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Role of STAT3 phosphorylation in mouse embryonic stem cell self-renewal and differentiation

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Role of STAT3 phosphorylation in mouse embryonic
stem cell self-renewal and differentiation

by
Guanyi Huang
Mentor: Qi-Long Ying, Ph.D

Genetics, Molecular and Cellular Biology
Keck School of Medicine
University of Southern California
May 2014

I

Epigraph
The opposite of a correct statement is a false statement. But the
opposite of a profound truth may well be another profound truth.
-- Niels Bohr

II

Dedication
I dedicate this body of work to my parents for their constant
inspiration, guidance and love over the years, and my love and
best friend Ying who walked into my life and made it great.

III

Acknowledgements
I am extremely grateful to Dr. Qi-Long Ying for providing me with the opportunity to
pursue my Ph.D studies in his laboratory. Qi-Long has shown me not just how to do
science but, more importantly, how to think like a scientist. Even though he was heavily
involved with many activities, he still found the time to keep in touch with me regularly
and gave excellent guidance. He was patient, supportive, available and inquisitive about
his student's interests. He always encouraged me to explore new directions in research
and granted me complete freedom to design projects. Qi-Long has helped tremendously
in my own research, leading to my project being awarded by the CIRM fellowship in
2010 and 2011. I sincerely thank him for the guidance and support that made the work in
this thesis possible.

My special thanks go to Dr. Chang Tong for his mentoring during my initial years in the
lab, his inspiring scientific discussions as a senior colleague, and for his help with
experiment as and when necessary. I respect him much for his profound scientific
expertise, dedicated work attitude, and wonderful personality. I would also thank my
present and past colleagues for their advice and support. Dr. Hexin Yan, Dr. Shoudong
Ye, Dr. Ping Li, soon-to-be Doctor Dongbo Qiu, and Xinliang Zhou have all helped me
not only with my experiments, but also by being my very good friends.

I am particularly lucky to have great dissertation committees who provided critical
comments and suggestions. Much gratitude to the members in the committee including
IV

Dr. Robert Maxson, Dr. Gregor Adams, Dr. Justin Ichida and Dr. Krzysztof Kobielak for
their help in finishing my thesis and with career development advice.

Nothing would have been possible without the unconditional love, wishes and patience of
my parents, Tianxue Huang and Xuehong Wu. They provided me with the best life
possible and did everything they could to make my life shine. They have given me all the
qualities that have played a critical role in my achievements. For that, I am eternally
indebted. To Ying Zhang, the love of my life: you deserve a big “thank you”. With my
family being so far away, your presence and support was often times a blessing. You
believed in me, shielded me during the rough times and inspired me to achieve new
heights. I cannot imagine a more special soulmate.

V

Table of Contents
Acknowledgements ............................................................................................................ III
List of Figures and Tables................................................................................................. VII
Part I: Role of STAT3 phosphorylation in mouse embryonic stem cell self-renewal
and differentiation ............................................................................................................ 1
Abstract ........................................................................................................................ 1
Introduction ................................................................................................................. 2
Materials and methods ............................................................................................... 21
Results ....................................................................................................................... 26
Establishment of inducible STAT3 expression system in STAT3
̶ / ̶ mESCs ..... 26
pY705 is absolutely required for STAT3-mediated mESC self-renewal ........... 31
pS727 supports mESC survival and proliferation, and is dispensable for STAT3-
mediated self-renewal ........................................................................................ 38
pS727 promotes optimal pluripotency by enhancing STAT3 transcriptional
activity ................................................................................................................ 44
STAT3 pS727 promotes neuronal differentiation of mESCs............................. 46
PD0325901 inhibition of serum-induced pS727 prevents mESC differentiation
............................................................................................................................ 51
Loss of STAT3 S727 phosphorylation enables LIF-induced mEpiSC
reprogramming ................................................................................................... 57
Proposed model for the role of differential STAT3 phosphorylation in mESC
fate determination ............................................................................................... 67
Discussion .................................................................................................................. 69
References ................................................................................................................. 72

VI

Part II: Sophisticated rat genome engineering by embryonic stem cell -based
homologous recombination ............................................................................................ 86
Abstract ...................................................................................................................... 86
Introduction ............................................................................................................... 87
Experimental design, Results and Discussion ........................................................... 99
Derivation and propagation of rat ESCs .......................................................... 101
Design of the targeting vector .......................................................................... 103
Introduction of gene-targeting vectors into rat ESCs. ...................................... 109
Isolation of gene-targeted rat ESC colonies ..................................................... 110
Characterization of gene-targeted rat ESC colonies ........................................ 112
Karyotyping and subcloning of correctly targeted rat ESCs ............................ 116
Production of knockout rats ............................................................................. 118
Strain combination ........................................................................................... 119
Generation of Cre-expressing rat lines ............................................................. 120
Concluding remarks and future directions ............................................................... 122
References ............................................................................................................... 125

VII

List of Figures and Tables
Figure 1.1.1 Different functional domains of STAT3 molecule…………………………..3
Figure 1.1.2 Canonical JAK-STAT3 pathway…………………………………………….3
Figure 1.1.3 The proposed model explaining how STAT3 coordinate with distinct gene
regulators to achieve diverse cellular functions…………………………………………...7
Figure 1.1.4 An illustration of promoter co-occupation of STAT3 and other transcription
factors in mESCs…………………………………………………………………………12
Figure 1.1.5 LIF activates STAT3, MAPK and PI3K/Akt pathways in mESCs...............14
Figure 1.1.6 LIF/STAT3 signaling must be activated at an appropriate level so that the
direct downstream targets can contribute to core pluripotency network regulation……..15
Figure 1.1.7 3i or 2i condition represents the ground state of mESC self-renewal that does
not require extrinsic stimuli……………………………………………………………...17
Figure 1.1.8 The experimental design of investigating the role of STAT3 phosphorylation
in mESC self-renewal and differentiation………………………………………………..20
Figure 1.2.1 Cytotoxicity of STAT3-Y705F transgene in STAT3
̶ / ̶ mESCs……………27
Figure 1.2.2 The expression level of STAT3-WT in STAT3
̶ / ̶ mESCs greatly exceeds the
endogenous level of STAT3 in wild-type 46C ESCs……………………………………27
Figure 1.2.3 The principle of ProteoTuner inducible system……………………………28
Figure 1.2.4 STAT3
-/-
+ DD-STAT3 transgenic mESCs in N2B27+2i medium display
typical mESC morphology on feeder cells………………………………………………29
Figure 1.2.5 Dose-dependent modulation of DD-STAT3 protein level by S1…..............30
VIII

Figure 1.2.6 Immuno-fluorescence analysis of STAT3 quantity modulated by S1……...30
Figure 1.2.7 Phosphorylation potential of STAT3
-/-
+ DD-STAT3 mutant mESCs…….31
Figure 1.2.8 STAT3
-/-
mESCs differentiate under mESC+LIF+S1 condition…………...33
Figure 1.2.9 Comparison of the self-renewal potential of different STAT3
-/-
+ DD-STAT3
cells under various culture conditions…………………………………………………....33
Figure 1.2.10 STAT3
̶ / ̶ + DD-STAT3-Y705F mESCs failed to self-renewal under mESC
+LIF+S1 condition……………………………………………………………………….34
Figure 1.2.11 STAT3
̶ / ̶ + DD-STAT3C mESCs self-renew in absence of LIF………....35
Figure 1.2.12 Short and long term self-renewal potential of STAT3
-/-
+ STAT3C-Y705F
and STAT3
-/-
+ STAT3C-Y705D mESCs in mESC+LIF+S1 condition………………...36
Figure 1.2.13 Subcellular distribution of different STAT3 mutants under mESC+LIF+S1
condition…………………………………………………………………………………37
Figure 1.2.14 qRT-PCR analysis of socs3 expression in different STAT3
-/-
+ DD-STAT3
mESCs after 1 hour of LIF stimulation………………………………………………..…37
Figure 1.2.15 Long-term self-renewal of STAT3
̶ / ̶ + DD-STAT3-S727A mESCs under
mESC+LIF+S1 condition………………………………………………………………..38
Figure 1.2.16 Proliferation rates of STAT3
-/-
+ DD-STAT3-WT and STAT3
-/-
+ DD-
STAT3-S727A mESCs in mESC+LIF+S1 and N2B27+2i………………………...……39
Figure 1.2.17 Substrate attachment assay of STAT3
-/-
+ DD-STAT3 mESCs..................40
Figure 1.2.18 TUNEL-FITC staining of different STAT3
-/-
+ DD-STAT3 mESCs in
mESC+LIF+S1 condition……………………………………………………………..…41
IX

Figure 1.2.19 The transcription level of Myc among different STAT3
̶ / ̶ + DD-STAT3
mutant cell lines in mESC+S1 after 1 hour of LIF stimulation………………………….42
Figure 1.2.20 Improved proliferation of STAT3
-/-
+ DD-STAT3-S727A mESCs with
forced expression of c-myc…………………………………………………………...….43
Figure 1.2.21 Western blot analysis showing differential expression of core pluripotency
factors among STAT3
̶ / ̶
+ DD-STAT3 mESCs………………………………………….44
Figure 1.2.22 Differential transcription of STAT3 target genes among STAT3
̶ / ̶
+ DD-
STAT3 mESCs after 1 hour of LIF stimulation……………………………………….…46
Figure 1.2.23 Immuno-fluorescence staining of different STAT3
-/-
+ DD-STAT3 mESCs
after neural differentiation…………………………………………………………...…..48
Figure 1.2.24 RT-PCR analysis of induction of neural precursor genes in differentiation
of STAT3
-/-
+ DD-STAT3-WT and STAT3
-/-
+ DD-STAT3-S727A cells…………...…50
Figure 1.2.25 Immuno-fluorescence staining of STAT3
-/-
+ DD-STAT3-S727D mESCs
after neural differentiation……………………………………………………………….50
Figure 1.2.26 Quantitative comparison of the pixel intensity of Nestin and Tuj1 staining
among different STAT3
-/-
+ DD-STAT3 mESCs…………………………………..……51
Figure 1.2.27 Phosphorylation of STAT3 S727 in mESCs was specifically inhibited by
PD0325901………………………………………………………………………………52
Figure 1.2.28 PD0325901 blocked serum-, but not LIF-, induced phosphorylation of
S727 in mESCs……………………………………………………………………..……53
Figure 1.2.29 AP staining and morphological observation of STAT3
-/-
+ DD-STAT3
mESCs in mESC+LIF+S1 condition with or without PD0325901………………….…..56
X

Figure 1.2.30 PD0325901 can augment Nanog expression in STAT3
-/-
+ DD-STAT3-
S727A mESCs……………………………………………………………………….…..57
Figure 1.2.31 Illustration of mESCs and mEpiSCs, their culture conditions, pluripotency
states, and developmental origins……………………………………………………..…58
Figure 1.2.32 Schematic picture showing the difference of GCSF/GRgp-Y118F and LIF
in mEpiSC reprogramming potency……………………………………………….…….61
Figure 1.2.33 Microscopic pictures of different STAT3
̶ / ̶ + DD-STAT3 mEpiSCs (p12)
cultured in mESC+FGF+Activin A.……………………………………………….…….62
Figure 1.2.34 RT-PCR analysis of gene expression profile of different STAT3
̶ / ̶ + DD-
STAT3 mEpiSCs………………………………………………………….……………..62
Figure 1.2.35 Morphology of STAT3
-/-
+ DD-STAT3 mEpiSCs in N2B27+S1 medium
on day 11 with or without administration of LIF……………………………….………..64
Figure 1.2.36 Relative expression of fgf5 and Rex1 in STAT3
-/-
+ STAT3-S727A cells
after being subject to N2B27+LIF+S1 reprogramming condition for 11 days……..……64
Figure 1.2.37 Morphology of mESC-like clones picked from reprogrammed STAT3
-/-
+
DD-STAT3-S727A d11 cells at different passages……………………………….……..65
Figure 1.2.38 RT-PCR analysis of fgf5 and Rex1 expression in a representative clone of
STAT3
-/-
+ STAT3-S727A d11 mESC-like cells (passage 6)………………..…………65
Figure 1.2.39 Opposing functions of STAT3 Y705 and S727 phosphorylation in mEpiSC
reprogramming………………………………………………………………..………….66
Figure 1.2.40 Proposed model for the role of differential STAT3 phosphorylation in
mESC fate determination…………………………………………………………….…..68
Table 1 Similarities and differences between mouse ESCs and EpiSCs…………..…….59
XI

Figure 2.1.1 ZFN-mediated knockout technology in the rat…………………….……….92
Figure 2.1.2 ESC-based gene-targeting in the rat………………………………………..97
Figure 2.2.1 Flowchart outlining how to generate gene knockout rats step by step…....100
Figure 2.2.2 Rat ESCs derived and maintained in the 2i condition………………….…102
Figure 2.2.3 Luciferase assay showing that the PGK promoter activity is much lower than
that of the CAG promoter in rat ESCs…………………………………………….……104
Figure 2.2.4 Schematic diagram showing the strategy for constructing a rat gene-targeting
vector……………………………………………………………………………………106
Figure 2.2.5 Amplification of genomic sequence of rat Oct4…………..………………107
Figure 2.2.6 Schematic diagram showing the gene-targeting strategy of generating Oct4-
GFP knockin and conditional knockout rats……………………………………………108
Figure 2.2.7 Flow chart of a modified method to pick up rat ESC colonies after
electroporation and drug selection……………………………………...………………111
Figure 2.2.8 PCR screening of gene-targeted rat ESC colonies after electroporation and
drug selection…………………………………………………………………………...113
Figure 2.2.9 A GFP-positive colony of gene-targeted rat ESCs selected from PCR
screening……………………………………………………………………………..…113
Figure 2.2.10 PCR amplification of the long and short homology arm from the selected
Oct4-GFP targeted rat ESC colony……………………………………………………..114
Figure 2.2.11 Sequence analysis of the selected Oct4-GFP targeted rat ESC colony….115
Figure 2.2.12 Oct4-GFP targeted rat ESCs differentiate and lose GFP fluorescence after
Cre-mediated Oct4 deletion…………………………………………………………….116
XII

