used in early establishments of mESC lines

(Evans and Kaufman, 1981). It has since been determined that MEFs support pluripotency by

providing LIF and factors involved in the Wnt and Bone Morphogenic Protein (BMP)

signaling pathways (Young, 2011). Interestingly, these pathways are all directly integrated

into the OSN TF network which controls pluripotency.

After its discovery and isolation, LIF was used to derive and maintain mESCs without the

use of MEFs (Nichols et al., 1990). LIF exerts its effect by binding to the LIF receptor, which

heterodimerizes with another class I cytokine receptor, gp130 (Davis et al., 1993). This

triggers phosphorylation/activation of JAK molecules (Narazaki et al., 1994), which in turn

phosphorylates/activates STAT3 (Niwa et al., 1998). When STAT3 is phosphorylated, it

forms a homodimer and translocates to the nucleus, acting as a TF (Ihle, 1996). As

mentioned earlier, STAT3 extensively co-localizes to OSN-bound MTLs (Chen et al., 2008),

illustrating a direct connection between external signal recognition and the core

transcriptional circuit underpinning pluripotency.

The importance of BMP signaling for pluripotency was highlighted when attempts to culture

pluripotent mESCs without MEFs in Fetal Bovine Serum (FBS)-free media supplemented

with LIF failed (Ying et al., 2003a). This highlighted the notion that LIF/STAT3 signaling

alone is not sufficient to maintain pluripotency, and that FBS/MEFs must be stimulating

5

other pathways integral for self-renewal. Another report had shown that while inhibition of

endogenous BMPs does not induce differentiation, addition of BMP4 to neural differentiation

media antagonized the development of neural precursors, instead generating non-neural

differentiated cells (Ying et al., 2003b). The addition of BMP4 to mESC cultures lacking

MEFs and FBS but containing LIF allows for long-term undifferentiated passaging,

derivation of new mESC lines as well as the ability to contribute to the generation of

chimeric mice (Ying et al., 2003a). In serum-free media containing LIF alone, mESCs have

an impaired capacity for self-renewal at low cell density and cultures eventually differentiate

into neuroectoderm. In BMP4 alone, mESCs quickly differentiate to non-neural fates. It is

currently believed that LIF and BMP4 work in tandem to prevent differentiation to specific

lineages, and if one of these components is removed, the resulting imbalance promotes

differentiation (Wray et al., 2010). The presence of BMP4 also causes increased

phosphorylation/activation of SMAD1 and increased expression of Id1 and Id3 (Inhibitor of

differentiation) genes in mESCs (Ying et al., 2003a). SMAD1 is another TF which was

studied by ChIP-seq and found to preferentially localized to OSN MTLs (Chen et al., 2008),

representing another direct assimilation between externally-derived signals and the

transcriptional regulation of mESCs.

It is interesting to note that a reduction in Oct4 (by RNA interference) causes a reduction in

the binding of SMAD1 and STAT3 TFs specifically on OCT4, SMAD1 and STAT3 cobound

sites, and that this reduction is not due to a drop in SMAD1 or STAT3 levels (Chen et al.,

2008). Furthermore, while withdrawal of LIF reduced STAT3 binding and withdrawal of

BMP4 reduced SMAD1 binding, OCT4 binding is not perturbed by either treatment (Chen et

al., 2008). Collectively, these results suggest the presence of a hierarchal relationship

between TFs involved in external signal transduction and TFs comprising the core regulatory

circuit of pluripotency.

In more recent times, the Wnt signaling pathway has become implicated in the maintenance

of pluripotency. The main components of Wnt signaling were active in human ESCs (Sato et

al., 2003) and the expression of a Wnt antagonist (Sfrp2) induces the formation of neural

progenitor cells in mESCs (Aubert et al., 2002). Wnt proteins bind to the Frizzled family of

6

receptor proteins, which results in downstream activation of Dishevelled proteins. Active

Dishevelled serves to inactivate the downstream APC/Axin/GSK3β complex, and when this

complex is inactive, β-catenin can accumulate in the nucleus to activate transcription. In its

active form, the APC/Axin/GSK3β complex phosphorylates β-catenin which results in

degradation (Okita and Yamanaka, 2006). Nuclear β-catenin has been shown to activate the

LEF/Tcf group of TFs (Okita and Yamanaka, 2006), of which Tcf3 has been shown to co-

occupy promoters with Oct4 and Nanog (Cole et al., 2008). Interestingly, pharmacological

inhibition of GSK3β using 6-bromoindirubin-3’-oxime (BIO) results in nuclear accumulation

of β-catenin, which in turn maintains the expression of pluripotency-associated genes even in

the absence of LIF (Sato et al., 2004).

The last pathway to be intensively studied with regards to pluripotency is the fibroblast

growth factor (FGF) signaling cascade. FGFs, through binding to FGF receptors, have been

shown to activate the Ras-Raf-MEK-ERK pathway (Thisse and Thisse, 2005). Both the FGF

(Thisse and Thisse, 2005) and ERK pathways (Roux and Blenis, 2004) have been implicated

in development, differentiation and cell proliferation. Fgf4-null mESCs are resistant to both

neural and mesodermal differentiation, as well as having greatly reduced levels of

active/phosphorylated ERK1/2, effects which are restored by the presence of FGF4 (Kunath

et al., 2007). ERK-null mESCs have been derived and these cells are resistant to mesoderm

formation, as they continue to express Oct4, Nanog and Rex1 after 4 days of differentiation

(Kunath et al., 2007). The impairments in differentiation to different lineages suggests that

Fgf4-induced ERK signaling is responsible only for the exit of pluripotency and not the

commitment to specific lineages (Wray et al., 2010).

The pathways described above and their widespread integration with the OSN transcription

factor network suggests that pluripotency is tightly linked to external stimuli. Recently,

highly specific chemical inhibitors of ERK- and GSK3-signaling have been developed (Bain

et al., 2007) and present research efforts have focused on how these molecules affect

pluripotency in vitro. The culture of mESCs in the presence of CHIR99021 (GSK3 inhibitor)

and PD0325901 (ERK inhibitor) is known as “dual inhibition” or 2i (Ying et al., 2008).

mESCs cultured in 2i media are proposed to be the closest in vitro representation of naïve

7

pluripotency, or precursor cells from the pluripotent epiblast (ICM) of pre-implantation

embryos (Nichols and Smith, 2009). 2i media also homogenizes expression of various

pluripotency factors such as Rex1 and Nanog, whereas under LIF + serum conditions,

expression can be quite variable (Wray et al., 2010). Following implantation, the ICM forms

two distinct lineages; the epiblast which generates the embryo, and the hypoblast which

generates primitive endoderm/the yolk sac (Yamanaka et al., 2006). Culturing 8-cell stage

mouse embryos in 2i prevents development of the hypoblast, with the ICM instead

developing entirely into Oct4, Sox2 and Nanog-expressing epiblast (Nichols et al., 2009).

Since the generation of the hypoblast seems to be dependent on FGF/ERK and Wnt

signaling, it is hypothesized that in the absence of external signaling, the default

developmental program for the ICM is to maintain pluripotency (Wray et al., 2010). These

results collectively point towards a “ground state” of pluripotency, and it is currently

believed that mESCs cultured in 2i are, for most intents and purposes, identical to pre-

implantation epiblast cells (Nichols et al., 2009).

Unique features of pluripotent mESCs

In addition to the OSN-mediated transcriptional network controlling pluripotency, mESCs

have been shown to display several distinct cellular features which play a role in both the

maintenance of self-renewal and permissiveness to differentiation (Young, 2011). In most

cases, these changes can be quickly induced by withdrawing LIF for 24 hours, suggesting

that mESCs undergo rapid, early time-point changes in response to differentiation signals.

Most relevant to the study proposed by this thesis is that pluripotent mESCs have been

shown to be transcriptionally hyperactive on a genome-wide scale (Efroni et al., 2008). When

compared to neuronal progenitor cells (NPCs, 7 day differentiation protocol), mESCs express

higher levels of both total and messenger RNA, as well as repeat sequences and transposable

and retroviral elements (Efroni et al., 2008). In addition, mESCs express low levels of

lineage committed genes (Efroni et al., 2008). However, following 24 hours of LIF

withdrawal from mESC cultures, the pluripotent-specific global up-regulation of

transcription is lost, with significant reductions in intergenic, intronic and exonic

transcription found in 9-15 of the 21 assayed chromosomes (Efroni et al., 2008). After

differentiation to NPCs, reductions of the same features are found in 12-17 chromosomes,

8

suggesting that progression of differentiation further down-regulates global transcription

(Efroni et al., 2008). When looking only at regions active in both mESCs and NPCs, 24 hours

of LIF withdrawal induces a down-regulation of intergenic, intronic and exonic transcription

in 14-19 chromosomes (Efroni et al., 2008). Taken together, these results signify that mESCs

have higher levels of transcription from more genomic regions, and that this feature is rapidly

lost following the initiation of differentiation (Efroni et al., 2008).

Supporting the findings of higher global transcription, mESCs and pluripotency in general

are associated with open chromatin structure (Ahmed et al., 2010). When a mouse embryo

reaches the 8-cell stage, chromatin is largely dispersed into a mesh of 10 nm fibres with

many visible ribonucleoproteins (RNP) (Ahmed et al., 2010). A similar chromatin state is

observed in both pre-implantation embryonic day 3.5 epiblast cells and mESCs (Ahmed et

al., 2010). In contrast, post-implantation embryonic day 5.5 epiblast cells showed many areas

of chromatin compaction of various sizes. 10 nm fibers were rarely observed and RNP

density was reduced (Ahmed et al., 2010). This pattern was mirrored in other cells in various

stages of differentiation such as in embryonic day 5.5 primitive endoderm, suggesting that

differentiation is associated with less transcriptionally-permissive chromatin (Ahmed et al.,

2010). In support of this, Oct4-null early epiblast cells also show widespread condensation of

chromatin (Ahmed et al., 2010).

Pluripotent mESCs also exhibit an increase in the dynamics of chromatin-associated proteins

when compared to mESCs cultured without LIF for 24 hours (Meshorer et al., 2006).

Nucleosomes are composed of a histone octamer; four heterodimers of the core histone

proteins H2A, H2B, H3 and H4. Histone octamers are linked together by H1, and the

interaction of the nucleosome, H1 and DNA are what drives the formation of condensed

chromatin (Sarma and Reinberg, 2005). Using Fluorescent Recovery After Photobleaching

(FRAP) techniques, it was determined that recovery of HP1 and H2B was significantly faster

in mESCs when compared to mESCs cultured without LIF for 24 hours (Sarma and

Reinberg, 2005). This result was not obtained due to an increase in histone protein expression

and suggests that pluripotent cells are quickly able to remodel chromatin in response to

differentiation.

9

Finally, mESC possess many instances of bivalent domains. The aforementioned core histone

proteins can be modified by the addition of numerous different molecules such as

phosphorylation, acetylation and methylation (Jenuwein and Allis, 2001). While there are

many potential histone modifications mediated by a wide variety of proteins, of particular

interest are the transcriptionally-permissive marking, methylation of lysine 4 on Histone H3

(H3K4) and the transcriptionally-repressive marking, methylation of lysine 27 on Histone H3

(H3K27) (Jenuwein and Allis, 2001). Many important developmentally-associated genes are

marked with both H3K4 and H3K27 in pluripotent mESCs (Bernstein et al., 2006). These

genes are expressed at very low levels in mESCs, and the bivalent chromatin markings are

said to repress high levels of transcription while keeping them poised for activation upon

differentiation (Bernstein et al., 2006). Several genes associated with neural differentiation

were determined to have bivalent markings in mESCs, but upon differentiation to neural

precursor cells, the repressive H3K27 signatures were erased as these genes became

expressed (Bernstein et al., 2006).

Specification of mesoderm and hematopoiesis in the murine embryo and mESCs

A critical event in the development of the murine embryo is the specification of the three

primary germ layers; mesoderm, endoderm and ectoderm (Murry and Keller, 2008).