Figure 2.2.13 Karyotyping and subcloning of correctly targeted rat ESCs………….....117
Figure 2.2.14 Production of gene-targeted knockout rats……….……………………...118
Figure 2.2.15 Gene-targeting design of the Rosa26 Cre-ERT2 knockin rat……………121

1

Part I: Role of STAT3 phosphorylation in mouse embryonic
stem cell self-renewal and differentiation
Abstract
STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine
727 residues. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF)
signaling maintains pluripotency by inducing JAK-mediated phosphorylation of STAT3
Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs
remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in
regulating mESC identities, using a small molecule-based system to post-translationally
modulate the quantity of transgenic STAT3 in STAT3
-/-
mESCs. We demonstrated that
pY705 is absolutely required for STAT3-mediated mESC self-renewal, while pS727 is
dispensable, serving only to promote proliferation and optimal pluripotency by enhancing
STAT3 transcription activity. S727 phosphorylation is regulated directly by FGF/Erk
signaling, a differentiation cue in mESCs, and is crucial in the transition of cells from
pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in
significantly reduced neural differentiation potential, which could be recovered by a S727
phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem
cells to naïve pluripotency, suggesting the opposing functions of STAT3 pY705 and
pS727 in reprogramming. Together, these data demonstrated a dynamic equilibrium of
STAT3 pY705 and pS727 in the control of mESC fate.
2

Introduction
JAK-STAT3 signaling
Signal transducer and activator of transcription 3 (STAT3) belongs to a family of several
proteins (STATs) that have critical functions in various cytokine-driven signaling
pathways (Bromberg and Darnell, 2000). STAT3 is a functionally latent cytoplasmic
transcription factor containing the following domains: tetramerization, coiled coil
(protein interaction), DNA binding, linker, dimerization, and transcriptional activation
domains (Figure 1.1.1) (Buettner et al., 2002). Upon binding of cytokines to their
respective receptors on the cell surface, receptors dimerize and activate associated
tyrosine kinases such as family of Janus kinases (JAKs). Subsequently, JAKs
phosphorylate the tyrosine residues within the cytoplasmic tail of cytokine receptors,
which in turn provide the critical docking site for recruitment of cytoplasmic STAT3
monomer via its SH2 domain, and then the recruited STAT3 molecules become
themselves substrates for JAK-mediated phosphorylation (tyrosine 705) (Wang et al.,
2012). Phosphorylated STAT3 dimerize through reciprocal SH2 interaction, and
translocate into the nucleus, where the homodimers activate target gene transcription
(Sasse et al., 1997). This JAK/STAT3 canonical pathway represents one of the most
common mechanisms of how extracellular signaling proteins regulate gene transcription
within the cell (Figure 1.1.2), and control cell behaviors (Levy and Darnell, 2002).

3

Figure 1.1.1 Different functional domains of STAT3 molecule (modified from (Buettner
et al., 2002)).

Figure 1.1.2 Canonical JAK-STAT3 pathway (modified from (Levy and Darnell, 2002)).

STAT3 phosphorylation
STAT3 can be phosphorylated at two important amino acid residues: tyrosine 705 (Y705)
and serine 727 (S727). Phosphorylation of tyrosine 705 (pY705) by JAKs is generally
believed essential for its dimerization, nuclear translocation, DNA binding, and gene
transcription (Aggarwal et al., 2009). On the contrary, the function of phosphorylated
serine 727 (pS727) remains controversial: while it was previously documented that
4

pS727 is a secondary event after pY705 and only enhances STAT3 transcriptional
activity (Lufei et al., 2007; Wen et al., 1995; Yokogami et al., 2000), more and more
evidences are now suggesting pS727 actually plays a more complex role than originally
expected. For example, several studies have demonstrated that pS727 can activate
STAT3 transcription independent of pY705 in various types of cells (Androutsellis-
Theotokis et al., 2006; Hazan-Halevy et al., 2010; Liu et al., 2003; Qin et al., 2008;
Sakaguchi et al., 2012). In addition, pS727 may also direct STAT3 to mitochondria to
regulate cellular respiration, a new function that differs from the canonical role of STAT3
as a transcription factor (Gough et al., 2009; Wegrzyn et al., 2009). Thus, it is very likely
that the exact function of STAT3 pY705 and pS727 varies among different types of cells,
as well as the environments these cells are exposed to.

Biological functions of STAT3
Activated by various types of cytokines, growth factors, oncogenes, carcinogens, and
viruses, STAT3 has been associated with inflammation, cellular transformation,
development, differentiation, survival, proliferation, invasion, angiogenesis, and cancer
metastasis (Aggarwal et al., 2009). Numerous studies have identified constitutive STAT3
activation in a wide variety of cancer cell lines and primary tumors, which is often a
consequence of persistent activity of associated tyrosine kinases including Src, epidermal
growth factor receptor (EGFR) and JAKs (Buettner et al., 2002). STAT3 regulates the
expression of many genes that are directly responsible for tumor progression. For
example, STAT3 up-regulates cyclin D, c-myc, fos and jun to promote proliferation
(Hirano et al., 2000; Masuda et al., 2002), bcl-xl and survivin to escape apoptosis (Aoki
5

et al., 2003), matrix metalloproteinases (MMPs) to enhance tumor invasion (Kesanakurti
et al., 2013; Zhang et al., 2009), vascular endothelial growth factor (VEGF) to support
tumor angiogenesis (Niu et al., 2002), and also immune suppressive factors such as
interleukin 10 (IL-10) and transforming growth factor-beta (TGFβ) to evade immune
surveillance. And these factors together contribute greatly to chemo-resistance and radio-
resistance of tumor cells. Therefore, STAT3 has been proposed as a promising tumor
therapeutic target and many molecular approaches for STAT3 inhibition are now been
investigated extensively for potential clinical applications.

Interestingly, several investigations have also revealed novel STAT3 functions that raise
the necessity of revisiting the supposed oncogenic role of STAT3. Chapman and
colleagues took advantage of a conditional STAT3 knockout mouse model to show that
STAT3 actually promotes mammary epithelium apoptosis in vivo (Chapman et al., 2000;
Chapman et al., 1999). Another study reported an anti-proliferative role of STAT3 in IL-
10 stimulated macrophages (O'Farrell et al., 1998). In terms of immune response, STAT3
can even exert opposing functions within the same cell type when different stimulating
cytokines are involved (Hutchins et al., 2013a). In dendritic cells, STAT3 could be
activated by stimulation of IL-6 or IL-10, and serves as a pro- or anti-inflammatory factor,
respectively (Braun et al., 2013). Importantly, there have been evidences that support the
unexpected tumor suppressive function of STAT3 in glioblastoma pathogenesis (de la
Iglesia et al., 2008a; de la Iglesia et al., 2008b). And Couto and colleagues also
demonstrated that STAT3 is a negative regulator of thyroid tumorigenesis (Couto et al.,
2012). These observations together highlighted the extreme complexity of STAT3
6

biological functions, and thus called for a better understanding of the underlying
mechanisms and contexts involved.

Recent technological advances in epigenetics and bioinformatics have provided an
unprecedented opportunity to unravel how a pleiotropic transcription factor can drive a
diversity of biological processes (Hutchins et al., 2013a). Using publicly available ChIP-
seq data, Hutchins and colleagues compared the genome-wide binding patterns of STAT3
in four different cell types: embryonic stem cells (ESCs), CD4
+
T cells, macrophages,
and AtT-20 corticotroph cells (Hutchins et al., 2013b). Their findings suggest that
STAT3 utilizes a universal set of transcriptional regulatory modules, in association with
E2F1 and myc, to fine-tune the activation level of JAK-STAT3 and promote proliferation.
But more importantly, STAT3 also recognizes distinct regulators to determine the cell-
type specific functions in different cells. For example, STAT3 coordinates with factors
including Oct4, Sox2, Klf4 to maintain pluripotency in ESCs, yet in CD4
+
T cells forms
protein complex with multiple GATA factors that are important in T cell development.
These findings establish that STAT3 recruits specific regulators and activates distinct
gene expression programs based on the genetic background, type and developmental
stage of the cell (Figure 1.1.3). Moreover, if we proceed to study the formation of
specific gene activation complexes and the subsequent involvement gene regulatory
elements, the exact role of STAT3 pY705 and pS727 in a given cellular context must be
examined meticulously due to their great significance in providing docking sites for
binding partners as well as determining DNA binding affinities for transcriptional
regulation (Durant et al., 2010; Langlais et al., 2012).
7

Figure 1.1.3 The proposed model explaining how STAT3 coordinate with distinct gene
regulators to achieve diverse cellular functions (adopted from (Hutchins et al., 2013b)).

STAT3 in mouse embryonic stem cell self-renewal: a context-specific study
Mouse embryonic stem cells (mESCs) are cell populations derived from the inner cell
mass (ICM) of an embryonic day 3.5 mouse blastocyst (Evans and Kaufman, 1981;
Martin, 1981). Under appropriate in vitro culture conditions, ESCs proliferate indefinitely
without differentiation, a property hereinafter referred to as “self-renewal”; and at the
same time retain the developing potential to generate nearly all cell types that originate
from the three primary germ layers endoderm, mesoderm and ectoderm, termed
“pluripotency” (Smith, 2001). Historically mESCs were maintained in co-culture with
mitotically inactivated feeder fibroblasts (Evans and Kaufman, 1981; Martin, 1981), or in
Buffalo rat liver cell-conditioned medium (Smith and Hooper, 1987), yet later efforts in
pinpointing the active component(s) in conditioned medium identified a single cytokine,
leukemia inhibitory factor (LIF), which supported self-renewal of mESCs from 129 strain
8

of mice in the absence of feeder cells (Smith et al., 1988; Williams et al., 1988). On the
contrary, LIF withdrawal resulted in rapid differentiation of mESCs into a mixed
population of mesoderm and endoderm cells (Niwa et al., 1998).

Interestingly, LIF belongs to the well-characterized IL-6 family of cytokines that mediate
inflammation, immune responses, haematopoiesis, neuronal regeneration and embryonic
development (Heinrich et al., 2003). LIF initiates signaling cascade by binding to a low
affinity LIF receptor (LIFR) in association with a common IL-6 family co-receptor
subunit glycoprotein 130 (gp130). The LIF-LIFR-gp130 trimeric complex recruits
receptor-associated kinases JAKs and subsequently STAT3 as described above (Figure
1.1.2). And it has been demonstrated that STAT3 activity is essential for LIF-mediated
mESC self-renewal and its constitutive activation replaces LIF-dependency (Matsuda et
al., 1999; Niwa et al., 1998).

STAT3 coordinators in mESC self-renewal
Apparently, neither LIF cytokine nor JAK-STAT3 pathway functions exclusively in stem
cell fate regulation. Thus, as described in Fig. 1.1.3, this conventional signal transduction
pathway must coordinate with other regulators to initiate mESC-specific transcription
programs for self-renewal, a function required only in stem cells. The identification of
these STAT3 coordinators and downstream target genes will greatly improve our
understanding of the underlying mechanisms.

9

Oct4, Sox2, and Nanog are the core transcriptional regulatory factors in mESC self-
renewal (Boyer et al., 2005). Oct4 is expressed in the embryo throughout the pre-
implantation period, and re-appears in germ cell precursors of adult mice (Kehler et al.,
2004). Oct4-deficient embryos survive the morula stage, but cannot form the ICM and
also fail to give rise to ESC colonies in vitro, indicating its necessity in stem cell
maintenance (Nichols et al., 1998). It binds to the octamer motif (5'-ATGCAAAT-3') of
DNA to control a number of genes involved in pluripotency, and in many cases works in
partnership with Sox2 (Loh et al., 2006). Oct4 is also one of the transcription factors used
to create induced pluripotent stem cells (iPSCs), together with Sox2, Klf4 and c-myc in
mouse, rat and human, demonstrating its capacity to induce an embryonic stem cell-like
state (Hamanaka et al., 2011; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu
et al., 2007). Interestingly, Oct4 expression level must be precisely regulated as both too
much and too little of Oct4 cause ESC differentiation (Niwa et al., 2000). Researchers
also found that mouse ESCs with reduced Oct4 expression by heterozygosity showed
increased genome-wide binding of Oct4, particularly at pluripotency associated
enhancers, more homogeneous expression of pluripotency factors, and enhanced self-
renewal (Karwacki-Neisius et al., 2013). In addition, Oct4 is not expressed exclusively in
the epiblast and therefore itself alone does not suffice pluripotency specification (Nichols
and Smith, 2009). These observations together bring more complexity in the exact role of
Oct4 in self-renewal.

Sox2 belongs to Sox family of transcription factors that have a highly conserved HMG
(high-mobility group) DNA-binding domain. Sox2 expression is widely distributed in the
10

developing embryo, including ICM, epiblast, neural tissues and extra-embryonic
ectoderm. Sox2-null embryos die immediately after implantation (Avilion et al., 2003;
Wood and Episkopou, 1999). Sox2 is essential for ESC self-renewal and pluripotency as
knockdown or conditional deletion of Sox2 results in trophoblast differentiation (Ivanova
et al., 2006; Masui et al., 2007). This phenotype is similar to that caused by Oct4 deletion
because Sox2 often acts as a heterodimer with Oct4 to regulate transcription of genes
such as fgf4 (Yuan et al., 1995), Nanog (Kuroda et al., 2005), lefty1 (Nakatake et al.,
2006) and themselves (Okumura-Nakanishi et al., 2005; Tomioka et al., 2002).