Following the implantation of an early blastocyst to the uterine wall, the ICM begins to

divide into two distinct cell populations; the extraembryonic endoderm and the epiblast

(Niwa, 2007). Extraembryonic endoderm eventually gives rise to all extraembryonic tissues,

while the epiblast yields the pluripotent primitive ectoderm (Niwa, 2007). Around embryonic

day 6.25, the epiblast epithelializes into a cup-like structure called the egg cylinder which is

composed of primitive ectoderm surrounded by primitive endoderm (Tam and Behringer,

1997). Shortly after (between embryonic day 6.5-7.5), formation of the primitive streak (PS)

occurs (Tam and Behringer, 1997) and the presence of this structure marks the beginning of

gastrulation (Murry and Keller, 2008). The PS is responsible for generating the anterior-

posterior body axis and facilitates the differentiation of primitive ectoderm (former epiblast)

to both the mesoderm and endoderm lineages (Downs, 2009). Interestingly, especially given

the importance of the PS, its structure and localization have not been fully elucidated

10

(Downs, 2009). It is currently known that the PS has three distinct regions (posterior, middle

and anterior) and gene expression patterns are different in each of these areas (Murry and

Keller, 2008). Brachyury, a specifier and marker of mesoderm, is expressed in all three

regions of the PS (Kispert and Herrmann, 1994). Genes such as Foxa2 and Goosecoid are

found in anterior regions, while genes such as HoxB1 and Evx1 are found in posterior regions

(Murry and Keller, 2008). To initiate formation of mesoderm, epiblast cells are mobilized,

and migrate through the posterior PS. The first such cells which transverse the PS are

responsible for forming the yolk sac (Murry and Keller, 2008), a structure composed of

extraembryonic mesoderm. Initiation of primitive hematopoiesis occurs in the yolk sac, and

the first detectable hematopoietic cells are found at embryonic day 7.5 (Haar and Ackerman,

1971). Primitive erythroblasts from the yolk sac are the predominant blood cells in early

development and synthesize the embryonic globin genes (Palis et al., 1999). Circulation is

established by embryonic day 8.5, and slightly prior to this, definitive hematopoiesis is

established in the para-aortic splanchnopleura region (Ferkowicz, 2003). Definitive

hematopoiesis is marked by the expression of the adult globin genes, and by embryonic day

9.5, the main site of definitive hematopoiesis is the fetal liver (Palis et al., 1999).

In order to eventually take advantage of the therapeutic potential pluripotency holds,

numerous mESC differentiation protocols have been developed in order to generate

mesoderm and various precursors of the hematopoietic lineage. In fact, the in vitro

establishment of hematopoiesis is one of the most studied developmental programs by use of

mESCs (Murry and Keller, 2008). The earliest differentiation protocols used chemical

treatments such as dimethyl sulfoxide (DMSO) to generate mixed populations of cells from

mESCs (Smith 1991). These early protocols involved a withdrawal of LIF for seven days,

followed by reintroduction of LIF in conjunction with an inducer of differentiation (Smith

1991). The next generation of differentiation protocols by and large used embryoid bodies as

a model of development (Keller et al., 1993). Embryoid bodies (EBs) are three-dimensional

aggregations of mESCs created by the withdrawal of LIF and a transition to growth in non-

adherent suspension culture (Keller, 1995). EBs are differentiated, containing cell lineages

from the mesoderm, endoderm and ectoderm lineage and are reported to be the closet in vitro

representation of mouse blastocyst development (Murry and Keller, 2008). Treatment of EBs

11

with 1% DMSO and withdrawal of LIF yields an 80% rate of commitment to the

hematopoietic lineage, with a stepwise acquisition of markers of mesoderm as well as early

and later markers of hematopoiesis (Lako et al., 2001). Current efforts to direct

differentiation involve the use of conditional expression of genes which induce

differentiation, addition of signaling ligands, complex cell sorting protocols as well as culture

on coated tissue culture flasks (stromal cells or extra-cellular matrix proteins) (Keller, 2005).

Transcription factories organize the nucleus in three-dimensional space

The colocalization of genes, the chromosomes they lie on and transcription machinery offers

the eukaryotic cell various opportunities for fine-tuned gene regulation. Past research has

highlighted the existence of foci of hyperphosphorylated RNA polymerase II (RNApolII)

within the nucleus, termed transcription factories (Iborra et al., 1996). Previous studies have

shown that these transcription factories persist upon inhibition of transcription initiation and

elongation and are not simply RNApolII aggregates on actively transcribed genes (Mitchell

and Fraser, 2008). This finding is in stark contrast to the canonical view of transcription, in

which the gene to be transcribed remains static and recruits transcriptional machinery to its

promoter. Furthermore, transcription factories can recruit several genes regulated by the

same TF for transcription, creating detectable interactions between genes located either close

or far away from each other in cis or even on other chromosomes (trans) (Schoenfelder et al.,

2010). Interestingly, occupancy of transcription factories by TFs seems to be selective. Klf1

is a TF expressed in definitive erythrocytes, yet it only occupies between 10-20% of

transcription factories (Schoenfelder et al., 2010). These findings suggest that transcription is

highly organized in the three-dimensional space of the nucleus.

Experimental Proposal

Pluripotent mESCs clearly have unique properties related to transcription and many of these

features have been shown to be affected by as little as 24 hours of differentiation. This

research project therefore proposes to examine both the short-term and long-term changes in

transcription and nuclear organization that pluripotent mESCs undergo as they are treated

with a simple differentiation protocol. Since the Mitchell Lab is ultimately interested in

identifying chromatin loops associated with pluripotency, EBs are not an applicable model

12

system. For this reason, the effects of 2i/LIF withdrawal and 1% DMSO treatment of

pluripotent mESCs will be investigated. As introns are co-transcriptionally spliced (Singh

and Padgett, 2009), both primary and processed transcripts will be quantified for a more

complete understanding of the changes in transcription induced by differentiation. Markers of

pluripotency will be monitored in order to determine which genes are up- or down-regulated

first, while markers of mesoderm and hematopoiesis will be monitored in order to determine

how differentiated mESCs become. Whereas finely mapping the early time-point changes in

Oct4, Sox2 and Nanog (and other associated genes) expression represents a novel result in

itself, a comprehensive elucidation of the response of mESCs to a specific differentiation

protocol will serve to inform future experiments. By knowing the precise window at which a

gene is either up- or down-regulated, future members of the Mitchell Lab will have critical a

priori knowledge into the time at which chromatin loops sustaining pluripotency are likely

disrupted.

13

Methods

Cell Culture

The cells used for all described experiments were E14TG2a (E14), a mouse embryonic stem

cell line available from the ATCC (CRL-1821). E14s were grown in DMEM containing 15%

FBS, LIF, 1mM sodium pyruvate, 1 mM non-essential amino acids, penicillin/streptomycin,

GlutaMAX and 0.1 mM 2-mercaptoethanol. This media was further supplemented with 3 µM

CHIR99021 (GSK3β inhibitor) and 1 µM PD0325901 (MEK inhibitor), referred to hereafter

as 2i media. Frozen stocks of E14s were established with mitotically-inactive mouse

embryonic fibroblast feeder cells (MEFs and media provided by Malgosia Kownacka). E14s

were subcultured every other day at a ratio between 1:6-1:10 and media was changed on the

days in between passaging. All cell-growing surfaces were coated in 0.1% gelatin (Bioshop

GEL771.100) solubilized in endotoxin-free cell culture water. MEFs were removed from

cultures before the beginning of experiments by allowing the resuspended pellet of

trypsinized cells to sit in solution for 20 minutes (causing MEFs to sink to the bottom of the

tube). The upper portion of media (which is enriched for smaller E14 cells) was plated and

passaged at least twice before starting differentiation to ensure that MEFs are effectively

removed.

Differentiation Protocol

E14 cultures (MEF-free) were plated onto 0.1% gelatin-coated growing surfaces in 2i media

for 12 hours prior to the initiation of differentiation (to ensure the treatment does not affect

cell attachment). Following this 12 hour period, cells were washed once with PBS

(containing no divalent cations) and treated with differentiation media. The time at which

cells received this media was designated as the start of treatments (0 hours/days).

Differentiation media was the same as described above, only without the addition of LIF and

2i. For initial experiments, 1% DMSO was added to differentiation media. For the final early

time-point experiment, 1% DMSO was not added to differentiation media.

RNA Isolation and cDNA Synthesis

Total RNA was isolated from E14 and differentiation-induced E14 cells using TRIzol

(according to manufacturer’s protocol, Invitrogen). 10,000 ng (as determined by NanoDrop

14

spectrophotometric quantification) of total RNA was treated with DNase-I (Fermentas) in a

100 µL reaction. This was then followed by an acidic phenol-chloroform extraction in order

to remove any remaining DNA and DNase-I. The resulting RNA was quantified by

NanoDrop again, and used for cDNA synthesis. 600 ng of purified RNA was processed into

cDNA using the iScript Select cDNA synthesis kit (Bio-Rad) in a 20 µL reaction. Random

hexamer primers were used in order to amplify both primary and processed mRNA. As a

control for DNA contamination, 600 ng of each RNA sample was put into a cDNA synthesis

reaction with no reverse-transcriptase. The conditions for cDNA synthesis thermocycling

were as follows: 25oC for 5 minutes, 42

oC for 30 minutes and 85

oC for 5 minutes. The

resulting 20 µL reaction was diluted 5x and 1 µL of diluted cDNA was used for every RT-

qPCR reaction.

Real-Time Quantitative PCR (RT-qPCR)

Reverse-transcribed cDNA samples were quantified using a standard curve to determine

absolute expression levels. For standard curve material, genomic DNA was extracted from

the same E14 cells used for experiments. Cells were pelleted, then resuspended in a DNA

extraction buffer containing 10 mM Tris-HCl (pH-8.0), 0.1 M EDTA (pH-8.0) and 0.5%

SDS. This was followed by Proteinase K treatment (BioShop) and phenol-chloroform

extraction. The concentration of resulting genomic DNA was accurately quantified using the

PicoGreen assay (Invitrogen). Genomic DNA was then subject to five five-fold serial

dilutions were made (25 ng/µL to .04 ng/µL). 1 µL of diluted genomic DNA was used for

every RT-qPCR reaction.

RT-qPCR was performed on the Bio-Rad CFX384 thermocycler. Each RT-qPCR reaction

contained 1x Bio-Rad iTaq SYBR green mastermix (no ROX), 0.3 or 0.75 pM of each primer

(forward and reverse) and 1 µL of template (either genomic DNA or cDNA). cDNA samples

were ran in triplicate, while genomic DNA standard curves were assayed in duplicate. Each

reaction had a total volume of 10 µL. The conditions for RT-qPCR were as follows: 94oC for

3 minutes, followed by 40 cycles of 94oC for 30 seconds then 62

oC for 30 seconds.

15

RT-qPCR Data Analysis

To standardize RT-qPCR expression levels, all data was normalized to Gapdh primary

transcript. The 5 day differentiation study was repeated three independent times. The two 1

day differentiation experiments were each performed two independent times, but on one of

these repetitions, two separate tissue culture plates were differentiated and harvested for each

given time-point. The graphs shown in the Results section represent the average (mean)

normalized expression value of these replicates. Error bars represent the standard error of the

mean. The significance of difference between RT-qPCR values from differentiated and

pluripotent cells over time were assessed by two-way ANOVA and Bonferroni post tests

using Sigma Plot 12. The results from the 1 day experiment which compares the effects of

DMSO at only one time-point were analyzed by one-way ANOVA using Sigma Plot 12.

Immunofluorescence Staining and Imaging

Fluorescence images for OCT4, SOX2, NANOG and RNApolII were obtained by seeding

pluripotent or differentiated cells onto sterile 0.1% gelatin-coated glass coverslips for

between 24 and 48 hours (antibody list and dilutions in Appendix). Cells were fixed at room

temperature for 20 minutes in neutral-buffered 10% formalin (Sigma-Aldrich). After washing

with PBS, cells were simultaneously blocked and permeabilized at room temperature for 30

minutes in 10% Fetal Bovine Serum in 0.1% Triton X-100 in PBS. For OCT4, SOX2 and

NANOG images, cells were incubated with primary antibodies at 4oC overnight in antibody

buffer (0.2% Fetal Bovine Serum in 0.1% Triton X-100 in PBS). Cells were washed with

PBS + 0.1% Tween, then were incubated with the RNApolII primary antibody for 30

minutes at room temperature (in antibody buffer). Cells were washed with PBS + 0.1%

Tween three times for five minutes each. After incubation in antibody buffer containing

secondary antibodies for 30 minutes at room temperature, cells were washed with PBS +

0.1% Tween four times for five minutes each. Cells were then counterstained in DAPI,

washed twice in PBS then washed once in distilled, molecular biology grade water (to

remove remaining PBS). Coverslips were then mounted onto glass microscope slides using

VECTASHIELD (Vector Labs) and edges sealed with transparent nail polish (Revlon).

Images were taken using a Leica SP5 Confocal or Zeiss + Metasystems camera.

16

In order to determine the number of cells within a colony with nuclei positive for OCT4,

SOX2 or NANOG, slide labels were covered up and placed onto the microscope (without

knowledge of the treatment or timepoint). At 40x magnification with the FITC (ab5131) or

Texas Red (ab4H8) filter selected, a colony was chosen and the number of cells with a strong

RNApolII signal was counted. The filter was then switched to Texas Red (OCT4 and SOX2)

or FITC (NANOG) and the number of cells with distinct nuclear staining was counted.

Approximately 500 cells were counted from two independently prepared coverslips.

Significance of results was analyzed by one-way ANOVA using SigmaPlot 12.

17

Results

Introduction

While changes in Oct4, Sox2 and Nanog expression have been characterized in numerous

differentiation protocols, early changes in primary transcript levels have not been

quantitatively measured as mESCs exit pluripotency. Additionally, the effects of 2i/LIF

withdrawal and DMSO treatment of mESCs maintained in the absence of feeder cells have

not been investigated. As mESCs are very sensitive to the conditions in which they are grown

and since there numerous different ways of maintaining pluripotency in vitro, it is important

to first characterize the differentiation process for mESCs maintained under different

conditions. In this body of work, I have investigated the dynamics of Oct4, Sox2 and Nanog

expression when mESCs cultured in media containing 2i, LIF and FBS are differentiated. For

a comprehensive view of how differentiation proceeds, both short-term (up to 24 hours) and

long-term (up to 5 days) investigations were made. The main goal of these experiments was

to obtain a general idea of the timeframe and the magnitude of changes to Oct4, Sox2 and

Nanog expression as mESCs exit the pluripotent state and are induced to differentiate.

Long-term differentiation induces changes to the mESC-specific gene expression profile

For preliminary insight into the changes in gene expression caused by the described

differentiation protocol, a long-term study was performed. Days 1, 2 and 5 were chosen as

timepoints for investigation into the progress of differentiation. Total RNA was harvested

and RT-qPCR for Oct4, Sox2, Nanog, Kit, Flk1, Brachyury, CD41 and GATA1 primary and

processed transcripts was performed. After 1 day of differentiation, significant reductions in

Nanog (Figure 1) and Kit (Figure 2) primary and processed transcript were discovered.

Following this initial drop, Nanog and Kit expression did not significantly change for the

remaining 4 days of differentiation. By day 5, significant decreases in Oct4 (Figure 3a)

primary transcript and Sox2 (Figure 4) were found. This was accompanied by significant

increases in Brachyury primary (70-fold) and processed (200-fold) transcript (Figure 5), a

marker of commitment to the mesodermal lineage. While significant decreases of Flk1

processed transcript were found in days 1 and 2 of differentiation (Figure 6b), these

expression values were extremely low (under 20-fold lower than Oct4 primary transcript

18

expression after 5 days of differentiation). No expression of CD41 or GATA1 was detected

(results not shown). Taken together, these results demonstrate that following 5 days of

differentiation, mESCs express lower levels of Oct4, Sox2, Nanog and Kit while Brachyury

becomes expressed at a comparatively high rate.

19

0

1

2

3

4

5

6

7

8

9

Day 1 Day 2 Day 5

Re

lati

ve N

an

og

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

A

0

2

4

6

8

10

12

14

16

18

Day 1 Day 2 Day 5

Re

lati

ve N

an

og

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

B

Figure 1. Nanog expression significantly drops after 1 day of differentiation

Nanog primary (1a) and processed (1b) transcript expression are found to be significantly down-regulated

in differentiated mESCs after 1 and 5 days. At 2 days, p = 0.07 for Nanog processed transcripts, (slightly

above the statistical cutoff), while primary transcripts remain significantly lower in differentiated cells.

Blue boxes represent Nanog expression in pluripotent mESCs, while red boxes represent mESCs subject

to 2i/LIF withdrawal and treatment with DMSO. Data points are an average of three independent

replicates, normalized to GAPDH expression. Error bars depict SEM. * = p<0.05, **= p<0.01

20

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Day 1 Day 2 Day 5

Re

lati

ve K

it A

bu

nd

ance

Day of Experiment

2i/Lif

DMSO

A

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Day 1 Day 2 Day 5

Re

lati

ve K

it A

bu

nd

ance

Day of Experiment

2i/Lif

DMSO

B

Figure 2. Kit expression significantly drops after 1 day of differentiation

Kit primary (2a) and processed (2b) transcripts are found to be significantly down-regulated in

differentiated mESCs after 1, 2 and 5 days. Blue boxes represent Kit expression in pluripotent mESCs,

while red boxes represent mESCs subject to 2i/LIF withdrawal and treatment with DMSO. Data points are

an average of three independent replicates, normalized to GAPDH expression. Error bars depict SEM.

**=P<0.01, ***= p<0.001.

21

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Day 1 Day 2 Day 5

Re

lati

ve O

ct4

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

A

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

Day 1 Day 2 Day 5

Re

lati

ve O

ct4

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

B

Figure 3. Oct4 primary transcript expression significantly drops after 5 days of differentiation

Oct4 primary transcript expression (3a) drops significantly after 5 days of differentiation, but Oct4

processed transcript (3b) is not similarly affected. Blue boxes represent Oct4 expression in pluripotent

mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal and treatment with DMSO. Data

points are an average of three independent replicates, normalized to GAPDH expression. Error bars depict

SEM. ** = p<0.01.

22

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

Day 1 Day 2 Day 5

Re

lati

ve S

ox2

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

Figure 4. Sox2 expression significantly drops after 5 days of differentiation

Sox2 (which contains only one exon) expression is significantly down-regulated after 5 days of differentiation.

Blue boxes represent Sox2 expression in pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF

withdrawal and treatment with DMSO. Data points are an average of three independent replicates, normalized

to GAPDH expression. Error bars depict SEM. * = p<0.05.

23

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

Day 1 Day 2 Day 5

Re

lati

ve B

rach

yury

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

A

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Day 1 Day 2 Day 5

Re

lati

ve B

rach

yury

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

B

Figure 5. Brachyury expression significantly increases after 5 days of differentiation

Brachyury primary (5a) and processed (5b) transcript expression is significantly up-regulated after 5 days

of differentiation. Blue boxes represent Brachyury expression in pluripotent mESCs, while red boxes

represent mESCs subject to 2i/LIF withdrawal and treatment with DMSO. Data points are an average of

three independent replicates, normalized to GAPDH expression. Error bars depict SEM. * = p<0.05.

24

0.00

0.01

0.01

0.02

0.02

0.03

Day 1 Day 2 Day 5

Re

lati

ve F

lk1

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

A

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Day 1 Day 2 Day 5

Re

lati

ve F

lk1

Ab

un

dan

ce

Day of Experiment

2i/Lif

DMSO

B

Figure 6. Flk1 transcription significantly drops after 1 day of differentiation

Flk1 processed transcripts (6b) are found to be significantly down-regulated in differentiated mESCs after

1 and 2 days of differentiation. No significant differences in Flk1 primary transcript expression were

found (6a). In either treatment, Flk1 is expressed at very low (likely biologically irrelevant) levels. Blue

boxes represent Flk1 expression in pluripotent mESCs, while red boxes represent mESCs subject to

2i/LIF withdrawal and treatment with DMSO. Data points are an average of three independent replicates,

normalized to GAPDH expression. Error bars depict SEM. * = p<0.05, **= p<0.01.

25

Long-term differentiation induces changes to mESC-specific cellular morphology

Brightfield microscopy images were also taken at days 1, 2 and 5 (Figure 7). After 1 day of

differentiation, colonies begin to lose the hallmark raised, 3-dimensional structure associated

with pluripotency (A and B from Figure 7). Individual nuclei become visible within colonies,

and cells begin to form protrusions which more readily enable contact with nearby colonies.

By day 2 of differentiation, the features described in day 1 become more pronounced (Figure

7C). Individual cells begin to physically separate themselves from other nearby cells. While

colonies still form, they are more irregular in shape (as opposed to the tightly packed circular

colonies present in pluripotent mESCs). Protrusions also become more prevalent and

generally extend further away from the cell which they project from (Figure 7C). On day 5, a

proportion of cells take on a very flattened, almost sunken-in morphology characteristic of

mesodermal precursor cells (Figure 7D). These cells lack protrusions and are capable of

growing in a monolayer sheet if plated at a high enough density. Interestingly, some cells still

maintain morphology resembling that of day 2 of differentiation (Figure 7D).

26

Figure 7. Brightfield microscopy images of pluripotent and

differentiated mESCs

Figure 7 shows brightfield images of pluripotent mESCs (A) and

mESCs withdrawn from 2i/LIF and treated with DMSO for 1, 2 and 5

days (B, C and D). Raised colony structure is largely lost after 1 day

of differentiation. Individual cells separate out more and have more

defined projections after 2 days of differentiation. At 5 days of

differentiation, select cells adopt a sunken-down morphology. Images

taken at 10x magnification. Scale bar represents 100 µm.

27

Long-term differentiation induces changes to the localization of Oct4, Sox2 and Nanog

proteins

Immunofluorescence images of cells stained for OCT4 (Figures 8-12), SOX2 (Figures 13-17)

or NANOG (Figures 18-22) with RNA Polymerase II were also taken in order to determine

how nuclear organization changes as cells differentiate. OCT4, SOX2 and NANOG are

present in the nucleus of pluripotent mESCs. As hypothesized earlier, each transcription

factor along with RNA Polymerase II displays a focal staining pattern, first visible at 40x

magnification. Cells are tightly packed within a colony and have very little cytoplasm, with

no OCT4, SOX2 or NANOG foci visible outside the boundary of the DAPI counterstain.

Cells in the process of mitosis (metaphase and anaphase) are readily visible and do not have

strong Oct4, Sox2, Nanog or RNA Polymerase II signals. Mirroring gene expression results,

the proportion of cells within a colony with a positive nuclear signal for OCT4 does not

significantly change until day 5 (Figure 8). Even at this later timepoint, nuclear OCT4

staining is still widespread, with an average of 83% of cells within a colony positive for

OCT4 (Figures 8 and 12). Interestingly, Sox2 staining in pluripotent cells is less bright than

OCT4 and NANOG, yet more focalized. While there is a slight yet significant decrease in the

proportion of SOX2-positive nuclei after 1 day of differentiation (Figures 13 and 15), this

ratio drops significantly on day 2 and again on day 5 (Figures 13, 16 and 17). By this time,

very few cells are positive for SOX2 (1.35%). The proportion of nuclei with NANOG signals

significantly drops after 1 day of differentiation and again after 2 days (Figures 18, 20 and

21). This drop is curbed by day 5 (Figures 18 and 22), where a statistically similar number of

cells within a colony to day 2 have nuclear positive signals (15%). By day 5 of

differentiation, cells have developed larger cytoplasms. At this time-point, SOX2 and

NANOG foci are no longer localized to the nucleus, instead adopting a uniform, colony-wide

weak staining pattern (Figures 16,17, 21 and 22).

28

0

10

20

30

40

50

60

70

80

90

100

2i 1d 2d 5d

% o

f C

ells

wit

hin

a C

olo

ny

Po

siti

ve f

or

Oct

4

Figure 8. Differentiation causes a loss of OCT4 staining

Figure 8 quantifies the loss of OCT4-positive cells following 1, 2 and 5 days of

differentiation. The percentage of cells within a colony positive for OCT4 significantly

drops after 5 days of differentiation. Error bars represent SEM. * = p<0.01

*

A

29

Figure 9. OCT4 immunofluorescence in pluripotent mESCs

Figure 9 depicts OCT4 (red), RNA Polymerase II (green) and DAPI (blue) staining in pluripotent mESCs.

Image was taken at 100x magnification, scale bar represents 25 µm.