Nanog functions in coordination with Oct4 and Sox2 to establish ESC identities. Nanog
expression level fluctuates greatly in mouse ESCs to contribute to population
heterogeneity (Kalmar et al., 2009; MacArthur et al., 2012). Over-expression of Nanog in
mouse ESCs stabilizes an undifferentiated state by constitutively conferring self-renewal
independent of growth factors or small molecules (Chambers et al., 2003; Mitsui et al.,
2003; Ying et al., 2003a); and in human ESCs allows feeder-free propagation for multiple
passages (Darr et al., 2006). Nanog-null embryos appear to be able to initially give rise to
pluripotent cells, yet these cells immediately differentiate into the extra-embryonic
endoderm lineage (Chambers et al., 2007). Nanog knockdown assay in mouse and human
ESCs results in similar phenotypes (Chambers et al., 2003; Hyslop et al., 2005; Mitsui et
al., 2003), which could partially be explained by a negative regulation on primitive
endoderm-inducer Gata6 (Mitsui et al., 2003). Genome-wide mapping of Nanog binding
sites has identified many pluripotency genes including Esrrb, Rif1, Foxd3 and REST (Loh
et al., 2006). Among them Esrrb is proved to be a direct Nanog target (Festuccia et al.,
11

2012): over-expression of Esrrb in Nanog
−/−
ESCs led to cytokine-independent self-
renewal, while its deletion abolished the effect of Nanog over-expression. Interestingly,
Nanog is not strictly required in maintenance or establishment of pluripotency as shown
by the derivation of Nanog
−/−
ESCs (Chambers et al., 2007) and iPSCs from Nanog
−/−

somatic cells (Carter et al., 2014; Schwarz et al., 2014). Oct4 has been reported to form a
heterodimer with Sox2, and bind to Nanog promoter region to activate transcription
(Rodda et al., 2005). Together these three factors co-occupy the promoter regions of
many other genes important for pluripotency (Boyer et al., 2005).

Using high-throughput ChIP-seq technologies, Chen and colleagues expanded the scope
to map the genomic occupation of 13 sequence-specific pluripotency factors, and
identified a protein cluster containing Nanog, Oct4, Sox2, Smad 1 and STAT3 (Chen et
al., 2008). The readouts show that 56.8% of STAT3 binding sites are associated with the
Oct4-Sox2-Nanog core factor-binding loci, and they share many common regulatory
targets including Klf4, Esrrb, c-myc, and Tcfcp2l1. This observation provides direct
evidence that LIF signaling supports self-renewal by strengthening core pluripotency
circuitry. Further analysis also revealed the existence of a synergistic transcriptional
activation among these pluripotency factors: in RNAi experiment, Oct4 depletion
compromises STAT3 binding ability, suggesting the pivotal role of Oct4 in stabilizing the
regulatory complex for transcription. These results together offer substantial insights on
how extrinsic LIF/STAT3 signaling is integrated into nuclear core pluripotency factors to
support mESC self-renewal (Figure 1.1.4).

12

Figure 1.1.4 An illustration of promoter co-occupation of STAT3 and other transcription
factors in mESCs. STAT3 was thus integrated into the core pluripotency circuitry to
support self-renewal (adopted from (Chambers and Tomlinson, 2009)).

STAT3 downstream targets in mESC self-renewal
A number of studies have been carried out to identify STAT3 downstream target genes as
potential candidates for key pluripotency factors (Bourillot et al., 2009; Cinelli et al.,
2008; Sekkai et al., 2005; Snyder et al., 2008; Xie et al., 2009). For example, STAT3
directly regulates the expression of myc transcription factor, and sustained expression of
myc supports mESC self-renewal in absence of LIF (Cartwright et al., 2005). myc and
STAT3 also co-occupies the promoter regions of many genes that are highly enriched in
mESCs, suggesting the existence of feed-forward loops for signal amplification (Kidder
et al., 2008). Pramel7 serves downstream of LIF/STAT3 as a non-transcription factor that
maintains pluripotency by suppressing extracellular signal-regulated kinase 1/2 (Erk1/2)
phosphorylation. RNAi knockdown of Pramel7 resulted in mESC differentiation,
13

whereas its over-expression partially liberated mESCs from LIF dependency (Casanova
et al., 2011). Among Krueppel-like family factors, Klf4 and Klf5, but not Klf2, are direct
downstream targets of LIF/STAT3 (Hall et al., 2009). In mESCs, Klf4 is primary
activated by JAK/STAT3, which in turn induces the expression of Sox2 and Nanog to
collectively promote self-renewal (Niwa et al., 2009). In addition, Klf4 is also one of the
first four transcriptional factors used to create iPSCs, indicating its significance in
pluripotency factor network (Takahashi et al., 2007; Takahashi and Yamanaka, 2006).

Importantly, besides the STAT3 signaling cascade, recruitment of JAKs to LIFR/gp130
receptor also results in the activation of two additional pathways: the mitogen-activated
protein kinase (MAPK) /Erk and the phosphatidylinositol 3-kinase (PI3K) /protein kinase
B (Akt) (Figure 1.1.5) (Graf et al., 2011). Therefore, Genes that are actively transcribed
upon LIF stimulation of mESCs are not necessarily STAT3 downstream targets. This
concern was resolved by using GRgp-Y118F mESCs in which only STAT3-specific
targets could be selectively activated (Burdon et al., 1999a). As a result, Gbx2, a novel
pluripotency regulator downstream of STAT3, was recently discovered (Tai and Ying,
2013). Forced expression of Gbx2 replaced the requirement of STAT3 activation in
mESC self-renewal. Gbx2 has also been proved to be able to boost up iPSC
reprogramming efficiency when used in combination with Yamanaka factors (Oct4, Sox2,
Klf4, c-myc). Furthermore, similar to Klf4, over-expression of Gbx2 alone was sufficient
to reprogram mouse epiblast stem cells (mEpiSCs) back to mESCs, a ground state of
pluripotency (Guo et al., 2009; Tai and Ying, 2013).

14

Figure 1.1.5 LIF activates STAT3, MAPK and PI3K/Akt pathways in mESCs (adopted
from (Graf et al., 2011)).

Through comparative transcriptome analysis intersected with genome location data, two
separate groups of investigators hit the same target by discovering a novel pluripotency
factor Tfcp2l1 that acts downstream of LIF/STAT3 to sustain self-renewal (Martello et
al., 2013; Ye et al., 2013). Its expression is abundant in mouse ICM and ESCs, while is
silenced abruptly after differentiation into epiblast or EpiSCs. Tcfcp2l1 is hardwired into
core pluripotency factor network through activation of Nanog and Tbx3, and its function
is further enhanced by Oct4, Sox2 and Esrrb. This observation provided a major
connection between extrinsic LIF/STAT3 signaling and intrinsic core pluripotency
circuitry because LIF does not directly regulate these factors (Figure 1.1.6). Many
experiments have been performed to confirm this hypothesis: Tfcp2l1 over-expression
15

supported mESC self-renewal in absence of LIF/STAT3 signaling; its knockdown
impaired self-renewal; and it largely recapitulated the contribution of LIF to EpiSC
reprogramming. Tfcp2l1 is highly expressed in the inner cell mass of human blastocysts,
but is significantly down-regulated during derivation of human ESCs (O'Leary et al.,
2012), and up-regulated during generation of naive state human ESCs by introducing
Klf2+Klf4 or Klf4+Oct4 (Hanna et al., 2010). Tfcp2l1 may therefore play an important
role in establishing and maintaining naïve pluripotency by acting downstream of
LIF/STAT3. However, it should be noted that the self-renewal promoting effect of
LIF/STAT3 through its downstream targets is also regulated by the total activation level
of STAT3 (Figure 1.1.6). We have recently discovered that insufficient STAT3 activation
failed to prevent differentiation into meso/endoderm cells, yet STAT3 signaling overload
led to mESC crisis and differentiation into trophoblast lineage as well (unpublished data).

Figure 1.1.6 LIF/STAT3 signaling must be activated at an appropriate level so that the
direct downstream targets can contribute to core pluripotency network regulation.
16

2i condition: the ground state of mESC self-renewal
Significant efforts have been put into further identification of active components in the
established “serum+LIF” culture condition for mESCs. One of the motivations came
from an urgent need of directed mESC differentiation for research and therapeutic
purposes (Ying and Smith, 2003). Therefore, it is essential to establish a refined serum-
free, as opposed to a complex multi-factorial, condition in which the role of each
component is clarified (Wray et al., 2010). The very first success was to use bone
morphogenetic protein 4 (BMP4) to replace serum (Ying et al., 2003a). Thus LIF and
BMP together constitute the first defined medium for mESC maintenance: the former
primarily inhibits meso-endoderm differentiation, while the latter prevents neuro-
ectoderm differentiation.

On the other hand, studies have shown that the autocrine fibroblast growth factor 4
(FGF4)/Erk signal is an active differentiation-driving force in mESCs (Kunath et al.,
2007; Stavridis et al., 2007). And interestingly, neither LIF nor serum/BMP blocks Erk
activation (Ying et al., 2003a). This observation offered a new perspective for mESC
self-renewal, which is to block intrinsic differentiating-inducing momentum rather than
to introduce extrinsic signal stimuli. Indeed, the addition of small molecular inhibitors
against FGF/Erk signaling (SU5402 and PD184352, or PD0325901 alone) resulted in
suppressed differentiation of mESCs, suggested by their continued self-renew for several
passages after LIF withdrawal (Ying et al., 2008). However, those cells showed poor
clonogenicity due to compromised growth and viability. This defect could be rescued by
using a glycogen synthase kinase-3 (GSK3) inhibitor CHIR99021, which consolidates
17

pluripotency by enhancing metabolic and biosynthetic ability and thus overall viability of
mESCs. In addition, optimization in basal culture medium led to the application of the
serum-free N2B27 medium to best support mESC growth nutritionally. Together, a new
chemically defined culture condition (3i: CHIR99021, SU5402 and PD184352 or 2i:
CHIR99021 and PD0325901, in N2B27 medium) was developed (Figure 1.1.7) (Ying et
al., 2008). 2i or 3i culture represents a true “stemness” condition that not only supports
derivation of ESC lines from completely recalcitrant mice strains, but also from the
species of rat (Buehr et al., 2008; Li et al., 2008; Nichols et al., 2009). This demonstrates
a conserved mechanism governing pluripotency in rodents.

Figure 1.1.7 3i or 2i condition represents the ground state of mESC self-renewal that does
not require extrinsic stimuli (adopted from (Huang et al., 2011)).

18

Notably, it was demonstrated that 3i or 2i condition does not activate STAT3 signaling
(Ying et al., 2008). To assess definitively whether STAT3 activation is required for
mESC self-renewal, STAT3
-/-
embryos were harvested by crossing STAT3 heterozygous
mice and cultured in 2i. STAT3
-/-
mESCs were subsequently isolated and they grew
robustly in 2i condition with a typical mESC morphology that was indistinguishable from
wild-type mESCs. When transferred to serum+LIF conventional mESC culture medium,
STAT3
-/-
cells rapidly differentiated and died, confirming the necessity of STAT3
activation for self-renewal in serum condition. Together, these data conclude that the
N2B27+2i condition supports mESC self-renewal by circumventing the otherwise
obligatory STAT3 pathway and thus enables the establishment of STAT3
-/-
mESCs (Ying
et al., 2008). These two distinct mESC culture conditions have thus offered us a unique
opportunity to interrogate STAT3 function in detail.

Rationale and experimental design of this study
STAT3 signaling pathway is activated in response to various extracellular cytokines and
growth factors and exerts diverse biological functions in the cell (Aggarwal et al., 2009).
High throughput genomic studies using ChIP-seq have provided significant insights on
the underlying mechanisms (Hutchins et al., 2013b). It has been proposed that STAT3
recruits different regulatory modules to activate cell type-specific gene expression
programs (Figure 1.1.3). STAT3 itself is also subject to a variety of post-translational
modifications (Hutchins et al., 2013a), among which phosphorylation is particularly
interesting for its potential role in transcription activation, DNA-binding affinity, and
association with distinct coordinators.
19

STAT3 plays an indispensable role in LIF-mediated mESC self-renewal. LIF treatment
results in the activation of the JAK/STAT3 pathway, and subsequently the expression of
pluripotency genes (Niwa et al., 1998; Raz et al., 1999). Phosphorylation of Y705 is
believed to be the key event in the transcriptional activation of STAT3. Phosphorylation
of S727, however, has not been functionally linked to any STAT3-mediated mESC fate
determination. In this study, we sought to investigate the exact function of STAT3
phosphorylation (STAT3 pS705 and pS727) in a cell-specific context: the self-renewal
and differentiation of mESCs. This work will have long-term and far-reaching
implications for our basic understanding of stem cell biology, which is critical to the
future of regenerative medicine.

Previous attempts in dissecting STAT3 function in mESCs were hampered due to the
necessity of STAT3 activation in maintaining pluripotency. Any disturbance in STAT3
signaling, e.g., loss-of-function assays, in mESCs may alter cell identities and lead to
confounding phenotypes. On the other hand, over-expression of STAT3 mutant proteins
in wild-type mESCs lacks physiological relevance. Fortunately, our recently developed
N2B27+2i medium enabled the generation of STAT3
-/-
mESCs, which allows fine
manipulation of STAT3 signaling and provides a powerful tool for investigating how
STAT3 functions in mESCs (Ying et al., 2008).