30


treatment for 1 day

Figure 10 depicts OCT4 (red), RNA Polymerase II (green) and DAPI (blue) staining in mESCs

differentiated for 1 day. Image was taken at 100x magnification, scale bar represents 25 µm.

31


treatment for 2 days


differentiated for 2 days. Image was taken at 100x magnification, scale bar represents 25 µm.

32

Figure 12. Oct4 immunofluorescence in mESCs subject to 2i/LIF withdrawal and DMSO treatment

for 5 days


differentiated for 5 days. Colonies grow almost exclusively in monolayers at high density. OCT4 staining

is finally lost in some cells, and examples of “nuclear positive” and “nuclear negative” cells are easily

visible in the red section of the top panel. Image was taken at 100x magnification, scale bar represents 25

µm.

33

0

10

20

30

40

50

60

70

80

90

100

2i 1d 2d 5d

% o

f C

ells

wit

hin

a C

olo

ny

Po

siti

ve f

or

Sox2

Figure 13. Differentiation causes a loss of SOX2 staining

Figure 13 quantifies the loss of Sox2-positive cells following 1, 2 and 5 days of

differentiation. The percentage of cells within a colony positive for SOX2

significantly drops after 1, 2 and 5 days of differentiation. Error bars represent SEM. *

= p<0.05, **=p<0.01

A

34

Figure 14. SOX2 immunofluorescence in pluripotent mESCs

Figure 14 depicts SOX2 (red), RNA Polymerase II (green) and DAPI (blue) staining in pluripotent

mESCs. Image was taken at 100x magnification, scale bar represents 25 µm.

35


treatment for 1 day

Figure 15 depicts SOX2 (red), RNA Polymerase II (green) and DAPI (blue) staining in mESCs


36

Figure 16. SOX2 immunofluorescence in mESCs subject to 2i/LIF withdrawal and DMSO treatment for 2

days


differentiated for 2 days. The colony-wide weak signal is first visible at this time-point. Image was taken

at 100x magnification, scale bar represents 25 µm.

37




differentiated for 5 days. Again, a colony-wide weak signal is visible, yet since distinct SOX2-positive

cells cannot be visualized, none are not counted as positive. Image was taken at 100x magnification, scale

bar represents 25 µm.

38

0

10

20

30

40

50

60

70

80

90

100

2i 1d 2d 5d

% o

f C

ells

wit

hin

a C

olo

ny

Po

siti

ve f

or

Nan

og

Days of Differentiation

Figure 18. Differentiation causes a loss of NANOG staining

Figure 18 quantifies the loss of NANOG-positive cells following 1, 2 and 5 days of

differentiation. The percentage of cells within a colony positive for NANOG significantly drops

after 1 and 2 but no further loss of NANOG signal is observed after 5 days of differentiation.

Error bars represent SEM. **=p<0.001

A

39

Figure 19. NANOG immunofluorescence in pluripotent mESCs

Figure 19 depicts NANOG (green), RNA Polymerase II (red) and DAPI (blue) staining in pluripotent

mESCs. Image was taken at 100x magnification, scale bar represents 25 µm.

40


treatment for 1 day

Figure 20 depicts NANOG (green), RNA Polymerase II (red) and DAPI (blue) staining in mESCs


41




differentiated for 2 days. The colony-wide weak signal is first visible at this time-point. This image shows

6 cells positive for PolII but only 2 positive for NANOG, again illustrating how the colony-wide weak

signal does not influence the cell counting results displayed in Figure 18b. Image was taken at 100x

magnification, scale bar represents 25 µm.

42




differentiated for 5 days. Again, a colony-wide weak signal is visible, yet since distinct NANOG-positive

cells cannot be visualized, none are not counted as positive. Image was taken at 100x magnification, scale

bar represents 25 µm.

43

The presence of DMSO has no significant effect on 2i/Lif withdrawal after one day of

differentiation

The results of the 5 day differentiation study indicate that the differentiation protocol causes

longer-term changes in Oct4, Sox2 and Brachyury expression while Kit and Nanog

expression is significantly attenuated within a 24 hour timepoint. Unfortunately, the results

described above do not address the fact that the applied differentiation protocol alters two

aspects of mESC culture at the same time; the withdrawal of 2i/LIF and the addition of

DMSO. In order to ensure that early down-regulation of Nanog and Kit was repeatable (and

therefore biologically relevant), and to distinguish between the effects of 2i/LIF withdrawal

and DMSO treatment, a 1 day differentiation experiment was performed. The same genes as

in the previous experiment will be quantified. mESCs were either maintained as pluripotent

cells or given one of two differentiation treatments; one without DMSO, 2i or LIF (-2i/LIF, -

DMSO) and one with DMSO but without 2i or LIF (-2i/LIF, +DMSO). The results of this

experiment will confirm the early timepoint conclusions drawn from the long-term

differentiation study while also determining the effects of DMSO on early changes in gene

expression.

After 1 day of differentiation, relative to pluripotent mESCs, no significant differences in

Oct4 or Sox2 expression were found. Nanog expression dropped significantly (14-fold for

both primary and processed transcript, Figure 25) while Kit showed a similar decrease in

expression (13-fold for primary transcript, 9-fold for processed transcript, Figure 26). This

therefore confirms the results obtained from the 1 day time-point of the long-term

differentiation experiment. Significant differences were found in the expression of Brachyury

and Flk1processed transcript, but it is important to note that both of these genes are expressed

at extremely low levels after 1 day of differentiation.

In the same timeframe, no significant differences in expression of Oct4, Sox2, Nanog, Kit,

Brachyury or Flk1 (Figures 23-28) were found between differentiation treatments lacking or

containing DMSO (-2i/LIF, -DMSO compared with -2i/LIF, +DMSO). These results indicate

44

that the presence of DMSO does not have a significant effect on the early time-point changes

in gene expression induced by 2i/LIF withdrawal.

45

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Re

lati

ve O

ct4

Ab

un

dan

ce

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

0

5

10

15

20

25

Re

lati

ve O

ct4

Ab

un

dan

ce

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

B

Figure 23. Oct4 expression is not significantly affected by 1 day of differentiation

Oct4 primary transcript (23a) and processed transcript (23b) expression is not significantly affected by 1

day of 2i/LIF withdrawal, either in the presence or absence of DMSO. Data points are an average of three

independent replicates, normalized to GAPDH expression. Error bars depict SEM.

A

46

0

5

10

15

20

25

30 R

ela

tive

So

x2 A

bu

nd

ance

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

Figure 24. Sox2 expression is not significantly affected by 1 day of differentiation

Sox2 expression is not significantly affected by 1 day of 2i/LIF withdrawal, either in the presence or absence of

DMSO. Data points are an average of three independent replicates, normalized to GAPDH expression. Error

bars depict SEM.

47

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

Re

lati

ve N

an

og

Exp

ress

ion

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

A

0

0.5

1

1.5

2

2.5

3

3.5

Re

lati

ve N

an

og

Exp

ress

ion

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

B

Figure 25. Nanog expression significantly drops after 1 day of differentiation, but the magnitude of

drop is not affected by the presence of DMSO

Nanog primary transcript (25a) and processed transcript (25b) expression is significantly down-regulated

after 1 day of 2i/LIF withdrawal, either in the presence or absence of DMSO. The presence of DMSO has

no significant effect on the expression of Nanog when mESCs are withdrawn from 2i/LIF for 1 day. Data


SEM. **=p<0.001

** = p<0.001.

48

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

Re

lati

ve K

it A

bu

nd

ance

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

A

0

0.5

1

1.5

2

2.5

3

3.5

4

Re

lati

ve K

it A

bu

nd

ance

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

B

Figure 26. Kit expression significantly drops after 1 day of differentiation, but the magnitude of

drop is not affected by the presence of DMSO

Kit primary transcript (26a) and processed transcript (26b) expression is significantly down-regulated by 1

day of 2i/LIF withdrawal, either in the presence or absence of DMSO. The presence of DMSO has no

significant effect on the expression of Kit when mESCs are withdrawn from 2i/LIF for 1 day. Data points

are an average of three independent replicates, normalized to GAPDH expression. Error bars depict SEM.

**=p<0.001

49

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

Re

lati

ve B

rach

yury

Ab

un

dan

ce

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

A

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

Re

lati

ve B

rach

yury

Ab

un

dan

ce

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

B

Figure 27. Brachyury processed transcript expression significantly drops after 1 day of

differentiation, but the magnitude of drop is not affected by the presence of DMSO

Brachyury processed transcript (27b) expression is significantly down-regulated by 1 day of 2i/LIF

withdrawal, either in the presence or absence of DMSO. No significant differences were found in

Brachyury primary transcript expression (27a). The presence of DMSO has no significant effect on the

expression of Brachyury when mESCs are withdrawn from 2i/LIF for 1 day. Data points are an average of

three independent replicates, normalized to GAPDH expression. Error bars depict SEM. **=p<0.001

50

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

0.0018

0.002

Re

lati

ve F

lk1

Ab

un

dan

ce

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

A

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Re

lati

ve F

lk1

Ab

un

dan

ce

2i/Lif

-2i/Lif, - DMSO

-2i/Lif, + DMSO

B

Figure 28. Flk1 processed transcript expression significantly drops after 1 day of differentiation,

but the magnitude of drop is not affected by the presence of DMSO

Flk1 processed transcript (28b) expression is significantly affected by 1 day of 2i/LIF withdrawal, either

in the presence or absence of DMSO. No significant differences were found in Flk1 primary transcript

expression (28a). The presence of DMSO has no significant effect on the expression of Flk1 when mESCs

are withdrawn from 2i/LIF for 1 day. Data points are an average of three independent replicates,

normalized to GAPDH expression. Error bars depict SEM. **=p<0.001

51

Fine-mapping of short-term differentiation uncovers significant changes in gene

expression as early as 6 hours after treatment

The results of the two previous experiments provided justification for finely-mapping the

drop in the expression of Nanog and Kit in order to determine when these genes are first

down-regulated. To better characterize the changes to the mESC-specific gene expression

profile occurring in the first 24 hours of differentiation, the number of genes to be quantified

by RT-qPCR was expanded. In addition to Oct4, Sox2, Nanog and Kit, primary and

processed transcripts for Tet1, Lefty2, Rex1, Fgf4, Tdgf1, Zic3, Dppa2, Klf4, cMyc, Sall4,

Stat3, Smad1 and Tcfcp2 were quantified after 6, 12 and 24 hours of differentiation. These

genes were chosen using BioGPS, a searchable online database containing microarray data

from many different cell types. Using the gene correlation function, BioGPS allows a

researcher to identify genes which have a similar expression profile to a query gene. For this

experiment, Nanog was chosen as a query as it is expressed almost exclusively in pluripotent

mESCs, thus identifying other genes which show pluripotent-specific expression. Genes of

interest with a correlation value of .90 or higher were selected for quantification by RT-

qPCR.

Following 6 hours of differentiation, Kit (Figure 29) and Klf4 (Figure 30) primary and

processed transcript as well as Stat3 processed transcript (Figure 31b) were significantly

down-regulated. These genes were expressed lower in differentiated cells for the remaining

18 hours of the experiment. Zic3 primary transcript expression was significantly higher in

differentiated cells after 6 hours and stayed this way until the end of the experiment (Figure

32a). There were no significant differences found in Zic3 processed transcript expression

(Figure 32b). Oct4 primary and processed transcript was significantly (1.5-fold) higher in

differentiated cells after 6 hours (Figure 33). Interestingly, the expression of Oct4 primary

and processed transcript dropped back to levels similar to pluripotent mESCs and stayed the

same for the remainder of the experiment. At the 12 hour timepoint, a significant (3-fold)

decline in the expression of Nanog primary and processed transcript is observed in

differentiating cells (Figure 34). This difference remains significant throughout the rest of the

experiment. A similar pattern is seen with Lefty2, with expression levels significantly

52

dropping first after 12 hours of differentiation and remaining low after 24 hours (Figure 35).

Tdgf1 primary and processed transcripts are both significantly higher in differentiated cells

after 24 hours of differentiation (Figure 36). While changes in c-Myc (Figure 37), Dppa2

(Figure 38) and Rex1 (Figure 39) were found, these differences were only significant at

p<0.1, and it is therefore difficult to conclude that these changes have any biological

significance. No significant changes were found in the expression of Tet1 (Figure 40a and b),

Fgf4 (Figure 40c and d), Sall4 (Figure 40 e and f), Smad1 (Figure 41 a and b), Tcfcp2 (Figure

41 c and d) or Sox2 (Figure 41 e).

53

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

6h 12h 24h

Re

lati

ve K

it E

xpre

ssio

n

Hours of Experiment

2i/Lif

-2i/Lif

A

0

1

2

3

4

5

6

7

8

9

6h 12h 24h

Re

lati

ve K

it E

xpre

ssio

n

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 29. Kit expression significantly drops after 6 hours of 2i/LIF withdrawal

Kit primary (29a) and processed (29b) transcript expression is significantly down-regulated after 6 hours

of differentiation. Expression remains significantly low for the remaining 18 hours of the experiment.

Blue boxes represent Kit expression in pluripotent mESCs, while red boxes represent mESCs subject to

2i/LIF withdrawal. Data points are an average of three independent replicates, normalized to GAPDH

expression. Error bars depict SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

54

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

6h 12h 24h

Re

lati

ve K

lf4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

1

2

3

4

5

6

7

8

9

6h 12h 24h

Re

lati

ve K

lf4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 30. Klf4 expression significantly drops after 6 hours of 2i/LIF withdrawal

Klf4 primary (30a) and processed (30b) transcript expression is significantly down-regulated after 6 hours

of differentiation. Expression remains significantly low for the remaining 18 hours of the experiment.

Blue boxes represent Klf4 expression in pluripotent mESCs, while red boxes represent mESCs subject to

2i/LIF withdrawal. Data points are an average of three independent replicates, normalized to GAPDH

expression. Error bars depict SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

55

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

6h 12h 24h

Re

lati

ve S

tat3

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

6h 12h 24h

Re

lati

ve S

tat3

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 31. Stat3 processed transcript expression significantly drops after 6 hours of 2i/LIF

withdrawal

Stat3 processed (31b) transcript expression is significantly down-regulated after 6 hours of differentiation.

Expression remains significantly low for the remaining 18 hours of the experiment. No significant

differences in Stat3 primary transcript expression (31a) were found. Blue boxes represent Stat3 expression

in pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal. Data points are an

average of three independent replicates, normalized to GAPDH expression. Error bars depict SEM. * =

p<0.05, ** = p<0.01, *** = p<0.001.

56

0

0.1

0.2

0.3

0.4

0.5

0.6

6h 12h 24h

Re

lati

ve Z

ic3

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

5

10

15

20

25

30

35

6h 12h 24h

Re

lati

ve Z

ic3

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 32. Zic3 primary transcript expression is significantly up-regulated after 6 hours of 2i/LIF

withdrawal

Zic3 primary (32a) transcript expression is significantly up-regulated after 6 hours of differentiation.

Expression remains significantly high for the remaining 18 hours of the experiment. No significant

differences in Zic3 processed transcript expression (32b) were found. Blue boxes represent Zic3

expression in pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal. Data


SEM. * = p<0.05, ** = p<0.01.

57

0

0.2

0.4

0.6

0.8

1

1.2

6h 12h 24h

Re

lati

ve O

ct4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

5

10

15

20

25

30

35

40

6h 12h 24h

Re

lati

ve O

ct4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 33. Oct4 expression is significantly up-regulated after 6 hours of 2i/LIF withdrawal

Oct4 primary (33a) and processed (33b) transcript expression is significantly up-regulated after 6 hours of

differentiation. Oct4 expression in differentiated cells drops back to levels not significantly different from

pluripotent mESCs at 12 and 24 hours. Blue boxes represent Oct4 expression in pluripotent mESCs, while

red boxes represent mESCs subject to 2i/LIF withdrawal. Data points are an average of three independent

replicates, normalized to GAPDH expression. Error bars depict SEM. * = p<0.05, *** = p<0.001

58

0

0.5

1

1.5

2

2.5

3

3.5

4

6h 12h 24h

Re

lati

ve N

an

og

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

+

A

0

1

2

3

4

5

6

6h 12h 24h

Re

lati

ve N

an

og

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 34. Nanog expression significantly drops after 12 hours of 2i/LIF withdrawal

Nanog primary (34a) and processed (34b) transcript expression is significantly down-regulated after 12

hours of differentiation. Processed transcript expression remains significantly low for the remaining 12

hours of the experiment. Nanog primary transcript expression after 24 hours is higher in pluripotent

mESCs, but the difference is right on the statistical cutoff (p=0.05). Blue boxes represent Nanog

expression in pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal. Data


SEM. + = p=0.05, * = p<0.05.

59

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

6h 12h 24h

Re

lati

ve L

efty

2 A

bu

nd

ance

Hours of Experiment

2i/Lif

-2i/Lif

A

0

1

2

3

4

5

6

6h 12h 24h

Re

lati

ve L

efty

2 A

bu

nd

ance

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 35. Lefty2 expression significantly drops after 12 hours of 2i/LIF withdrawal

Lefty2 primary (35a) and processed (35b) transcript expression is significantly down-regulated after 12

hours of differentiation. Expression remains significantly low for the remaining 12 hours of the

experiment. Blue boxes represent Lefty2 expression in pluripotent mESCs, while red boxes represent

mESCs subject to 2i/LIF withdrawal. Data points are an average of three independent replicates,

normalized to GAPDH expression. Error bars depict SEM. ** = p<0.01, *** = p<0.001.

60

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

6h 12h 24h

Re

lati

ve T

dg

f1 A

bu

nd

ance

Hours of Experiment

2i/Lif

-2i/Lif

A

0

0.5

1

1.5

2

2.5

3

6h 12h 24h

Re

lati

ve T

dg

f1 A

bu

nd

ance

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 36. Tdgf1 expression is significantly up-regulated after 1 day of differentiation

Tdgf1 primary and processed transcripts are found to be significantly up-regulated in differentiated

mESCs after 24 hours of differentiation. Blue boxes represent Tdgf1 expression in pluripotent mESCs,

while red boxes represent mESCs subject to 2i/LIF withdrawal. Data points are an average of three

independent replicates, normalized to GAPDH expression. Error bars depict SEM. ** = p<0.01.

61

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

6h 12h 24h

Re

lati

ve c

-Myc

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

1

2

3

4

5

6

7

6h 12h 24h

Re

lati

ve c

-Myc

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 37. c-Myc processed transcription is up-regulated after 1 day of differentiation

c-Myc processed transcript (37b) expression is found to be up-regulated in differentiated mESCs after 1

day of differentiation, but this value is not statistically significant (p = .066). No significant changes in c-

Myc primary transcript (37a) expression were detected. Blue boxes represent c-Myc expression in

pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal. Data points are an

average of three independent replicates, normalized to GAPDH expression. Error bars depict SEM.

62

0

0.1

0.2

0.3

0.4

0.5

0.6

6h 12h 24h

Re

lati

ve D

pp

a2

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

1

2

3

4

5

6

7

6h 12h 24h

Re

lati

ve D

pp

a2

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 38. Dppa2 processed transcription is up-regulated after 1 day of differentiation

Dppa2 processed transcript (38b) expression is found to be up-regulated in differentiated mESCs after 1

day of differentiation, but this value is not statistically significant (p = .083). No significant changes in c-

Myc primary transcript (38a) expression were detected. Blue boxes represent Dppa2 expression in

pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal. Data points are an

average of three independent replicates, normalized to GAPDH expression. Error bars depict SEM.

63

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

6h 12h 24h

Re

lati

ve R

ex1

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

5

10

15

20

25

30

35

40

6h 12h 24h

Re

lati

ve R

ex1

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

Figure 39. Rex1 primary transcript expression significantly drops after 1 day of differentiation

Rex1 primary transcript (39a) expression is found to be significantly down-regulated in differentiated

mESCs after 1 day. Rex1 processed transcript (39b) expression is down-regulated in differentiated cells

after 24 hours, but not significantly (p=0.057). Blue boxes represent Rex1 expression in pluripotent

mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal. Data points are an average of

three independent replicates, normalized to GAPDH expression. Error bars depict SEM. * = p<0.05.

64

0

0.1

0.2

0.3

0.4

0.5

6h 12h 24h Re

lati

ve T

et1

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

5

10

15

20

6h 12h 24h Re

lati

ve T

et1

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

0

0.5

1

1.5

2

6h 12h 24h Re

lati

ve F

gf4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

C

0

2

4

6

8

10

12

14

6h 12h 24h Re

lati

ve F

gf4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

D

0

0.05

0.1

0.15

0.2

6h 12h 24h Re

lati

ve S

all4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

E

0

5

10

15

20

25

30

35

6h 12h 24h Re

lati

ve S

all4

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

F

Figure 40. Tet1, Fgf4 and Sall4 expression does not significantly change after 1 day of

differentiation

Following 24 hours of 2i/LIF withdrawal, Tet1 primary transcript (40a) and processed transcript (40b) as

well as Fgf4 primary transcript (40c) and processed transcript (40d) and Sall4 primary transcript (40e) and

processed transcript (40f) expression does not significantly change. Blue boxes represent Tet1/Fgf4

/Sall4expression in pluripotent mESCs, while red boxes represent mESCs subject to 2i/LIF withdrawal

and treatment with DMSO. Data points are an average of three independent replicates, normalized to

GAPDH expression. Error bars depict SEM.

65

Figure 41. Smad1, Tcfcp2 and Sox2 expression does not significantly change after 1 day of

differentiation

Following 24 hours of 2i/LIF withdrawal, Smad1 primary transcript (41a) and processed transcript (41b)

as well as Tcfcp2 primary transcript (41c) and processed transcript (41d) and Sox2 (41e) expression does

not significantly change. Blue boxes represent Smad1/Tcfcp2/Sox2 expression in pluripotent mESCs,

while red boxes represent mESCs subject to 2i/LIF withdrawal and treatment with DMSO. Data points are

an average of three independent replicates, normalized to GAPDH expression. Error bars depict SEM.

0

0.02

0.04

0.06

0.08

0.1

6h 12h 24h

Re

lati

ve S

ma

d1

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

A

0

0.2

0.4

0.6

0.8

1

1.2

6h 12h 24h

Re

lati

ve S

ma

d1

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

B

0

0.005

0.01

0.015

0.02

0.025

0.03

6h 12h 24h

Re

lati

ve T

cfcp

2 A

bu

nd

ance

Hours of Experiment

2i/Lif

-2i/Lif

C

0

0.1

0.2

0.3

0.4

0.5

6h 12h 24h

Re

lati

ve T

cfcp

2 A

bu

nd

ance

Hours of Experiment

2i/Lif

-2i/Lif

D

0

10

20

30

40

50

60

6h 12h 24h Re

lati

ve S

ox2

Ab

un

dan

ce

Hours of Experiment

2i/Lif

-2i/Lif

E

66

Discussion

Summary of Results

It was found that 2i/LIF withdrawal in combination with DMSO treatment causes significant

down-regulation of the expression of Nanog and Kit following only one day of

differentiation. Oct4 primary transcript and Sox2 expression was down-regulated after 5 days

of differentiation while Brachyury was significantly up-regulated over the same timeframe.

Differentiation did not cause an up-regulation of Flk1, CD41 or GATA-1. Differentiation

caused changes in cellular morphology visible after only 1 day, as well as a change in the

localization of OCT4, SOX2 and NANOG TFs. Further experimentation showed that DMSO

does not have an effect on the early time-point changes in expression caused by 2i/LIF

withdrawal. An expanded look into the early changes in gene expression associated with

2i/LIF withdrawal revealed significant differences in the expression of Kit, Klf4, Zic3, Stat3

and Oct4 following 6 hours of differentiation. Changes in the expression of Nanog and

Lefty2 were found after 12 hours, while Tdgf1 expression was significantly up-regulated

following 24 hours of differentiation.