We created various transgenes carrying different STAT3 phosphorylation mutations and
introduced them into STAT3
-/-
mESCs. We obtained the stable transgenic cell lines and
maintained them in N2B27+2i condition. Upon experimentation, however, we could
20

manipulate the STAT3 signaling pathway and functionally test the phenotypic outcomes
by transferring these cells to STAT3-dependent culture condition (serum+LIF) (Figure
1.1.8). This experimental design precludes the influence of endogenous STAT3, and the
resultant phenotypes will solely depend on the expression of STAT3 phosphorylation
mutant transgenes. Using this unique system, we demonstrated the presence of a dynamic
equilibrium of STAT3 Y705 and S727 phosphorylation in controlling mESC identities.

Figure 1.1.8 The experimental design of investigating the role of STAT3 phosphorylation
in mESC self-renewal and differentiation.

21

Materials and methods
mESC cell culture. 46C ESCs, R1 ESCs and STAT3
̶ / ̶ mESCs were cultured on plates
pre-coated with 0.1% gelatin or mitotically inactive mouse embryonic fibroblasts (MEFs).
46C mESCs and R1 mESCs were grown in Glasgow Minimum Essential Medium
(GMEM, Sigma) supplemented with 10% Fetal bovine serum (FBS, Hyclone), 2 mM L-
glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), 0.1 mM β-mercaptoethanol (a formulation hereinafter referred to as “mESC
medium”), and 1000 Units/ml murine LIF (Stemgent). STAT3
̶ / ̶
mESCs were grown in
N2B27 medium supplemented with 3 µM CHIR99021 and 1 µM PD0325901 as
previously described (Tong et al., 2011). CHIR99021 and PD0325901 were synthesized
in Division of Signal Transduction Therapy, University of Dundee, UK. C-myc inhibitor
10058-F4 was purchased from EMD Millipore and used at the concentration of 50 µM.

Preparation of N2B27 medium
To make 10 ml of 100× N2 stock, 0.67 ml of 75 mg/ml BSA, 33 µl of 0.6 mg/ml
progesterone solution, 100 µl of 160 mg/ml putrescine solution, 10 µl of 3 mM sodium
selenite solution, 1 ml of 100 mg/ml apo-transferrin, and 1 ml of 10 mg/ml insulin were
added into 7.187 ml DMEM/F12 medium. DMEM/F12-N2 medium was prepared by
adding 1 ml of 100× N2 stock into 100 ml of DMEM/F12. To make neurobasal/B27
medium, 2 ml of B27 and 0.5 ml of 200 mM L-glutamine were added into 100 ml of
neural basal medium. N2B27 medium was subsequently made by mixing DMEM/F12-
N2 with neurobasal/B27 medium at a ratio of 1:1, with the addition of β-mercaptoethanol
to the final concentration of 0.1 mM.
22

Transgenesis of STAT3 mutant cell lines. Various STAT3 mutant genes were
generated using the QuikChange® Site-Directed. Mutagenesis Kit according to
manufacturer’s instructions (Stratagene), and fused with the destabilizing domain (DD) at
the STAT3 N-terminus. The DD-STAT3 mutants were then cloned into a CAG-ires-puro
vector between the XhoI and NotI sites. Plasmids were electroporated into STAT3
̶ / ̶
mESCs. Transfected STAT3
̶ / ̶ mESCs were plated onto drug-resistant DR4 feeder cells,
and allowed to propagate for two days before puromycin (1 µg/ml, Invitrogen) selection
was initiated. Positive clones were expanded and transgene expression was confirmed by
western blotting. Mutant cell lines were routinely maintained in N2B27+2i medium. For
c-myc over-expression, the coding region of c-myc was cloned and inserted into the
PiggyBac expression vector, which was later transfected together with transposase vector
into STAT3
̶ / ̶ + DD-STAT3-S727A cells.

Protein and RNA analysis. For protein analysis, cell lysis buffer was prepared by
adding 50 µl of β-mercaptoethanol to 950 µl of Laemmli Sample Buffer (BioRad) and
1000 µl of water. Cells were washed with PBS and lysed in the culture wells. Samples
were collected and heated to 95 °C before being subjected to electrophoresis on BioRad
Tris-HCl precast gels. Electrotransfer to PVDF membranes was performed and blots were
probed with primary antibodies overnight at 4 °C, and then for 1 hour at room
temperature with appropriate secondary antibodies. Pierce ECL Western Blotting
Substrate was used to detect the signal. The primary antibodies used include STAT3
(1:2000, BD Biosciences), P-STAT3-Y705 (1:2000, Cell Signaling), P-STAT3-S727
(1:2000, BD Biosciences), c-myc (1:500, Calbiochem), α-Tubulin (1:4000, Invitrogen),
23

Oct4 (1:3000, Santa Cruz), Sox2 (1:1000, Santa Cruz), Klf4 (1:1000, R&D Systems), and
Nanog (1;1000, R&D Systems). For RNA analysis, total RNA was isolated using the
RNeasy Mini Kit (Qiagen), and 1 µg of the corresponding RNA yield was used to
synthesize cDNA (QuantiTect Transcription Kit, Qiagen). The quantitative real-time
PCR mixtures were prepared using SYBR Green PCR Master Mix and run on an
ABI7900HT Fast Real-Time PCR System (Applied Biosystems). RT-PCR was carried
out by using the Paq5000 DNA polymerase (Agilent). Relative expression levels of
pertinent genes were analyzed and normalized against gapdh.

Self-renewal assays. To determine the self-renewal potential of STAT3 mutant cell lines,
we plated cells in 12-well plates at a density of 1000 cells/well and cultured them in
N2B27+2i medium overnight. Different culture conditions were applied the next day.
After 7 days, cells were washed, fixed and stained for alkaline phosphatase (AP) activity
in accordance with manufacturer’s instructions (Sigma). Colonies in each well were
counted and scored in three categories: undifferentiated, mixed, and differentiated. Each
treatment was performed in triplicate. For long-term tests of self-renewal, cells were
plated at a density of 1.2 × 10
4
cells/cm
2
in 12-well plates and cultured for at least six
passages before morphological observation.

Neural differentiation and immunostaining. To induce neural differentiation, we
trypsinized, collected by centrifugation, and resuspended 3× 10
6
-5× 10
6
mESCs in 10 ml
of mESC medium with 1 µM of Shield1 (S1, Clontech). This single-cell suspension was
deposited onto a 10 cm non-adhesive bacteriological petri-dish. After 2 days in this
24

condition, cells aggregated in suspension and formed embryoid bodies (EBs). Medium
was changed every other day and retinoic acid (RA, sigma) was added to a final
concentration of 1 µM for days 4-8. On day 9, EBs were collected and trypsinized into a
single-cell suspension, which was plated into 2 wells of a BD Matrigel™ Basement
Membrane Matrix (BD Biosciences)-coated 12-well plate and cultured in the mESC+S1
condition. Immunostaining was performed according to a standard protocol, using
primary antibodies including Nestin (Santa Cruz, 1:200) and Tuj1 (Covance, 1:1000).
Alexa Flour fluorescent secondary antibodies (Invitrogen) were used at a dilution of
1:2000 and nuclei were stained with DAPI. For RT-PCR analysis, STAT3
̶ / ̶ + DD-
STAT3-WT and STAT3
̶ / ̶ + DD-STAT3-S727A cells were collected 0, 2, 4, 6, 8, 10 and
12 days after the onset of differentiation, for RNA isolation.

Cell proliferation assay. Cells were plated on feeders in 12-well plates at a starting
density of 2 × 10
4
cells/well in mESC medium supplemented with 1000 U/ml LIF and 1
µM of S1. After 24, 48, 72, or 96 hours, cells were harvested and counted under a
microscope. The experiment was performed in quadruplicate and paired student’s t tests
were used for statistical analysis. Doubling times were calculated by the software
available from www.doubling-time.com.

Kinase inhibitor assay. STAT3
̶ / ̶ + DD-STAT3-WT or R1 mESCs were plated in 12-
well plates at a density of 100,000 cells per well and starved in basal medium with S1
overnight. In the next morning, cells were pre-treated by different serine/threonine kinase
inhibitors for 2 hours, and subsequently stimulated with serum, LIF or serum+LIF for 1
25

hour. Cell lysates were then collected for western blot analysis. Inhibitors used in the
experiment include JNK inhibitor SP600125 (50 µM, Sigma), Erk 1/2 inhibitor
PD0325901 (1 µM), mTOR inhibitor rapamycin (100 nM, Calbiochem), CDK inhibitor
olomoucine (50 µM, Sigma), PI3K inhibitor LY294002 (50 µM, Calbiochem), PKC
inhibitor Go6983 (1 µM, Calbiochem) and JAK inhibitor 1 (5µM, Calbiochem).

26

Results
Establishment of inducible STAT3 expression system in STAT3
̶ / ̶ mESCs
To evaluate the role of STAT3 Y705 and S727 phosphorylation in regulating mESC self-
renewal, we introduced full-length STAT3 (STAT3-WT), STAT3β (an isoform lacking
55 amino acid residues in the C-terminal domain), STAT3-Y705F (a mutant in which
tyrosine 705 is replaced by phenylalanine), and STAT3-S727A (a mutant in which serine
727 is replaced by alanine) transgenes into STAT3
̶ / ̶ mESCs. Initially, we used the CAG
promoter to drive constitutive and uncontrolled expression of the transgenes. However,
we were unable to establish reliable STAT3
̶ / ̶ + STAT3-Y705F mESC lines using this
method. The majority of STAT3
̶ / ̶ mESCs transfected with STAT3-Y705F vector died
soon after the first passage, and the surviving colonies expressed the transgene only
weakly (Figure 1.2.1), suggesting that STAT3-Y705F has a cytotoxic effect in mESCs. In
contrast, STAT3
̶ / ̶ + STAT3-WT, β and S727A mESC lines could all be efficiently
generated. We also found that the expression level of STAT3-WT in STAT3
̶ / ̶ mESCs
greatly exceeded the endogenous level of STAT3 in wild-type 46C ESCs (Figure 1.2.2),
and that STAT3-WT-expressing STAT3
̶ / ̶ mESCs were able to self-renew in the absence
of LIF for several passages. These observations led us to surmise that the phenotypes
exhibited by these cell lines might lack physiological relevance. Therefore, we sought to
control the expression of the STAT3 mutants to obtain more meaningful data regarding
their functions in ESCs.
27

Figure 1.2.1 Cytotoxicity of STAT3-Y705F transgene in STAT3
̶ / ̶ mESCs. Various
transgene plasmids were transfected into STAT3
̶ / ̶ mESCs, followed by two weeks of
drug selection. Positive clones were expanded and then subject to western blotting
analysis using antibodies including STAT3, P-STAT3-Y705, P-STAT3-S727, α-Tubulin.
Wild-type 46C mESCs were used as positive control.

Figure 1.2.2 The expression level of STAT3-WT in STAT3
̶ / ̶ mESCs greatly exceeds the
endogenous level of STAT3 in wild-type 46C ESCs

28

We next adopted an inducible system, the ProteoTuner induced protein stabilization, to
achieve a functional regulation of STAT3 transgene expression (Banaszynski et al., 2006).
The sequences encoding various STAT3 transgenes (WT, Y705F and S727A) were
modified to include a N-terminal ligand-responsive destabilizing protein domain (DD).
The resulting DD-STAT3 fusion protein, after its translation in the cytoplasm, will be
readily degraded through proteasome pathway. This degradation can be prevented by
adding Shield1 (S1), a stabilizing ligand, to the medium. S1 binds specifically to DD and
protects DD-tagged proteins from degradation. This fast regulation acts directly on the
protein level, enables the accumulation of protein in hours, and prevents disturbance of
gene transcriptional control. As compared to other technologies that regulate mRNA
expression or stability, this method is more convenient, specific, and reversible.

Figure 1.2.3 The principle of ProteoTuner inducible system. (A) Diagram showing the
DD-STAT3 expression vector used in the following experiments. The 12kd DD sequence
was inserted into the N-terminus of STAT3 mutant transgene sequence using XhoI and
AgeI sites. (B) Schematic picture showing the inducible protein stabilization.

29

Using this system, we were able to establish STAT3
̶ / ̶ + DD-STAT3-Y705F mESC lines
that could be maintained in the 2i condition as robustly as other STAT3 transgenic lines.
They form round, compact, dome-shape mESC colonies on MEF feeder cells (Figure
1.2.4). When S1 was added to the culture medium, DD-STAT3 protein started to
accumulate in the cells and was dose-dependent to the amount of S1 used (Figure 1.2.5).
Immunofluorescence staining also confirmed a stable level of DD-STAT3 expression in
the presence of S1; in its absence, no DD-STAT3 leak expression was apparent. Next, we
tested if the STAT3 phosphorylation mutations were correctly introduced. As shown in
Figure 1.2.7, DD-STAT3-WT was phosphorylated at both S727 and Y705 in LIF-
stimulated STAT3
̶ / ̶
+ DD-STAT3-WT mESCs, whereas DD-STAT3-Y705F and DD-
STAT3-S727A were phosphorylated only at S727 and Y705 sites, respectively, in cells
expressing either of the corresponding transgenes, confirming site-specific loss of
phosphorylation potential.

Figure 1.2.4 STAT3
-/-
+ DD-STAT3 transgenic mESCs in N2B27+2i medium display
typical mESC morphology on feeder cells. Scale bar = 50 µm.
30

Figure 1.2.5 Dose-dependent modulation of DD-STAT3 protein level by Shield 1 (S1).
Cells were cultured in N2B27+2i medium and treated with various concentrations of S1
overnight. DD-STAT3-WT expression in STAT3
-/-
mESCs is depicted as an example.
The two bands in the STAT3 blot represent STAT3 from MEF feeder cells (lower band)
and DD-STAT3 fusion protein (upper band).

Figure 1.2.6 Immuno-fluorescence analysis of STAT3 (green) quantity modulated by S1.
DAPI stains the nuclei (blue). STAT3
̶ / ̶ + DD-STAT3-Y705F mESCs were treated with
or without S1 overnight before analysis. Scale bar = 50 µm.