DMSO, 2i/LIF withdrawal and changes in gene expression after 5 days of

differentiation

Two major issues arise when attempting to place the drop in Oct4, Sox2 and Nanog

expression from long-term differentiation into the context of relevant literature. The first

problem is the relative age of DMSO differentiation protocols. DMSO was found to induce

the differentiation of mouse erythroleukemia cells to hemoglobin-producing precursors of the

erythroid lineage (Friend et al., 1971). A second wave of differentiation studies using DMSO

followed in the 1990’s (Dani et al., 1997). However, since Nanog was not identified until

2003 (Chambers et al., 2003), all studies involving DMSO prior to 2003 do not measure

Nanog expression. The second issue is the use of DMSO is most commonly applied to

embryoid bodies (EBs), which contain subsets of differentiated cells such as precursors from

the blood and vascular systems (Kennedy and M. Keller, 2003). Even though the study on

which this thesis is based reports that DMSO contributes to the development of the

mesoderm and more specifically, the hematopoietic lineage (Lako et al., 2001), this

publication fails to distinguish whether DMSO promotes the differentiation of pluripotent

67

cells to the hematopoietic lineage, or whether DMSO responsiveness is dependent on a pre-

existing commitment to the mesoderm lineage. The same problem arises even when the

expression of markers of pluripotency (such as Oct4) is quantified (Adler et al., 2006).

Adding another level of complexity to fully understanding published results is the fact that

the removal of LIF is the first step in the generation of EBs (Keller et al., 1993). In some

experiments, it is hard to discern whether “-LIF 48hrs” refers to the withdrawal of LIF from

plated mESCs, or mESCs in the process of being differentiated to EBs (Trouillas et al.,

2009).

Even though some modern studies have used DMSO to induce differentiation in non-EB

mESCs, the results are again not easily interpreted. One study reports a 0.18 fold reduction in

Nanog and a .7 fold reduction in Oct4 expression following DMSO treatment, yet this was

after 4 days of growth in media containing only 1% FBS in addition to a chemically defined

serum replacement (Torres et al., 2012). Another study quantifies Oct4, Sox2 and Nanog

expression in mESCs treated with DMSO, albeit in the presence of LIF and only at one time-

point (Adamo et al., 2009). DMSO is also often used as a vehicle for delivering treatments.

The 2i chemicals are solubilized in DMSO, as are other small molecule activators, inhibitors

and treatments such as retinoic acid (Santostefano et al., 2012). Some studies occasionally

quantify pluripotency-associated genes in DMSO-treated mESCs, yet only at a concentration

of DMSO used for other treatments in the publication (often orders of magnitude below 1%

DMSO). There are some studies which have looked at the effects of DMSO treatment on

human ESCs (Pal et al., 2012), but the transcriptional networks associated with pluripotency

(especially with regards to Nanog) in human ESCs is marked with key differences when

directly compared to mESCs. Aside from these reports, there are very few, if any,

publications which measure Oct4, Sox2 and Nanog gene expression upon the withdrawal of

2i/LIF and the addition of DMSO to non-EB mESCs.

In comparison to the publication which formed the basis of this investigation (Lako et al.,

2001), 2i/LIF withdrawal and DMSO treatment of pluripotent mESCs does not achieve

significant progression through to the hematopoietic lineage. In the study by Lako et al.

2001, EBs (following aggregation by hanging drop for 48 hours) were treated with DMSO in

68

the absence of LIF for 6 days. Quantification of markers of hematopoiesis was performed on

each day by reverse-transcriptase PCR (RT-PCR). Step-wise expression of hematopoietic

markers was seen. Brachyury is expressed only at day 2 after down-regulation of activin β,

recapitulating the transition seen in embryos from primitive ectoderm to mesoderm (Lako et

al., 2001). Expression of Flk1 and Kit (markers of primitive hematopoiesis) peaked at day 3

of DMSO treatment. Finally, expression of GATA-1 and globin βH1 (markers of further

commitment to hematopoiesis) is seen at days 5 and 6 (Lako et al., 2001). Whereas simply

removing LIF from EBs (no DMSO treatment) for the same 6 day time period achieves

expression of all mentioned genes, the process is in a less co-ordinated/step-wise manner

(Lako et al., 2001). Brachyury is expressed for a total of 4 days as opposed to just one while

Flk1 is expressed robustly for 3 days as opposed to one. For the experiments I performed,

after 5 days of 2i/LIF withdrawal and DMSO treatment administered to pluripotent mESCs,

only Brachyury is significantly upregulated. No appreciable expression of Flk1, GATA-1 or

CD41 was detected. In agreement with Lako et al. 2001, expression of Kit is greatly reduced

after one day of differentiation but in contrast, Kit is not up-regulated at later timepoints. The

comparison of previous results with newly obtained results indicates that three-dimensional

aggregation is necessary for the expression of hematopoietic markers, and that DMSO is not

significantly contributing to hematopoietic commitment of pluripotent mESCs.

Another report describes changes in expression of Oct4 and Brachyury similar to the

obtained long-term differentiation results. Using RT-PCR (a less quantitative assay than RT-

qPCR) to assay mESCs following 5 days of LIF withdrawal without DMSO treatment, the

authors found that Oct4 expression declines gradually, but is ultimately still robustly

expressed (Sauter et al., 2005). The same holds true of Oct4 expression through 16 days of

EB culture (Sauter et al., 2005). Brachyury expression was upregulated following 3 days of

LIF withdrawal and its expression increases again over the next two days. (Sauter et al.,

2005). Also in corroboration of obtained results, microarray analysis of pluripotent mESCs

subject to LIF withdrawal for 1 and 2 days identified significant drops in Nanog at both

indicated time-points (Trouillas et al., 2009). No significant changes in either Oct4 or Sox2

were detected. Interestingly, a change in Kit expression was not described (Trouillas et al.,

2009). Additionally, one report has described the changes in Oct4, Sox2 and Nanog following

69

withdrawal of LIF and BMP (Thomson et al., 2011). Following 2 days of differentiation,

relative to pluripotent mESCs, Oct4 expresson is reduced by 26%. Sox2 expression is

reduced by 71% while Nanog expression is reduced by 95% (Thomson et al., 2011). While

these changes are greater than found in my experiments, it is important to note that BMP4 is

still present in my differentiation protocol by way of FBS, and is likely playing a role in

maintaining the expression of Oct4, Sox2 and Nanog at higher levels than Thomson et al.

2011.

Even though the observed pattern of Kit expression in differentiated mESCs is somewhat

counter-intuitive, it has been previously described. Kit encodes the Kit ligand, which binds to

the Stem Cell Factor receptor (Iemura et al., 1994). Kit has been shown to play roles in

hematopoiesis (Bernex et al., 1996) and mast cell development (Iemura et al., 1994). Kit is

expressed in mESCs (Lako et al., 2001) and in the yolk sac of embryonic day 7.5-9.5

embryos (Bernex et al., 1996). Homozygous-null mutant embryos develop to term but these

mice die shortly after birth due to severe anemia (Bernex et al., 1996). Interestingly, 1 day of

EB formation and LIF withdrawal (in either the presence or absence of DMSO) causes a

sharp down-regulation of Kit expression which is regained after an additional day of

differentiation (Lako et al., 2001). The expression of Kit and subsequent rapid down-

regulation induced by differentiation could be explained by the aforementioned widespread

transcription (and the early time-point down-regulation induced by LIF withdrawal) in

pluripotent mESCs, although this hypothesis is speculative at best without further

experimental validation.

Comparison of cell morphology after 5 days of differentiation indicates that 2i/LIF

withdrawal and addition of DMSO enables BMP4-mediated non-neural differentiation

A survey of relevant literature reveals striking morphological similarities between mESCs

subject to 5 days of my differentiation protocol and pluripotent mESCs cultured in N2B27

media supplemented with BMP4 (Ying et al., 2003b). mESCs grown in N2B27 without

BMP4 or LIF develop into neural precursors expressing Sox1 and Nestin, markers of neural

ectoderm (Ying et al., 2003b). If BMP4 is added, mESCs differentiate into distinctly non-

neural precursors, with a mixed-flattened morphology very similar to mESCs after 5 days of

70

my differentiation protocol (compare figure 7d with figure 4b of Ying et al., 2003b).

Furthermore, N2B27+BMP4 can also yield confluent monolayers similar in appearance to 5

days of 2i/LIF withdrawal and DMSO treatment (compare figure 7d with the bottom right

panel of figure 1a from Ying et al., 2003a). Indeed, one report has established a link between

LIF withdrawal and BMP action, indicating that in the absence of LIF, BMP function

switches from promoting self-renewal to inducing mesoderm and endoderm (Ying et al.,

2003a). This same report concludes that LIF prevents the non-neural differentiation induced

by BMP4 (Ying et al., 2003a). Since FBS (which contains BMP4) is still present in my

differentiation protocol, it is likely that BMP4 is playing a significant role in the changes

caused by 5 days of differentiation by 2i/LIF withdrawal and DMSO treatment.

Immunofluorescence uncovers changes in Oct4, Sox2 and Nanog localization following

2i/LIF withdrawal and DMSO treatment

While the publication of fluorescence images of OCT4, SOX2 and NANOG in both

blastocysts (Keramari et al., 2010) and mESCs (Sustackova et al., 2012) is common, very

few (if any) reports have shown large, high-magnification images of either the pluripotency

factors themselves or in conjunction with RNA Polymerase II staining. These images are

usually included to confirm the presence of pluripotent cells in differentiation or

reprogramming experiments. In addition to the scarcity of high resolution OCT4, SOX2 and

NANOG images, mESC culture in 2i/LIF has been shown to increase the proportion of cells

within a pluripotent colony bi-allelically expressing Nanog (Miyanari and Torres-Padilla,

2012), meaning NANOG staining in 2i/LIF might yield different patterns than mESCs

cultured in LIF alone. Therefore, it is somewhat difficult to place the staining results obtained

into the context of relevant literature.

With regards to focalization of pluripotency-associated TFs, one report has published high-

magnification images of a green fluorescent protein-tagged (GFP) OCT4 which are distinctly

focal (Sustackova et al., 2012). From examining the localizations of OCT4, SOX2 and

NANOG proteins following 5 days of 2i/LIF withdrawal and DMSO treatment (Figures 8-

22), it is clear that differentiation causes a change in the nuclear-specific localization of these

TF foci as seen in pluripotent mESCs. It is difficult to determine what the SOX2 and

71

NANOG colony-wide weak signal visible in between nuclei after 2 and 5 days of

differentiation represent (Figures 16, 17, 21 and 22). Since transcription of both Sox2 and

Nanog is significantly lower than mESCs following 5 days of differentiation, and since

NANOG has a half-life of 120 minutes (Ramakrishna et al., 2011), it is likely that these

colony-wide cytoplasmic foci are artifacts of the immunofluorescence protocol. Indeed, one

report shows a similarly noisy Sox2 stain in embryonic day 5 blastocysts which is not seen in

OCT4 stainings (Keramari et al., 2010). Another report, albeit at low magnification, shows a

cytoplasmic staining pattern of NANOG in 3T3 mouse embryonic fibroblast cells (Chambers

et al., 2007). This pattern is not observed in OCT4 stainings of 3T3 cells (Chambers et al.,

2007). Interestingly, a putative nuclear export signal has been found in the human NANOG

protein, and forced expression of a GFP-NANOG construct with its nuclear localization

signals deleted in COS-7 cells causes a localization of NANOG to the cytoplasm (Park et al.,

2012). Without further experimental examination (such as protein quantification by Western

blot), it is impossible to discount the possibility (however unlikely it may be) that SOX2 and

NANOG proteins remain in the cytoplasm after differentiation. It is important to note that

these cytoplasmic foci do not affect the cell-counting results as a cell within a colony is only

counted as positive if it possesses a strong nuclear OCT4, SOX2 or NANOG signal.

The presence of DMSO has no significant effects on the changes of gene expression

induced by 1 day of 2i/LIF withdrawal

As indicated by my results (figures 23-28), the presence of DMSO has no effect on the

changes in gene expression induced by 1 day of 2i/LIF withdrawal. Expression of Brachyury

and Flk1 processed transcript was significantly higher in mESCs even though they are

markers of differentiation. This is most likely due to the generally higher levels of

transcription in mESCs as these genes are expressed at extremely low levels in mESCs (100-

fold lower than Oct4 processed transcript). In contrast, Kit processed transcript expression in

mESCs is only 6-fold lower than Oct4 processed transcript, indicating that Kit expression is

not likely due to globally high levels of transcription in mESCs. It is tempting to speculate

that DMSO treatment does not have an effect on the long-term (i.e. 2 and 5 days) changes in

gene expression induced by differentiation. This must be experimentally verified before such

a claim can be made. It is also important to note that the Oct4, Sox2 and Nanog gene

72

expression results obtained from this 1 day differentiation experiment were in agreement

with the 1 day time-point of the long-term differentiation experiment.

Changes in the expression of Kit, Oct4, Zic3, Klf4 and Stat3 are detected as early as 6

hours after withdrawal of 2i/LIF

In line with the reports that indicate early time-point changes in the expression profile of

pluripotent mESCs, changes in the expression of Kit, Klf4, Stat3, Zic3 and Oct4 are detected

6 hours after the withdrawal of 2i/LIF. Kit processed transcript expression is reduced over 4-

fold when compared to mESCs and this drop is maintained for the remaining 18 hours of the

experiment. As discussed earlier, the levels at which Kit is expressed is likely too high to

represent the leaky transcription associated with pluripotency. The potentially counter-

intuitive expression of Kit might warrant further experimental investigation. Oct4 expression

is significantly up-regulated after 6 hours of differentiation, as Oct4 primary and processed

transcripts are 1.5-fold higher in differentiated cells when compared to mESCs. This is likely

biologically relevant as previous reports have shown a similar increase in Oct4 causes

differentiation to mesoderm and endoderm lineages (Niwa et al., 2000). This transient

increase (and subsequent return to levels similar to mESCs) could represent an initial

transcriptional response to trigger differentiation to the mesoderm lineage. It is interesting to

note that another publication which quantifies the change in Oct4 expression following 6

hours of LIF withdrawal does not report a similar spike (Zhang et al., 2010). Zhang et al.

2010 also report an upregulation of Sox2 expression following 24 hours of LIF withdrawal.

These findings indicate that the response of mESCs to 2i/LIF withdrawal is different than

that of mESCs cultured in and withdrawn from LIF alone.

Zic3 is a zinc finger transcription factor necessary for the maintenance of pluripotency in

mESCs (Lim et al., 2007). Experimental knockdown of Zic3 causes a significant drop in the

expression of Nanog, and cells consequently up-regulate markers of the endoderm lineage

(Lim et al., 2007). The promoter of Zic3 is bound by both OCT4 and NANOG (Loh et al.,

2006) and siRNA against Oct4, Sox2 or Nanog causes a decrease in the expression of Zic3

(Lim et al., 2007). Furthermore, ZIC3 has been shown to occupy and activate the Nanog

promoter independently of OCT4 and SOX2 and Zic3 over-expression during 4 days of LIF

73

withdrawal maintains Nanog expression (Lim et al., 2010). Taken together, these results

demonstrate that Zic3 is extensively connected to the core transcriptional circuit controlling

pluripotency. In light of the role Zic3 plays in pluripotency, the fact that Zic3 primary

transcripts are significantly more highly expressed in differentiated cells was surprising. At

the same time, there was no significant difference found in the expression of Zic3 processed

transcripts between pluripotent and differentiated cells. While these results indicate a

potential increase in the rate of Zic3 transcription, this finding is likely not biologically

relevant due to the lack of change in the expression of Zic3 processed transcript.

Klf4 (a member of the Krüppel-like family) is another zinc finger transcription factor which

is involved in the regulation of cell proliferation (Chen et al., 2001) and differentiation

(Klaewsongkram et al., 2007). Klf4 is necessary for pluripotency, as experimental

knockdown of Klf4 leads to a reduction in the levels of Oct4, Sox2 and Nanog as well as

differentiation (Zhang et al., 2010). Klf4 knockdown also causes a 5-fold upregulation of

Brachyury (Zhang et al., 2010). Over-expression of Klf4 in N2B27 media lacking both LIF

and BMP4 maintains the expression of Oct4, Sox2 and Nanog, indicating that the

pluripotency-maintaining action of Klf4 is downstream of the LIF/Stat3 and BMP4/Smad

pathways (Zhang et al., 2010). Along with Oct4, Sox2 and c-Myc, Klf4 was one of the four

factors virally transduced into MEFs in the first publication reporting the reprogramming of

non-stem cells back to pluripotency (Takahashi and Yamanaka, 2006). While it is not

necessary for reprogramming, Klf4 is commonly included in the combination of genes

delivered to somatic cells in the generation of induced pluripotent stem cells (Gonzalez et al.,

2011). ERK1 and ERK2, which are specifically inhibited by the 2i component PD0325901

(Bain et al., 2007), have been shown to inactivate Klf4 by phophorylation (Kim et al., 2012).

Furthermore, the addition of LIF to mESCs cultured in 2i and cyclohexamide (an inhibitor of

protein synthesis) caused an up-regulation of Klf4 (Hall et al., 2009). Taken together, these

results demonstrate a significant link between Klf4 expression and 2i/LIF, explaining why

Klf4 levels drop (and continue to do so) after 6 hours of 2i/LIF withdrawal. A previous

publication reports a 0.15-fold reduction of Klf4 following 6 hours of LIF withdrawal (Zhang

et al., 2010). My findings show that 2i/LIF withdrawal causes a 3.5-fold drop in both Klf4

primary and processed transcript in the same timeframe. While this previous report

74

corroborates my findings, the difference in the fold-change of Klf4 expression when

compared to this publication again signifies that the transcriptional response of mESCs to

2i/LIF withdrawal is different than LIF withdrawal alone.

As described in previous sections, LIF binds to the LIF receptor which homodimerizes with

the gp130 receptor (Davis et al., 1993). This results in the phosphorylation and activation of

STAT3, which translocates to the nucleus to act as a TF (Niwa et al., 1998). STAT3

extensively colocalizes with Oct4, Sox2 and Nanog binding sites, indicating a direct

connection between external signaling pathways and the core regulatory circuit of

pluripotency (Chen et al., 2008). One report shows that both Stat3 and phosphorylated Stat3

proteins are less abundant in mESCs following only 15 minutes of LIF withdrawal (Mitsui et

al., 2003). Trouillas et al. 2009 also show that LIF withdrawal causes a significant reduction

in Stat3 transcription following 24 hours of LIF withdrawal. It is therefore not surprising that

expression of Stat3 processed transcript was found to be significantly down-regulated after

only 6 hours of 2i/LIF withdrawal.

Changes in the expression of Nanog and Lefty2 are detected as early as 12 hours after

withdrawal of 2i/LIF, while Tdgf1 changes are detected after 24 hours

After a further 6 hours of differentiation by withdrawal of 2i/LIF, two additional genes,

Nanog and Lefty2 were found to be significantly down-regulated when compared to mESCs.

Lefty2 is a member of the TGF-β superfamily and is involved in left-right patterning in the

mouse embryo (Tabibzadeh and Hemmati-Brivanlou, 2006). Lefty2 is expressed in both

pluripotent mESCs as well as mESCs treated with retinoic acid for 24 and 48 hours (Oulad-

Abdelghani et al., 1998). While Lefty2 is commonly used as a marker for pluripotency, there

has been little research into its potential targets in mESCs. It is therefore expected that 2i/LIF

withdrawal would cause a significant down-regulation of Lefty2.

One report demonstrates that the drop in Nanog expression is likely due to the preceding

down-regulation of Klf4. Klf4 has been shown to bind two sites in the Nanog promoter

(Zhang et al., 2010). Knockdown of Klf4 has been shown to reduce the expression of Nanog

(Zhang et al., 2010). Interestingly, this result is a direct parallel to the expression profile of

75

cells withdrawn from 2i/LIF from 0-12 hours. After 2 hours of LIF withdrawal, Klf4 is down-

regulated more rapidly than Nanog (Zhang et al., 2010). Zhang et al. 2010 also looked at the

effects of re-introducing LIF following 16 hours of LIF withdrawal. The subsequent up-

regulation of Klf4 precedes that of Nanog, and this result is most pronounced between 0-12

hours following the re-introduction of LIF (Zhang et al., 2010). The combination of these

results with my results demonstrates that Nanog expression is at least partially reliant on the

expression of Klf4. In contrast to my results though, Zhang et al. 2010 report only a 0.05-fold

reduction in Nanog expression after 12 hours of LIF withdrawal. This result, as well as the

findings that 2 days of LIF withdrawal results in a 2-fold up-regulation of Sox2 (Zhang et al.,

2010) and 6 hours of LIF withdrawal does not result in a spike in Oct4 (Zhang et al., 2010)

signifies considerable differences in the changes in expression of Oct4, Sox2 and Nanog

when 2i/LIF withdrawal is compared to LIF withdrawal alone.

Tdgf1 (also known as Cripto-1) has been referred to as a marker of pluripotency (Zhang et

al., 2010). Its promoter is bound by both OCT4 and NANOG, and it is a downstream effector

in the WNT/β-catenin signaling pathway (Bianco et al., 2010). It is therefore difficult to

place the up-regulation of Tdgf1 found in differentiated cells into the context of the literature.

TDGF1 has been studied in human cancer, and results often indicate that its expression is

associated with enhanced tumourigenesis in human cancers (de Castro et al., 2010). It is

possible that Tdgf1, similar to Lefty2, plays a role in both the maintenance of pluripotency

and in lineage specification.

Changes in expression were also found in c-Myc, Dppa2 and Rex-1 following 24 hours of

2i/LIF withdrawal, but the P-values for the differences were all between 0.05 and 0.1. Given

that this experiment was only performed three times, and also considering the fact that all of

the genes discussed above had P-values well below 0.05, more repetitions should be

performed before concluding that 2i/LIF withdrawal causes a significant difference in the

expression of these genes.

76

Future Direction

The results described in this have outlined several avenues potentially worthy of future

experimentation. The most logical follow-up to the expanded gene expression study would

be to sequence RNA extracted from mESCs differentiated for 6, 12 and 24 hours. This would

allow for a complete elucidation of genome-wide changes in expression caused by

differentiation. It would also be interesting to examine the degree of pluripotency that

mESCs differentiated for 1 day exhibit. While cells subject to this treatment no longer

express Nanog, they continue to express Oct4 and Sox2. An embryoid body formation assay

similar to the one described by Lako et al., 2001 could be performed. If mESCs withdrawn

from 2i/LIF for 24 hours are still pluripotent, their ability to form embryoid bodies should not

be compromised. The levels of lineage commitment achieved by any given period of 2i/LIF

withdrawal could also be assessed by the re-introduction of 2i/LIF. The time at which the

expression of a gene significantly changes could also be examined by the chromosome

confirmation capture (3C, or one of its variants, e4C or 5C). If there is a chromatin loop

responsible for the transcription of a gene, it is likely disrupted upon the down-regulation of

said gene. This thesis has identified several candidates for 3C analysis within the first 24

hours of the induction of differentiation

Conclusions

Pluripotency is a unique and transient cellular characteristic which is crucial for mammalian

development. While the nature of pluripotency has nearly limitless potential for personal

therapeutic use, the molecular underpinnings of both self-renewal and differentiation must be

better understood before this potential can be fully harnessed. This thesis describes the

transcriptional changes mESCs undergo as they are induced to differentiate towards the

mesoderm lineage. The above results and relevant literature search show that in the long-

term, 2i/LIF withdrawal in the presence of DMSO was sufficient to cause mESCs to exit

pluripotency. Nanog and Kit expression was found to be significantly down-regulated after 1

day of differentiation, while Sox2 and Oct4 primary transcript expression was significantly

down-regulated after 5 days. While Brachyury expression was highly upregulated after 5

days (signifying commitment to the mesoderm lineage), expression of early markers of

hematopoiesis was not achieved. When compared to the findings of Lako et al., 2001, these

77

results confirm the notion that 3-dimensional aggregation, as in EBs, is likely critical for the

in vitro differentiation of mESCs to the hematopoietic lineage. Differentiation as seen by 5

days was likely BMP4-mediated, given a similar morphological appearance of my cells to

other reports (Ying et al. 2003b). 5 days of differentiation also alters the nuclear-specific

localization of OCT4, SOX2 and NANOG. My experiments have also shown that the

presence of DMSO does not significantly affect the changes in Nanog and Kit expression

found after 1 day of differentiation by 2i/LIF withdrawal. Significant changes in Stat3, Klf4,

Kit, Zic3 and Oct4 were found after 6 hours of 2i/LIF withdrawal. The rapid loss of

expression of Stat3 and Klf4 likely causes a subsequent down-regulation of Nanog after 12

hours of differentiation, while the up-regulation of Oct4 could be playing a role in the

ultimate commitment to the mesoderm lineage. This study has therefore characterized both

short-term and long-term changes to the transcriptional networks underlying pluripotency in

mESCs treated with a simple differentiation protocol.