31

Figure 1.2.7 Phosphorylation potential of STAT3
-/-
+ DD-STAT3 mutant mESCs.
STAT3
-/-
, DD-STAT3-WT, DD-STAT3-Y705F and DD-STAT3-S727A mESCs were
cultured in N2B27+2i+S1 medium and stimulated with LIF for 1 hour before lysates
were collected for immunoblotting with pSTAT3 antibodies.

The above results together demonstrated that we established a robust inducible system to
control STAT3 transgene expression in STAT3
-/-
mESCs cultured in N2B27+2i. Next, we
attempted to transfer these different mutant cell lines to a STAT3-dependent medium
(serum+LIF). Under this condition, STAT3 function would directly affect how these cells
behave and therefore contribute to the cellular phenotypes.

pY705 is absolutely required for STAT3-mediated mESC self-renewal
To investigate the exact roles of STAT3 S727 and Y705 phosphorylation in mESC fate
regulation, we performed functional rescue assays on different STAT3 transgenic lines.
We started by plating those cells at single cell density on feeder coated 12-well plates
(1000 cells/ well). And immediately after overnight culture in the N2B27+2i condition to
allow cell attachment, we exposed DD-transgenic STAT3
̶ / ̶ mESCs to three different
conditions: N2B27+2i, the positive control that supports self-renewal independent of
32

LIF/STAT3; mESC serum medium only, the negative control that fails to support self-
renewal; and mESC+LIF+S1, the experimental condition in which STAT3 is induced and
activated by LIF stimulation. We kept culturing the cells for 7 days, a time that allows
single pluripotent cell to form colonies if the condition is appropriate, and then performed
alkaline phosphatase (AP) staining that specifically marks self-renewing mESCs.

In mESC+LIF+S1 group, as expected, STAT3
̶ / ̶ mESC colonies started to collapse 3-4
days after the switch, and the peripheral cells gradually differentiated and died. On day 7,
there was barely any remaining mESCs in the well, as shown in both microscopic
observation and AP-staining assay (Figure 1.2.8, 1.2.9). In other groups, all cells grown
in N2B27+2i condition remained mESCs, confirming STAT3 is not required; while cells
grown in mESC-only medium fully differentiated (Figure 1.2.9). The introduction of
STAT3-WT was able to fully rescue this phenotype: STAT3
̶ / ̶ + DD-STAT3-WT mESCs
formed a substantial amount of colonies that tested positive for AP activity. STAT3
̶ / ̶ +
DD-STAT3-S727A mESCs were also able to self-renewal, despite fewer clones emerged
in the well as compared to STAT3
̶ / ̶ + DD-STAT3-WT mESCs. On the other hand,
STAT3
̶ / ̶ + DD-STAT3-Y705F mESCs failed to form AP-positive colonies in this
condition, consistent with previous reports that pY705 is indispensable for STAT3-
mediated self-renewal in mESCs (Niwa et al., 1998). STAT3
̶ / ̶ + DD-STAT3-Y705F
mESCs grew into highly differentiated, morphologically flat colonies consisting of
squamous epithelial-type cells within one passage and could not be further expanded in
the mESC+LIF+S1 condition (Figure 1.2.10).
33

Figure 1.2.8 STAT3
-/-
mESCs differentiate under mESC+LIF+S1 condition. Cells were
plated at 1000 cells per well of a 12-well feeder plate and allowed to grow for 7 days.
Top: microscopic observation; Bottom: AP staining assay. Scale bar = 50 µm.

Figure 1.2.9 Comparison of the self-renewal potential of different STAT3
-/-
+ DD-STAT3
cells under various culture conditions: N2B27+2i, mESC, and mESC+LIF+S1. Cells
were plated at 1000 cells per well of a 12-well feeder plate and allowed to grow for 7
days. We counted the numbers of differentiated, undifferentiated and mixed colonies
these mESCs formed in the mESC+LIF+S1 culture condition.
34

Figure 1.2.10 STAT3
̶ / ̶ + DD-STAT3-Y705F mESCs failed to form AP-positive colonies
in mESC+LIF+S1 condition, and those cells could not be maintained beyond one passage.
Scale bar = 50 µm.

As described in Figure 1.1.2, Y705 phosphorylation mediates both the dimerization and
transcriptional activation of STAT3. We next attempted to investigate its necessity in
each event. It was previously demonstrated that STAT3 monomers contact in the C-
terminus SH2 domains via the phosphorylation of STAT3 Y705 within (Figure 1.1.1).
The introduction of two cysteines at residues 662 and 664 in the SH2 domain thus may
allow the formation of sulfhydryl bonds between STAT3 monomers, and consequently
enable STAT3 auto-dimerization independent of Y705 phosphorylation (Bromberg et al.,
1999). The resulting STAT3 mutant (STAT3C) was also proved to render constitutively
transcriptional activation in absence of ligand binding. We created a DD-STAT3C vector
and introduced this transgene into STAT3
̶ / ̶
mESCs to examine the phenotype. When
using S1 to induce STAT3C expression, we noticed its strong self-renewal promoting
effect: STAT3
̶ / ̶ + DD-STAT3C cells remained in an undifferentiated state over many
passages even in the absence of LIF (Figure 1.2.11). This observation confirmed that
STAT3C induces an autonomous and constant activation of STAT3 signaling.
35

Figure 1.2.11 STAT3
̶ / ̶ + DD-STAT3C mESCs self-renew in absence of LIF. Cells were
plated at 1000 cells per well of a 12-well feeder plate and allowed to grow for 7 days
before AP staining assay.

We next asked whether STAT3 Y705 phosphorylation is truly required for transcriptional
activation. We generated STAT3
̶ / ̶ + DD-STAT3C-Y705F mESCs and exposed them to
mESC+S1+LIF condition: these cells could be maintained initially for a few days, but
died soon after being passaged, indicating a defect in self-renewal capacity (Figure
1.2.12). On the contrary, a transgene encoding a phospho-mimetic mutation (DD-
STAT3C-Y705D) supported long-term self-renewal in STAT3
̶ / ̶
mESCs (Figure 1.2.12).
Taken together, these data suggested that Y705 phosphorylation is still required for
STAT3-mediated self-renewal despite STAT3C endows auto-dimerization of STAT3
monomers.

To further support this conclusion, we examined the subcellular localization of DD-
STAT3 mutants using nuclear fractionation assay (Figure 1.2.13). After LIF stimulation,
we were unable to detect the accumulation of DD-STAT3-Y705F transgene in nuclear
lysates, confirming that Y705 phosphorylation is required for dimerization and
36

translocation into the nucleus. Due to the auto-dimerization effect from STAT3C, DD-
STAT3C-Y705F transgene entered into the nucleus normally like DD-STAT3-WT and
DD-STAT3C. However, when checking the transcriptional activity of these transgenes,
we found that DD-STAT3C-Y705F failed to activate Socs3, one of the primary STAT3
downstream targets; whereas STAT3
̶ / ̶ + DD-STAT3C mESCs contained ~5 fold more of
Socs3 transcripts than STAT3
̶ / ̶-
mESCs did. STAT3
̶ / ̶ + DD-STAT3C-Y705D mESCs,
on the other hand, restored the transcriptional activity of STAT3 (Figure 1.2.14). These
results establish that STAT3 Y705 phosphorylation is essential for target gene
transcription and thus absolutely required for STAT3-mediated mESC self-renewal.

Figure 1.2.12 Phase contrast images showing the short and long term self-renewal
potential of STAT3
-/-
+ STAT3C-Y705F and STAT3
-/-
+ STAT3C-Y705D mESCs in
mESC+LIF+S1 condition. Scale bar = 50 µm.

37

Figure 1.2.13 Subcellular distribution of different STAT3 mutants under mESC+LIF+S1
condition. Oct4 and Tubulin are used as nuclear and cytoplasmic control, respectively.

Figure 1.2.14 qRT-PCR analysis of socs3 expression in different STAT3
-/-
+ DD-STAT3
mESCs after 1 hour of LIF stimulation. STAT3
-/-
mESCs serve as negative control.

38

pS727 supports mESC survival and proliferation, and is dispensable for STAT3-
mediated self-renewal
As shown in Figure 1.2.9, STAT3
̶ / ̶ + DD-STAT3-S727A mESCs were able to self-
renew under mESC+LIF+S1 condition. We also counted the total numbers of AP staining
positive colonies of different STAT3 mutant cell lines under this condition and
categorized the colonies into three groups: differentiated, undifferentiated and mixed. For
example, the vast majority of the STAT3
̶ / ̶ and STAT3
̶ / ̶ + DD-STAT3-Y705F mESCs
were differentiated, while more than 90% of the STAT3
̶ / ̶ + DD-STAT3-WT mESC
colonies remained undifferentiated. As to STAT3
̶ / ̶ + DD-STAT3-S727A mESCs,
despite the total number of AP positive colonies decreased when compared to STAT3
̶ / ̶ +
DD-STAT3-WT mESCs, the quality of individual colonies were not compromised as
undifferentiated cells still dominated the whole population. In fact, a considerable
proportion of the STAT3
̶ / ̶ + DD-STAT3-S727A mESCs died within the first two days of
being switched to mESC+LIF+S1 condition. But the surviving colonies were uniformly
AP positive, and could be maintained continuously while retaining an undifferentiated
morphology resembling that of the STAT3
̶ / ̶ + DD-STAT3-WT mESCs (Figure 1.2.15).

Figure 1.2.15 Long-term self-renewal of STAT3
̶ / ̶ + DD-STAT3-S727A mESCs under
mESC+LIF+S1 condition.
39

Previous reports have shown that STAT3 S727 phosphorylation is associated with cell
survival and mitogenicity in various tumor and primary cells (Gartsbein et al., 2006;
Miyakoshi et al., 2014; Sakaguchi et al., 2012). We evaluated the cell proliferation rates
of two STAT3 mutant cell lines, and observed that STAT3
̶ / ̶ + DD-STAT3-S727A
mESCs grew more slowly than did STAT3
̶ / ̶ + DD-STAT3-WT mESCs under
mESC+LIF+S1 condition, with doubling times of 19.71 and 16.87 hours, respectively. In
contrast, these two cell lines proliferated at similar rates under N2B27+2i condition,
suggesting that LIF/STAT3 signaling is likely responsible for this phenotypic difference
(Figure 1.2.16).

Figure 1.2.16 Proliferation rates of STAT3
-/-
+ DD-STAT3-WT and STAT3
-/-
+ DD-
STAT3-S727A mESCs in mESC+LIF+S1 and N2B27+2i.

40

During passaging, we found that STAT3
̶ / ̶ + DD-STAT3-S727A mESCs underwent
extensive cell death. In a substrate attachment assay, both STAT3
̶ / ̶
and STAT3
̶ / ̶
+ DD-
STAT3-S727A mESCs adhered poorly to gelatin-treated plates and were unable to form
compact colonies; whereas the expression of DD-STAT3-WT transgene fully rescued the
defect of STAT3
̶ / ̶ mESCs: these cells attached strongly to the plate and aggregated into
typical mESC colonies (Figure 1.2.17).

Figure 1.2.17 Substrate attachment assay of different STAT3
-/-
+ DD-STAT3 mESCs.
Cells were plated at a density of 1 × 10
4
cells/cm
2
on gelatin-coated plates and cultured
under mESC+LIF+S1 overnight. Scale bar = 50 µm.

Furthermore, STAT3
̶ / ̶
and STAT3
̶ / ̶
+ DD-STAT3-S727A, but not STAT3
̶ / ̶
+ DD-
STAT3-WT, mESCs gave strong apoptotic signals as detected by TUNEL-FITC staining
(Figure 1.2.18), indicating that the reduced cell adhesion might be the reason for STAT3
̶
/ ̶ + DD-STAT3-S727A mESC death during passaging in mESC+LIF+S1. Together, these
results support that S727 phosphorylation plays an important role in mESC survival.

41

Figure 1.2.18 TUNEL-FITC staining of different STAT3
-/-
+ DD-STAT3 mESCs in
mESC+LIF+S1 condition. Scale bar = 50 µm.

To gain more molecular details that mediate the increased mESC proliferation and
survival via STAT3 S727 phosphorylation, we first looked through several STAT3
downstream targets. Myc is a key transcription factor involved in cell proliferation, cell
growth and apoptosis. In mESCs, myc serves as a downstream target of LIF/ STAT3
signaling to promote pluripotency (Cartwright et al., 2005; Kidder et al., 2008), but does
so mostly by stimulating proliferation in a manner independent of the core pluripotency
factor network (Kim et al., 2008; Kim et al., 2010). Moreover, Myc is not absolutely
required in iPSC formation, but only enhances the efficiency by promoting proliferation
and thereby enriching successfully reprogrammed cells (Nakagawa et al., 2008; Wernig
et al., 2008). The impaired proliferation yet efficient self-renewal exhibited by STAT3
̶ / ̶
42

+ DD-STAT3-S727A mESCs prompted us to investigate whether differential expression
of Myc plays a role in the associated phenotype.

To test this hypothesis, we first examined the transcription level of Myc among different
STAT3
̶ / ̶ + DD-STAT3 mutant cell lines (Figure 1.2.19). Following LIF stimulation of
STAT3
̶ / ̶ + DD-STAT3-WT mESCs, c-myc transcripts were increased 10-fold and n-myc
transcripts, 3.5 fold. In contrast, no change in c-myc or n-myc transcription was detected
in STAT3
̶ / ̶ or STAT3
̶ / ̶ + DD-STAT3-Y705F mESCs. C-myc and n-myc transcription in
STAT3
̶ / ̶ + DD-STAT3-S727A mESCs increased only slightly, suggesting compromised
myc transactivation.

Figure 1.2.19 The transcription level of Myc among different STAT3
̶ / ̶ + DD-STAT3
mutant cell lines in mESC+S1 after 1 hour of LIF stimulation.

43

Next, we attempted to rescue the survival and proliferation defect of STAT3
̶ / ̶ + DD-
STAT3-S727A mESCs. We cloned the coding region of c-myc gene and inserted it into
the PiggyBac expression vector for transfection. Forced expression of c-myc in STAT3
̶ / ̶
+ DD-STAT3-S727A mESCs led to increased proliferation rate under mESC+LIF+S1
condition. Consistently, this improvement could be abrogated by treatment with c-myc
inhibitor 10058-F4, suggesting that differential c-myc expression is the direct reason
responsible for the phenotype (Figure 1.2.20).

Figure 1.2.20 Improved proliferation of STAT3
-/-
+ DD-STAT3-S727A mESCs with
forced expression of c-myc: left: western blot showing over-expression of c-myc; right:
cell proliferation rate under mESC+LIF+S1, 10058-F4: c-myc inhibitor (50 µM).

44

pS727 promotes optimal pluripotency by enhancing STAT3 transcriptional activity
We analyzed the expression of core pluripotency factors in different STAT3
-/-
+ DD-
STAT3 mESCs to further clarify the minimal requirement for self-renewal. Oct4
expression was the same among different cell lines cultured under mESC+LIF+S1, yet
Sox2 and Nanog expression was greatly suppressed in STAT3
̶ / ̶
+ DD-STAT3-Y705F
mESCs (Figure 1.2.21), which also exhibited significant downregulation of Klf4, a direct
STAT3 downstream target gene. These results were consistent with the observation that
STAT3
̶ / ̶
+ DD-STAT3-Y705F mESCs could not be maintained under mESC+LIF+S1
(Figure 1.2.9). STAT3
̶ / ̶ + DD-STAT3-S727A mESCs showed expression of all core
pluripotency factors, but to a much lesser degree than did the STAT3
̶ / ̶ + DD-STAT3-
WT and STAT3
̶ / ̶ + DD-STAT3C mESCs (Figure 1.2.21).

Figure 1.2.21 Western blot analysis showing differential expression of core pluripotency
factors among STAT3
̶ / ̶
+ DD-STAT3 mESCs.
45

A number of previous studies have shown that pS727 enhances STAT3 transcriptional
activity in tumor and primary cells (Lufei et al., 2007; Wen et al., 1995; Yokogami et al.,
2000). Despite Sox2 and Nanog are not direct STAT3 downstream targets, their
expression levels, as hallmarks of pluripotency, are closely related to STAT3 activation
level. In addition, two known STAT3 targets, Klf4 and c-myc, showed reduced
expression in STAT3
̶ / ̶ + DD-STAT3-S727A mESCs (Figure 1.2.21, 1.2.19). These
results prompted us to explore the possibility that pS727 may support optimal
pluripotency by enhancing STAT3 transcriptional activity in mESCs. Therefore, we
examined the transcription level of six well-characterized STAT3 downstream target
genes (socs3, jun, gbx2, fos, klf5 and pim3) in different STAT3
̶ / ̶
+ DD-STAT3 mESCs
after 1 hour of LIF stimulation (Figure 1.2.22). Among these genes, socs3 is involved in
negative feedback of STAT3 signaling; gbx2 and klf5 are related to pluripotency; and jun,
fos, and pim3 are related to cell proliferation. The results indicated that DD-STAT3-
Y705F transgene is unresponsive to LIF stimulation, and DD-STAT3-S727A transgene
possesses impaired transcriptional activity. These data confirmed the results of the self-
renewal assays (Figure 1.2.9), and suggested that pS727 confers optimal mESC self-
renewal by enhancing STAT3 transcriptional activity, but is not essential for minimal
pluripotency.

46

Figure 1.2.22 Differential transcription of STAT3 target genes (socs3, jun, gbx2, fos, klf5
and pim3) among STAT3
̶ / ̶
+ DD-STAT3 mESCs after 1 hour of LIF stimulation.

STAT3 pS727 promotes neuronal differentiation of mESCs
In mESCs, LIF/STAT3 signaling maintains long-term self-renewal by suppressing
spontaneous differentiation. STAT3 also plays important roles in neurogenesis (Qin and
Zhang, 2012), neural stem cell fate determination (Cao et al., 2010). It was shown that
JAK2/STAT3 mediates NMDA receptor-induced synaptic transmission through Y705
phoshorylation and dimerization (Nicolas et al., 2012). In PC12 cells and primary
hippocampal neurons, nerve growth factor (NGF) activates receptor-tyrosine kinase
(TrkA) that in turn phosphorylates STAT3 at S727 residue to support neurogenesis (Ng et
al., 2006). STAT3 interacts with SH2B1β to promote fibroblast growth factor 1 (FGF1)-
induced neurite outgrowth (Chang et al., 2014). Snyder et al demonstrated that STAT3
directly activates its downstream target gene sox6 during neuronal differentiation (Snyder
et al., 2011). Importantly, however, there are also several studies stating that STAT3
47

activation inhibits neurogenesis. For example, LIF was reported to inhibit neuronal
differentiation in mouse postnatal neuronal development through STAT3 activation
(Moon et al., 2002). And in cultured neural stem cells, STAT3 was found to promote
astrogliogenesis at the expense of neurogenesis (Gu et al., 2005). Finally, the biological
functions of STAT3 in neuronal regeneration after injury have drawn much attention as
well. In response to traumatic nerve injury, JAK and protein kinase C (PKC)
phosphorylate STAT3 at Y705 and S727 residues, respectively, to support neurite growth
in dorsal root ganglion (Tsai et al., 2007). And several treatments for neurological
disorders function by activating pathways involving STAT3 pS727 phosphorylation and
subsequent augmentation of Hes3 gene expression that enhance the survival of injured
neurons (Androutsellis-Theotokis et al., 2006; Androutsellis-Theotokis et al., 2009).

Limited information has been obtained regarding the exact role of STAT3 in neural
differentiation of mESCs. Ying et al reported that STAT3 inhibits meso/endoderm, but
not neuroectoderm, differentiation to promote mESC self-renewal (Ying et al., 2003a),
implying the involvement of STAT3 signaling in neural lineage specification. However,
LIF cytokine, which activates STAT3 signaling in mESCs, is removed in most neural
differentiation protocols, leaving the impression that STAT3 is not important in this
process. Our system using different STAT3
̶ / ̶
+ DD-STAT3 mutant cell lines can provide
a refined control of STAT3 activation and therefore may shed light on how STAT3
functions in neural differentiation of mESCs.

48

Figure 1.2.23 Immuno-fluorescence staining of different STAT3
-/-
+ DD-STAT3 mESCs
with neural precursor cell marker Nestin (green) and neuron marker Tuj1 (red). Nuclei
were stained with DAPI (blue). Cells were fixed and stained with above antibodies on
day 18 of differentiation. Scale bar = 50 µm.

We adopted the method of embryoid body (EB) formation to evaluate the potential of
STAT3
̶ / ̶ + DD-STAT3 mESCs to undergo differentiation. The differentiation medium
contained S1 to ensure expression of different STAT3 mutants. LIF was however
removed to induce exit from pluripotency, and retinoic acid (RA) was added on
differentiation day 4-8 to direct neural specification. We found that STAT3
̶ / ̶ and STAT3
̶
/ ̶ + DD-STAT3-Y705F mESCs produced massive numbers of neural precursor cells and
efficiently differentiated into neurons afterwards. STAT3
̶ / ̶
+ DD-STAT3-WT mESCs
initially showed a delay in neural progenitor production, but by day 18 had given rise to
about the same number of neurons that STAT3
̶ / ̶ mESCs had by this day (Figure 1.2.23).
49

These observations establish that loss of STAT3 pY705 results in early exit from the
pluripotent state but appears to exert no effect on neuronal fate determination. Strikingly,
STAT3
̶ / ̶ + DD-STAT3-S727A mESCs gave rise to far fewer neural progenitor cells than
did the other STAT3
̶ / ̶ + DD-STAT3 mESC lines. These STAT3
̶ / ̶ + DD-STAT3-S727A
mESC-derived neural progenitors exhibited low expression of the neural stem cell marker
Nestin and produced Tuj1-positive neurons only sporadically, indicating an impeded
differentiation process.

To further dissect the cell identity changes in the above EB differentiation process, we
proceeded to divide the whole course into several small time windows (differentiation
day 0, 2, 4, 6, 8, 10, 12) and compare the exact time of induction of neural precursor
genes in STAT3
̶ / ̶ + DD-STAT3-WT and STAT3
̶ / ̶ + DD-STAT3-S727A mESCs (Figure
1.2.24). Among the cell identity markers we chose, sox1 represented early neural lineage
cell population that usually emerges before nestin-postive cells; while Oct4 was here used
to mark the remaining pluripotent cells in the mixed population. Starting from day 6 (2
days after addition of RA, the differentiation-inducing agent), sox1 and nestin mRNA
expression was observed in STAT3
̶ / ̶ + DD-STAT3-WT cells, indicating efficient
initiation of neural differentiation. Correspondently, Oct4 expression level gradually
dropped as differentiation proceeded, and became undetectable on differentiation day 10.
On the other hand, in STAT3
̶ / ̶ + DD-STAT3-S727A cells, sox1 and nestin expression
was not only delayed (occurring at day 8 for sox1 and day 10 for nestin), but also
weakened. Oct4 expression level in STAT3
̶ / ̶ + DD-STAT3-S727A cells also suggested
inefficient differentiation as pluripotent cells were still present at day 12.
50

Figure 1.2.24 RT-PCR analysis of induction of neural precursor genes (sox1, nestin) in
differentiation of STAT3
-/-
+ DD-STAT3-WT and STAT3
-/-
+ DD-STAT3-S727A cells.

To further confirm that STAT3 S727A mutation is responsible for the compromised
neural differentiation efficiency, we used a transgene encoding DD-STAT3-S727D,
which contains a phosphomimetic mutation at S727. STAT3
̶ / ̶ + DD-STAT3-S727D
mESCs underwent neuronal differentiation more efficiently than did STAT3
̶ / ̶ + DD-
STAT3-S727A mESCs (Figure 1.2.25, 1.2.26).

Figure 1.2.25 Immuno-fluorescence staining of STAT3
-/-
+ DD-STAT3-S727D mESCs
with neural precursor cell marker Nestin (green) and neuron marker Tuj1 (red). Nuclei
were stained with DAPI (blue). Cells were fixed and stained with above antibodies on
day 18 of differentiation. Scale bar = 50 µm.

51

Figure 1.2.26 Quantitative comparison of the pixel intensity of Nestin and Tuj1 staining
among different STAT3
-/-
+ DD-STAT3 mESCs. The measurements were averaged on at
least two different sets of images.

PD0325901 inhibition of serum-induced pS727 prevents mESC differentiation
Many growth factors and cytokines, including LIF, induce phosphorylation of S727
through different serine/threonine kinases. Considering that S727 is an important event
for efficient mESC neural differentiation after LIF withdrawal, we believe that S727 is
phosphorylated by other kinases instead of LIF/JAK in this process. To identify the
kinase(s) responsible for S727 phosphorylation in mESCs, we treated STAT3
̶ / ̶ + DD-
STAT3-WT mESCs with different kinase inhibitors and monitored the changes in pS727.
As shown in Figure 1.2.27, overnight starvation of STAT3
̶ / ̶ + DD-STAT3-WT mESCs
in basal medium led to a clean background of pS727, while stimulation of serum+LIF,
the conventional mESC culture condition, for 1h resulted in a dramatically elevated level
of pS727. The kinase inhibitors used include the following: JNK inhibitor SP600125, Erk
52

1/2 inhibitor PD0325901, mTOR inhibitor rapamycin, CDK inhibitor olomoucine, PI3K
inhibitor LY294002, PKC inhibitor Go6983 and JAK inhibitor 1.

Of the various kinase inhibitors tested, only the Erk1/2 inhibitor PD0325901 abrogated
the increase of S727, but not Y705, phosphorylation caused by serum+LIF stimulation.
Noticeably, JAK inhibitor 1, which completely prevented Y705 phosphorylation, had no
effect on the level of pS727, suggesting that phosphorylation of Y705 and S727 is
regulated via independent mechanisms (Figure 1.2.27). To rule out the possibility that
this phenomenon is due to unwanted effects of transgene overexpression, we obtained
similar results in wildtype R1 mESCs (Figure 1.2.27). These data demonstrate Erk 1/2
kinase is primarily responsible for STAT3 S727 phosphorylation in mESCs.

Figure 1.2.27 Phosphorylation of STAT3 S727 in mESCs was specifically inhibited by
PD0325901. After overnight starvation with basal medium+S1, STAT3
-/-
+ DD-STAT3-
WT mESCs (left) and R1 mESCs (right) were pre-treated with various inhibitors for 2
hours and then stimulated with mES+LIF for 1h.
53

LIF stimulation activates a number of different signaling molecules, including STAT3,
PI3K-Akt and MAPK/Erk (Figure 1.1.5) (Graf et al., 2011). To elucidate if LIF
contributes to the elevated level of STAT3 pS727, we stimulated cells with either serum
or LIF and tested the effect of PD0325901 addition on STAT3 phosphorylation. The
addition of PD0325901 effectively inhibited serum-induced phosphorylation of S727,
while Y705 phosphorylation was unaffected. In comparison, LIF stimulation led to a
significant increase in the level of pY705, but not pS727 and treatment with PD0325901
had no effect (Figure 1.2.28). Similar results were obtained using wild-type R1 mESCs
(Figure 1.2.28). Based on these observations we conclude that serum and LIF stimulation
function separately by inducing STAT3 phoshporylation on S727 and Y705, respectively.