78

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Appendices

Primer Name Description Sequence Chromosome Start End

Amplicon

Size

cMyc-EX2La Exon 2 Left CCTGACGACGAGACCTTCAT 15 61819434 61819453

cMyc-EX2Ra

Exon 2

Right GCTTCTCCGAGACCAGCTT 15 61819506 61819524 91 bp

cMyc-I1E2L

Intron 1

Left CCAACCAGAGCTGGATAACTC 15 61819009 61819029

cMyc-I1E2R

Exon 2

Right AAATAGGGCTGTACGGAGTCG 15 61819124 61819144 136 bp

Dppa2-EX2L Exon 2 Left TTTTAACGCTGGTCCCCTTT 16 48311725 48311744

Dppa2-EX2R

Exon 2

Right CTGGTGGTGTGTGGTCTAAGG 16 48311803 48311823 99 bp

Dppa2-I1E2L

Intron 1

Left GCCTGCTTCCTGAGGTTATCT 16 48311596 48311616

Dppa2-I1E2R

Exon 2

Right CGTAGTCTGTGTTTGGCTCCT 16 48311755 48311775 180 bp

Fgf4-EX1L Exon 1 Left CCTTTCTTTACCGACGAGTGT 7 152048670 152048690

Fgf4-EX1R

Exon 1

Right GTTCTTACTGAGGGCCATGAAC 7 152048756 152048777 108 bp

Fgf4-I2E3L

Intron 2

Left TGACACTCCGCCTACCTAAGA 7 152048589 152048609

Fgf4-I2E3Ra

Exon 3

Right CGTAGGCGTTGTAGTTGTTGG 7 152048713 152048733 145 bp

Flk1-EX3L Exon 3 left CTCAGCGTGATTCTGAGGAAA 5 76368425 76368445

Flk1-EX3R

Exon 3

right CGGTACGAGCACTTGTAGGC 5 76368319 76368338 127 bp

Flk1-I1E1L Intron 1 left CACCCTACTTCTCCGCCTTAC 5 76374084 76374104

Flk1-I1E1R

Exon 1

right GCTGCTAGCTGTCGCTCTGT 5 76374160 76374179 96 bp

GAPDH-E1I1L Intron 1 left GCACCAGCATCCCTAGACC 6 125115479 125115496

GAPDH-E1I1R

Exon 1

right CTTCTTGTGCAGTGCCAGGTG 6 125115566 125115587 109 bp

Kit-E8I8L Exon 8 left TGACGTACGACAGGCTCATA 5 76033334 76033353

Kit-E8I8R

Intron 8

right CGCAAGTCCGAAAACACA 5 760335213 760335230 187 bp

Kit-EX3L Exon 3 left TTCCCTCATCGAGTGTGATG 5 76005395 76005414

Kit-EX3R

Exon 3

right GCTTCACGTTTTTGATGGTGAT 5 76005465 76005486 92 bp

Appendix 1. Chart of primers used for RT-qPCR

List of primers used for RT-qPCR in this thesis. All primers (except for Oct4, Sox2 and Nanog

primary transcript) were used at a 3 µM concentration. Oct4, Sox2 and Nanog primary transcript

primers were used at a 7.5 µM concentration

88

Klf4-E2I1L Exon 2 Left GGGAAGTCGCTTCATGTGAG 4 55543817 55543836

Klf4-E2I1R

Intron 1

right GGCAACAGGGTGCTACAATTA 4 55543887 55543907 91 bp

Klf4-E2L Exon 2 Left GAAGACGAGGATGAAGCTGAC 4 55543587 55543607

Klf4-E2R

Exon 2

Right TGGACCTAGACTTTATCCTTTCC 4 55543658 55543680 94bp

Lefty2-E1I2L Exon 1 Left CAAGAGGTTCAGCCAGAATTT 1 182823539 182823559

Lefty2-E1I2R

Intron 2

Right AGGACTTGGTCACACCTCTCA 1 182823654 182823674 136bp

Lefty2-EX1L Exon 1 Left CGATGACCGAGGAACAGGT 1 182823376 182823394

Lefty2-EX1R

Exon 1

Right ACCTCACGTGGGTAGGGATG 1 182823470 182823489 114 bp

Nanog-E1I1L Exon 1 left TTGCTGTGCCAACATTTTCT 6 122657876 122657895

Nanog-E1I1R

Intron 1

right TTTTATATTTGAGATTGTAT 6 122657940 122657959 84 bp

Nanog-EX4L Exon 4 left CATAACTTCGGGGAGGACTTT 6 122663400 122663420

Nanog-EX4R

Exon 4

right AGGCTTGTGGGGTGCTAAAAT 6 122663506 122663526 127 bp

Oct4-E1I1L Exon 1 left ACGAGTGGAAAGCAACTCA 17 35643331 35643350

Oct4-E1I1R

Intron 1

right GCCAAGTCCCTTCACTTAC 17 35643428 35643447 116 bp

Oct4-EX5L Exon 5 left ATGAGGCTACAGGGACACCTT 17 35647309 35647330

Oct4-EX5R

Exon 5

right GTGAAGTGGGGGCTTCCATA 17 35647388 35647408 100bp

Rex1-E4I3L Exon 4 Left CACTTGTCTTTGCCGTTTTCT 8 44381765 44381785

Rex1-E4I3R

Intron 3

Right TGGGCTCTCGTATTGGAGTAT 8 44381917 44381937 173 bp

Rex1-EX4L Exon 4 Left GCACCAGAAAATGTCGCTTTA 8 44381156 44381176

Rex1-EX4R

Exon 4

Right GATAAAACCGCCCTGAGGAAG 8 44381250 44381270 115 bp

Sall4-EX2L Exon 2 Left GTTGCCTTTCGTGGTGAAG 2 168577960 168577978

Sall4-EX2R

Exon 2

Right AGAACTTCTCGTCTGCCAGTG 2 168577978 168578066 107 bp

Sall4-I2E2L

Intron 2

Left TGAACCAATGGATGTTCTCTG 2 168577893 168577913

Sall4-I2E2R

Exon 2

Right CCTTTCGTGTGTAACATATGCG 2 168577986 168578007 115 bp

Smad1-EX2La Exon 2 Left AGTGGTAGGGGTTGATGCAG 8 81895704 81895723

Smad1-EX2Ra

Exon 2

Right TCATTTATTGCCGTGTGTGG 8 81895807 81895826 123 bp

Smad1-I2E2L

Intron 2

Left GCTTGTTACAGCGAGGGAAG 8 81895507 81895526

Smad1-I2E2R

Exon 2

Right CTATAAGCGAGTGGAGAGCCC 8 81895685 81895705 199 bp

Sox2L Sox2 left ACGCCTTCATGGTATGGTC 3 34549479 34549498

89

Sox2R Sox2 right CGGACAAAAGTTTCCACTC 3 34549574 34549593 114 bp

Stat3-EX21L

Exon 21

Left CTCGGGCCTACAGTACTTTCC 11 100754419 100754439

Stat3-EX21R

Exon 21

Right TGTAGAGCCATACACCAAGCA 11 100754561 100754581 163 bp

Stat3-I3E3L

Intron 3

Left ACAAGACAGGCGTCACTTAGC 11 100769873 100769893

Stat3-I3E3R

Exon 3

Right CCAGCAATATAGCCGATTCC 11 100770033 100770052 180 bp

Tcfcp2-EX7L Exon 7 Left TGTACTCTCCGTTCCCGTTC 15 100351040 100351059

Tcfcp2-EX7R

Exon 7

Right CAGCACTGAGTTCACCATGAG 15 100351115 100351135 98 bp

Tcfcp2-

I13E13L

Intron 13

Left CCCAGAACAACCTACCGAAG 15 100342625 100342644

Tcfcp2-

I13E13R

Exon 13

Right AGGAGTCGTTGCAGTTGAGG 15 100342710 100342729 105 bp

Tdgf1-E5I4L Exon 5 Left CAGCCAGGTAGAAAGGTCTGA 9 110844835 110844855

Tdgf1-E5I4R

Intron 4

Right AGTGCTGTTGAGAGGGAACAG 9 110845010 110845030 196 bp

Tdgf1-EX4L Exon 4 Left TGTTCACAGTTGCGTCCATAG 9 110845049 110845069

Tdgf1-EX4R

Exon 4

Right GTCGCTTAATAAAACTTGCTGTC 9 110845122 110845144 96 bp

Tet1-EX1L Exon 1 Left ATGTAATTCTACGCCGCCAAT 10 62341069 62341089

Tet1-EX1R

Exon 1

Right AGTAGGTGCACTCCCCACAGT 10 62340989 62341009 101 bp

Tet1-I3E3L

Intron 3

Left TCCCCATGACCACGTCTACT 10 62303053 62303072

Tet1-I3E3R

Exon 3

Right GTAGACATTCAGCGGGTGGT 10 62303155 62303174 122 bp

T-EX2L Exon 2 left ACCACCGCTGGAAATATGTG 17 8628145 8628164

T-EX2R

Exon 2

right CCATTGAGCTTGTTGGTGAGT 17 8628290 8628310 166 bp

T-I2E3L Intron 2 left TGCTGCTGCGGTATTAAGGT 17 8628912 8628931

T-I2E3R

Exon 3

right CACGATGTGAATCCGAGGTT 17 8629004 8629023 112 bp

Zic3-E1I1L Exon 1 Left GAAGATTTTTGCCCGCTCT X 55285362 55285380

Zic3-E1I1R

Intron 1

Right AGCGCTTGGTACTGAAAGGTC X 55285533 55285553 192 bp

Zic3-E3L Exon 3 Left ATGTGAATTCGAAGGCTGTGA X 55286555 55286575

Zic3-E3R

Exon 3

Right ATATAGGGCTTGTCCGAGGTG X 55286624 55286644 90 bp

90

Appendix 2. Gel electrophoresis of RT-qPCR primer products

Amplicons of all primers used in this thesis and outlined in Appendix 1. Ladder is 2-Log ladder (100

bp-10,000 bp), available from New England Biolabs. (Note, T is another gene name for Brachyury)

List of Primers for top gel, Lanes 1-30:

2-Log ladder, Oct4-EX5, Oct4I1E1, Sox2, Nanog-EX4, Nanog E1I1, Tet1-E3I3, Tet1-EX1, Lefty2-

EX1, Lefty2-E1I1, Zic3-EX3, Zic3-E1I1, Rex1-EX4, Rex1-E4I3, Tdgf1-E5I4, Tdgf1-EX4, Fgf4-

I2E3, Fgf4-EX1, Dppa2-I1E2, Dppa2-EX2, Klf4-E2I1, Klf4-EX2, cMyc-I1E2, cMycEX2a, Sall4-

I2E2, Sall4-EX2, Smad1-EX2a, Stat3-I3E3, Stat3-EX21, 2-Log ladder

List of Primers for bottom gel, Lanes 31-42:

2-Log ladder, Smad1-E3I2, Tcfcp2-I13E13, Tcfcp2-EX7, Kit-E8I8, Kit-EX3, T-I2E3, T-EX2, blank,

Flk1-I1E1, Flk1-EX3, GAPDH-E1I1

Lane 1 Lane 30

Lane 31

91

Protein Antibody Isotype

Dilution

Used

OCT4 Santa Cruz Sc-5279 Mouse IgG 1/100

SOX2 R&D Systems MAB2018 Mouse IgG 1/100

NANOG

Cosmo Bio RCAB0002P-

F

Rabbit

Polyclonal 1/100

RNA Polymerase

II Abcam 4H8 Mouse IgG 1/500

RNA Polymerase

II Abcam ab5131 Rabbit IgG 1/500

Secondary Antibody Isotype

Dilution

Used

FITC Sigma F5137

Goat anti-

Rabbit 1/100

Alexa 594

Invitrogen A-

11005

Goat anti-

Mouse 1/500

Appendix 3. Antibodies used for immunofluorescence

Appendix 3a lists the primary antibodies used for immunofluorescence while Appendix 3b lists the

secondary antibodies used for immunofluorescence.

A

B

Documents

Characterizing changes in the transcriptional …...Michael Schwartz Master of Science Department of Cell and Systems Biology University of Toronto 2012 Abstract Mouse embryonic stem