Figure 1.2.28 PD0325901 blocked serum-, but not LIF-, induced phosphorylation of
S727. Serum and LIF stimulation was applied to STAT3
-/-
+ DD-STAT3-WT mESCs
(left) and R1 mESCs (right) separately before immunoblot analysis.

54

In mESCs, the Erk 1/2 kinase is primarily activated by FGF signaling, which is triggered
by a ligand-receptor interaction that leads to the auto-phosphorylation of tyrosine
residues in the intracellular domain of FGF receptor (FGFR), followed by activation
Grb2-mediated Ras-MEK-Erk cascade. FGF is among the first several growth factors
expressed in early mouse embryonic development, from 1-cell stage to blastocyst, egg
cylinder and primitive streak (Niswander and Martin, 1992; Rappolee et al., 1994). FGF
signaling regulates the early differentiation processes in the mouse blastocyst. Fgf4
-/-
embryos appear normal up to blastocyst stage yet die after implantation (Feldman et al.,
1995), likely due to a primary defect in blastocyst outgrowth: the formation of
trophectoderm and primitive endoderm. Surprisingly, in embryonic day (E) 3.5 and 4.5
blastocysts, FGF4 is produced by the ICM but not the trophectoderm or primitive
endoderm which requires FGF4 to grow and proliferate (Niswander and Martin, 1992;
Rappolee et al., 1994). FGF4 is also required for the derivation and maintenance of
trophoblast stem cells (TSCs) from E3.5 blastocysts(Tanaka et al., 1998), and is now
routinely added to extra-embryonic endoderm (XEN) cell cultures (Kunath et al., 2005).
Culture of blastocysts or isolated ICMs with exogenous FGF4 leads to an increased
number of parietal endoderm-like cells. Over-expression of activated H-RAS, a
downstream effector of FGF4 signaling, induces ESC differentiation into primitive
endoderm (Rappolee et al., 1994; Tanaka et al., 1998; Yoshida-Koide et al., 2004),
whereas genetic ablation of Grb2, which couples the FGF receptor to MEK-ERK
pathway, results in blastocysts that lack hypoblast (primitive endoderm) (Cheng et al.,
1998), suggesting an essential role of FGF4 in specification of primitive endoderm..

55

Mouse ESCs produce FGF4 and activate FGF/MEK/Erk signaling in an autocrine manner.
FGF4 is shown dispensable for mESC maintenance as fgf4-null cells, unlike fgf4-null
ICMs, do not display defect in proliferation in vitro under LIF condition (Wilder et al.,
1997). However, fgf4-null mESCs resist neural and mesodermal induction, and addition
of exogenous FGF4 could restore the lineage commitment potential, indicating that
FGF/MEK/Erk acts as a differentiation cue and is essential for exiting from self-renewal
(Kunath et al., 2007). Fgf4 deletion leads to a massive reduction in steady-state Erk1/2
phosphorylation; and Erk2
-/-
mESCs also differentiate inefficiently in adherent culture
(Kunath et al., 2007). Similar results were observed in FGFR and MEK inhibitor-treated
ESCs (Kunath et al., 2007; Ying et al., 2008). Inspired by these results, derivation of
mESC lines from recalcitrant strains were achieved using the selective MEK inhibitor
PD184352 in combination with LIF and BMP4 (Batlle-Morera et al., 2008).

Our results establish a new connection between FGF/Erk signaling pathway with the
transcription factor STAT3. In mESCs, LIF/JAK and FGF/Erk signaling pathways
converge on a single molecule, STAT3, to regulate stem cell fate determination: under
self-renewal conditions, LIF/JAK signaling is activated to phosphorylate STAT3 at Y705
to strengthen pluripotency network; yet upon differentiation, FGF/Erk signaling takes
over to phosphorylate STAT3 at S727 to induce neural differentiation. Consequently, the
intracellular pool of STAT3 is switched from a LIF/JAK/pY705-dominat status to a
FGF/Erk/pS727-dominant status. Experimentally, this conversion could be automatically
achieved by LIF withdrawal from the medium, leaving the autocrine FGF signal
functioning alone.
56

Activation of Erk1/2 signaling leads to mESC differentiation and its suppression can
promote self-renewal (Burdon et al., 1999b; Hamazaki et al., 2006). We next tested
whether the self-renewal potential of different STAT3
̶ / ̶ + DD-STAT3 mESCs could be
improved when Erk1/2 signaling was blocked. STAT3
̶ / ̶ and STAT3
̶ / ̶ + DD-STAT3-
Y705F mESCs were able to grow under mESC+LIF+S1+PD0325901 condition. Cultured
on feeder layers, they formed flattened, AP staining positive colonies before eventually
differentiating after 3-5 passages (Figure 1.2.29). This result suggested that PD0325901
could reduce dependence on STAT3 pY705 in the maintenance of pluripotency by
blocking pS727. On the other hand, administration of PD0325901 to STAT3
̶ / ̶ +
STAT3-S727A mESCs cultured under LIF+S1 resulted in improved cell survival and
consequently more AP-positive colonies (Figure 1.2.29).

Figure 1.2.29 AP staining and morphological observation of STAT3
-/-
+ DD-STAT3
mESCs in mESC+LIF+S1 condition with or without PD0325901. Scale bar = 50 µm.

57

However, it should be noted that the self-renewal promoting effect of PD0325901 is not
limited to inhibition of STAT3 pS727. Several studies pointed out that PD0325901
treatment led to increased expression of many pluripotency-associated genes in mESCs,
such as Nanog, Tfcp2l1 and Klf4; and forced expression of Nanog or Tfcp2l1 could
reproduce the self-renewal rendered by PD0325901 (Kim et al., 2012; Silva et al., 2009;
Ye et al., 2013). Similarly, we observed that PD0325901 augmented Nanog expression in
STAT3
̶ / ̶ + STAT3-S727A mESCs (Figure 1.2.30). Therefore, PD0325901 may promote
mESC self-renewal by blocking STAT3 pS727-induced differentiation and strengthening
core factor-sustained pluripotency

Figure 1.2.30 PD0325901 can augment Nanog expression in STAT3
-/-
+ DD-STAT3-
S727A mESCs.

Loss of STAT3 S727 phosphorylation enables LIF-induced mEpiSC reprogramming
Speaking from a developmental perspective, the differentiation from embryonic stem
cells to neural lineage cells is a long and complex process consisting of several critical
events. As depicted in Figure 1.2.31, it has been proposed that pluripotency contains two
successive yet distinct states as embryonic development proceeds: naive (mouse ESCs)
58

and primed (human ESCs and mouse EpiSCs) (Colman and Dreesen, 2009; Nichols and
Smith, 2009). It remains unclear, however, how their transcriptional and epigenetic status
result in the phenotypic differences and whether they share common and conserved
mechanisms that govern self-renewal.

Figure 1.2.31 Illustration of mESCs and mEpiSCs, their culture conditions, pluripotency
states, and developmental origins (adopted from (Colman and Dreesen, 2009; Nichols
and Smith, 2009)).

Importantly, although both mESCs and mEpiSCs can self-renew and stay pluripotent,
they differ in many aspects, especially in culture conditions: mESCs are cultured under
serum (or BMP) +LIF; while mEpiSCs are cultured under FGF2+Activin A (Table 1).
This change in culture condition indicates that the switch of STAT3 phosphorylation
status, from LIF/JAK/pY705 to FGF/Erk/pS727, happens when mESCs differentiate into
mEpiSCs. Additionally, STAT3
̶ / ̶ + DD-STAT3-S727A mESCs were resistant to
59

neuronal differentiation after LIF withdrawal (Figure 1.2.23, 1.2.24). Taken together, we
hypothesized that pS727 may play an important role in mEpiSC stage, a necessary
transition state for mESC undergoing differentiation.

Table 1 Similarities and differences between mouse ESCs and EpiSCs.
mESCs mEpiSCs Reference
Pluripotency
State
Naive Primed
(Evans and Kaufman, 1981)
(Martin, 1981)
(Tesar et al., 2007)
(Brons et al., 2007)
(Nichols and Smith, 2009)
Colony
Morphology
Dome Flat
(Tesar et al., 2007)
(Brons et al., 2007)
Clonogenecity Good Poor
(Kim et al., 2013)
(Tesar et al., 2007)
(Brons et al., 2007)
Pluripotency
Markers
Oct4, Sox2, Nanog
Klf 2/4, Rex1,
Oct4, Sox2, Nanog
Fgf5
(Tesar et al., 2007)
(Brons et al., 2007)
X chromosome
Inactivation
XaXa XaXi
(Hanna et al., 2010)
(Tesar et al., 2007)
(Brons et al., 2007)
Teratoma
Formation
Yes Yes
(Hanna et al., 2010)
(Tesar et al., 2007)
(Brons et al., 2007)
Germline
Contribution
Yes No
(Tesar et al., 2007)
(Brons et al., 2007)
Culture
Condition
Serum/LIF;
LIF/BMP;
N2B27+2i
bFGF+Activin A;
CHIR+XAV
(Smith et al., 1988)
(Ying et al., 2003a)
(Ying et al., 2008)
(Kim et al., 2013)
(Tesar et al., 2007)
(Brons et al., 2007)
60

In the recent years, substantial efforts have been put into understanding the conditions
that enable the inter-conversion between mESCs and mEpiSCs. For example, mEpiSCs
were reported to be generated from mESCs via embryoid body formation, a process that
partially mimics post-implantation embryonic development (Zhang et al., 2010). In
addition, it has been shown that mESCs can be differentiated into mEpiSCs by simply
switching culture conditons to FGF+Activin A, while the reversion from mEpiSCs to
mESCs is much more diffcult, and usually requires transgene expression such as Klf4,
Klf2, or Nanog (Guo et al., 2009; Hall et al., 2009; Hanna et al., 2009; Silva et al., 2009).

Since high STAT3 activation level is generally considered as a hallmark of naive
pluripotency, several studies also investigated whether LIF/JAK/STAT3 plays an active
role in the process of mEpiSCs reprogramming to mESCs. Yang et al expressed a
chimeric receptor GRgp-Y118F in mEpiSCs to achieve highly increased, sustained and
exclusive activation of JAK/STAT3/pY705 signaling (Yang et al., 2010). GRgp-Y118F
is fused by the extracellular ligand binding domain of granulocyte colony stimulating
factor (GCSF) and the transmembrane/cytoplasmic domains of the LIFR gp130. The
tryosine 118 residue of gp130 is also mutated to phenylalanine, which destroys the
docking site of SHP2 for Ras/MAPK and PI3 kinase activation (Figure 1.1.5). This
modification enables exclusive JAK/STAT3/pY705 signal activation using GCSF and
avoids complication of using LIF that activates multiple pathways (Burdon et al., 1999a).
Moreover, Y118F also abolishes Socs3-mediated negative feedback loop of STAT3
signaling by preventing Socs3 from binding to the receptor, thus leading to highly
increased and sustained signal activation (Burdon et al., 1999b; Schmitz et al., 2000).
61

Using this system, two studies in Austin Smith's group have demonstrated that those
mEpiSCs expressing GRgp-Y118F could be reprogrammed back to naive pluripotency by
using N2B27+GCSF condition that leads to hyper-activation of JAK/STAT3 (Figure
1.2.32) (van Oosten et al., 2012; Yang et al., 2010). Importantly, because N2B27 medium
is originally developed for neural differentiation and possesses strong differentiation-
inducing effect, GCSF/GRgp-Y118F condition is thus sufficient and dominant for
establishment of naive pluripotency (van Oosten et al., 2012).

Figure 1.2.32 Schematic picture showing the difference of GCSF/GRgp-Y118F and LIF
in mEpiSC reprogramming potency.

On the other hand, N2B27+LIF condition has been proved unable to reprogram mEpiSCs
to mESCs (Figure 1.2.32). In fact, LIF could only enhance the efficiency of transgene-
induced reprogramming such as Klf4 and Nanog (Yang et al., 2010). Compared with
GCSF/GRgp-Y118F condition, LIF is less potent in mEpiSC reprogramming, likely due
to two reasons: a) LIF only moderately activates JAK/STAT3/pY705; b) LIF also
activates other pathways that lead to pS727, despite to a much lesser extent than pY705
62

by JAK. To support this explanation, we took advantage of different STAT3
̶ / ̶ + DD-
STAT3 mutant cell lines to study how they behave under N2B27+LIF condition. We
cultured STAT3
̶ / ̶ + DD-STAT3 mESCs under FGF2/Activin A condition to convert
them to mEpiSCs (Figure 1.2.33). Cultured on feeder layers, the cells formed large, flat
mEpiSC colonies and expressed a high level of EpiSC-specific marker fgf5, a low level
of ESC-specific marker Rex1, and a reduced level of the pluripotency marker Oct4
(Figure 1.2.34), indicating a true mEpiSC state.

Figure 1.2.33 Microscopic pictures of different STAT3
̶ / ̶ + DD-STAT3 mEpiSCs (p12)
cultured in mESC+FGF+Activin A. Scale bar = 50 µm.

Figure 1.2.34 RT-PCR analysis of gene expression profile of different STAT3
̶ / ̶ + DD-
STAT3 mEpiSCs. fgf5: EpiSC-specific; Rex1: ESC-specific; Oct4: shared.

63

We then carried out a serum-free neural induction protocol by exposing monolayer
mEpiSCs to N2B27 medium (Ying and Smith, 2003; Ying et al., 2003b) supplemented
with S1. Consistent with the observation in Figure 1.2.23, in STAT3
̶ / ̶ , STAT3
̶ / ̶ + DD-
STAT3-Y705F, and STAT3
̶ / ̶ + DD-STAT3-WT mEpiSCs, neural-like cell populations
began to emerge on day 6, and were predominant on day 11, whereas STAT3
̶ / ̶ + DD-
STAT3-S727A mEpiSCs remained largely undifferentiated (Figure 1.2.35, bottom panel),
suggesting STAT3 pS727 is essential to high efficiency of neural specification from both
mESCs and mEpiSCs. Furthermore, because N2B27 medium could not induce neural
differentiation of STAT3
̶ / ̶ + DD-STAT3-S727A mEpiSCs, the N2B27+LIF condition
may relieve those mEpiSCs from the primed state of pluripotency and constitute a
reprogramming permissive environment. Indeed, under N2B27+LIF, STAT3
̶ / ̶ , STAT3
̶ / ̶
+ DD-STAT3-Y705F, and STAT3
̶ / ̶ + DD-STAT3-WT mEpiSCs still differentiated
into neural progenitor cells, presumably due to the strong neural-inductive effect of
N2B27; and only STAT3
̶ / ̶ + DD-STAT3-S727A mEpiSCs remained undifferentiated
(Figure 1.2.35, top panel), and gradually regained a mESC identity as indicated by the
increased expression of Rex1 and decreased expression of fgf5 by day 11 (Figure 1.2.36).
64

Figure 1.2.35 Morphology of STAT3
-/-
+ DD-STAT3 mEpiSCs in N2B27+S1 medium
on day 11 with or without administration of LIF. Scale bar = 50 µm.

Figure 1.2.36 Relative expression of fgf5 and Rex1 in STAT3
-/-
+ STAT3-S727A cells
after being subject to N2B27+LIF+S1 reprogramming condition for 11 days. 46C-
mEpiSCs, 46C-mESCs, and STAT3
-/-
+ STAT3-S727A mESCs were used as controls.

65

We isolated several colonies from STAT3
-/-
+ STAT3-S727A day 11 cells and continued
to culture them under N2B27+LIF+S1. On passage 6, these colonies developed into the
small, round, dome shape-like morphology typical of mESC colonies (Figure 1.2.37), and
exhibited a mESC-like gene expression pattern (Figure 1.2.38).

Figure 1.2.37 Morphology of mESC-like clones picked from reprogrammed STAT3
-/-
+
DD-STAT3-S727A d11 cells at different passages.

Figure 1.2.38 RT-PCR analysis of fgf5 and Rex1 expression in a representative clone of
STAT3
-/-
+ STAT3-S727A d11 mESC-like cells (passage 6).
66

These observations together demonstrated that LIF-induced Y705 phosphorylation
enables reprogramming of mEpiSCs into the naive state of pluripotency when S727
phosphorylation is absent. We thus proposed that STAT3 pY705 and pS727 exert
opposing functions in mEpiSC reprogramming: In GRgp-Y118F mEpiSCs, GCSF
stimulation leads to hyperactivation of JAK/STAT3/pY705 that enables reprogramming
(Figure 1.2.39 A); while in wildtype mEpiSCs, LIF cannot overcome the reprogramming
barrier because it activates JAK/STAT3/pY705 only moderately, and also causes slight
increase of STAT3 pS727, which secures a lineage primed state of pluripotency (Figure
1.2.39 B); In STAT3
-/-
+ STAT3-S727A mEpiSCs, LIF is able to solely revert mEpiSCs
to mESCs as the inhibitory effect of STAT3 pS727 is absent (Figure 1.2.39 C).

67

Figure 1.2.39 Opposing functions of STAT3 Y705 and S727 phosphorylation in mEpiSC
reprogramming: (A) GCSF/GRgp-Y118F mEpiSCs. (B) LIF/wildtype mEpiSCs. (C) LIF/
STAT3
-/-
+ STAT3-S727A mEpiSCs

Proposed model for the role of differential STAT3 phosphorylation in mESC fate
determination
Our results showed the presence of a shifting equilibrium of STAT3 phosphorylation that
controls mESC fates (Figure 1.2.40). In an environment conducive to self-renewal
(serum+LIF), LIF-induced activation of JAK/STAT3/pY705 is essential to counteract
differentiation cues from FGF/Erk signaling; while pS727 is dispensable, serving only to
promote proliferation and optimal pluripotency. On the other hand, in differentiative
culture, a high level of STAT3 pS727 is critical for the initiation of mESC differentiation
(into mEpiSCs) and neural commitment. Experimentally, mESC differentiation can be
triggered by LIF withdrawal, which results in a switch from LIF/JAK-mediated
phosphorylation of Y705 to FGF/Erk-mediated phosphorylation of S727. In mEpiSCs,
the re-establishment of high STAT3 pY705 enables the return back to mESCs. On the
other hand, high STAT3 pS727 stabilizes mEpiSCs by inhibiting STAT3 pY705-induced
reprogramming to naive pluripotency, and at the same time promotes differentiation
when environments are permissive.

68

Figure 1.2.40 Proposed model for the role of differential STAT3 phosphorylation in
mESC fate determination

69

Discussion
The LIF/ STAT3 pathway has been shown to play an important role in pluripotency
maintenance of mESCs cultured in serum-supplemented medium. In this study we
demonstrated, for the first time, the involvement of STAT3 pS727 in regulating the
homeostasis of mESCs. Using STAT3
̶ / ̶ mESCs maintained in N2B27+2i, we were able
to manipulate the STAT3 signaling pathway and functionally test the phenotypic
outcomes by transferring cells to LIF-dependent culture condition. The results suggest the
presence of a dynamic equilibrium of STAT3 Y705 and S727 phosphorylation in
controlling the stem cell identities (Fig. 1.2.40).

Foshay and colleagues recently reported that CCE mESCs overexpressing STAT3β, a
STAT3 isoform lacking the S727 residue, produced about 40% fewer neural progenitor
cells than wildtype CCE cells did (Foshay and Gallicano, 2008). In our experiments, we
observed a greater reduction of neural differentiation potential in the absence of S727
(Fig. 1.2.23-26), likely due to lack of endogenous STAT3 in the STAT3
̶ / ̶ mESCs.
Considering that STAT3β was previously described as a dominant-negative regulator in
STAT3 transactivation (Caldenhoven et al., 1996), the decrease in neural differentiation
was thus interpreted as evidence of the necessity of STAT3 activity in this process, while
leaving any specific contribution from pS727 unexamined. Our results demonstrate that
pS727 actually plays a key role in mESC neural differentiation.

In the absence of other self-renewal inputs, mESCs exit pluripotency after LIF
stimulation ceases, and can be directed to a neuroectodermal fate after progressing to the
70

mEpiSC stage. This transition is often considered to be the result of a switch in signaling
dependence from LIF to FGF. Consistent with the finding that FGF-induced Erk1/2
activation is required for neural specification and lineage commitment of pluripotent
mESCs (Kunath et al., 2007; Stavridis et al., 2007), our results showed that Erk1/2 is the
primary kinase controlling STAT3 S727 phosphorylation in mESCs, thus further
demonstrating the association between S727 phosphorylation and mESC neural
differentiation. It has also been suggested that neural induction of ESCs represents a cell-
intrinsic fate commitment in a differentiation-permissive culture condition of minimized
exogenous signals (Kamiya et al., 2011). This model would seem to account for the
observed efficiency with which STAT3
̶ / ̶ mESCs underwent differentiation into neural
progenitor cells in our experiments. Our results indicated that neither STAT3 pY705, nor
STAT3 itself, was required for the induction of differentiation. However, in ESCs with
abundant STAT3 expression, the STAT3 pool needs to be switched from LIF-induced
pY705-dominant status to FGF/Erk-induced pS727-dominant status to initiate the
differentiation programs, a change that is typically achieved by the removal of
supplemental LIF from the culture environment. These findings suggest an underlying
mechanism in which LIF signaling and FGF signaling converge on a single molecule,
STAT3, to regulate the self-renewal and fate commitment of mESCs.

Previous studies showed that mEpiSCs engineered to induce highly increased, sustained
and exclusive activation of JAK/STAT3/pY705 signaling could be reprogrammed back to
a state of naïve pluripotency under N2B27 condition. LIF treatment, on the other hand,
led only to modest activation of JAK/STAT3, along with MAPK and PI3K pathways, and
71

failed to reprogram mEpiSCs (van Oosten et al., 2012; Yang et al., 2010). This was
verified by our observation that STAT3
̶ / ̶ + STAT3-WT mEpiSCs still differentiated in
the N2B27+LIF condition. However, loss of STAT3 S727 phosphorylation endowed LIF
the capacity to reprogram mEpiSCs to naïve pluripotency, indicating the opposing
functions of STAT3 Y705 and S727 in this process. We therefore propose that loss of
STAT3 S727 phosphorylation relieves the requirement for hyperactivation of
JAK/STAT3/pY705 to overcome the EpiSC reprogramming block.

Mouse ES cells grown in the mES+LIF condition exhibit greater morphological and
transcriptional heterogeneity than those grown in the chemically defined N2B27+2i
condition (Marks et al., 2012). Given that LIF stimulation antagonizes differentiation
cues induced by FGF/Erk signaling in the serum condition, phosphorylation of STAT3,
which is dynamically regulated by both pathways, might contribute to the population
heterogeneity. Moreover, several studies have reported that the expression level of Nanog,
in Oct4-positive mESCs, regulates the equilibrium between naïve pluripotency and
lineage-primed states (Chambers et al., 2007; Galvin-Burgess et al., 2013; Singh et al.,
2007). STAT3
̶ / ̶ + STAT3-S727A mESCs showed lower level of Nanog expression than
STAT3
̶ / ̶ + STAT3-WT mESCs, despite being Oct4-positive and morphologically
indistinguishable from those cells. It is thus possible that STAT3 pS727 enables the
maximal transcription of pluripotency factors while also preserves susceptibility to
differentiation triggers.

72

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Part II: Sophisticated rat genome engineering by embryonic
stem cell -based homologous recombination
Abstract
The rat has long been a prime model organism in physiological, pharmacological and
neuro-behavioral studies. However, when it comes to genetically modified models, rats
lag far behind mice. Recent advances of Zinc Finger Nuclease (ZFNs), Transcription
Activator-Like Effector Nucleases (TALENs), Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPRs) and rat ESCs are diminishing the gap of between rat and
mouse with respect to reverse genetic approaches. Importantly, compared to ZFNs,
TALENs and CRISPRs, ESC–based gene targeting still own unique advantages in terms
of restricting the genetic modifications to a desired group of tissues or to a chosen period
during the development of the animal. We describe here a proof-of-principle study for
sophisticated genome engineering in rats by ESC–based gene targeting. This method
comprises the following procedures: derivation and expansion of rat ESCs, construction
of gene-targeting vectors, generation of gene-targeted rat ESCs and finally, production of
gene-targeted rats. This technique allows sophisticated genetic modifications to be
performed in the rat, as many laboratories have been doing in the mouse for the past two
decades. The entire process requires ~1 year to complete, from derivation of ESCs to
generation of knockout rats. This technology in combination with unique advantages of
rats provides new exciting opportunities to create animal models that mimic human
diseases more faithfully.
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Introduction
Biological relevance of rat models
The laboratory rat (Rattus norvegicus) was the first animal species bred and kept for pure
scientific purposes, and is also one of the most widely used animal models in biomedical
research as reflected by the number of scientific publications on it (Dwinell, 2010; Krinke,
2000). As early as 1828, albino rat mutants, which were selected from the offspring of
rats trapped in rat-baiting sports in Europe, were being used in fasting studies by
physiologists (Baker et al., 1979). Rat genetic studies were also launched shortly after the
rediscovery of Mendel’s laws in 1900, with one of the outcomes being the identification
of rat coat color as a Mendelian trait (Baker et al., 1979). Since the development of the
first inbred rat strain in 1909, more than 700 strains of laboratory rats have been bred for
experimentation (Baker et al., 1979). For example, the Zucker rat strain, which bears a
mutation in the leptin receptor gene, is sugar metabolic deficient and insulin resistant and
thus serves as a spontaneous genetic obesity model (Osmond et al., 2010). The
spontaneously hypertensive rat (SHR) has been the most widely studied model of
hypertension, and the availability of the SHR genomic sequence will greatly facilitate the
mapping of genetic traits for hypertension (Atanur et al., 2010).

Historically, the rat has been a superior model organism for a variety of human health-
related research fields, including physiology, pharmacology, neurobiology and drug
discovery (James and Lindpaintner, 1997). The rat shares a similar pathway with humans
for eradicating toxins, providing an ideal model for drug toxicology tests. Given its
physiological similarities to humans, the rat is currently the primary animal model in
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many preclinical tests, especially those related to cardiovascular disease (Aitman et al.,
2008), diabetes (Lee et al., 2010), breast cancer (Smits et al., 2007), chronic
inflammatory diseases (Holmdahl et al., 2001), and age-related diseases (Buffenstein,
2008). Rats are approximately ten times larger than mice, allowing investigators to
perform procedures such as nerve recordings, collection of tissue from small structures
and serial blood sampling more easily. Furthermore, most behavioral studies have been
performed on rats because, compared with mice, their behavior is more social, intelligent,
complex and skilled (Jacob, 1999). Scientists have managed to teach rats complex
behavioral paradigms and tasks. As a result, rats are now also entering the field of
cognitive neuroscience, where the use of monkeys is predominant (Kepecs et al., 2008;
Uchida and Mainen, 2003).

Over the past two decades, however, the development of rat models lag far behind mice,
mainly due to significant advances in mouse genetic toolboxes. In the 1980’s, embryonic
stem cells (ESCs) were successfully derived from mice, and later combined with gene-
targeting technology (Evans and Kaufman, 1981; Martin, 1981; Thomas and Capecchi,
1990). Ever since then, this ESC-based gene-targeting strategy has been extensively used
to create loss-of-function mutations and gene replacement on a predetermined gene locus
in mice. Thousands of mice gene knockout models have been generated and they have
become powerful tools for investigating gene function and relevant phenotypes. This
technology was previously limited to mice only because of the absence of germline-
competent rat ESC lines. In 2008, we developed a chemically-defined basal culture
system that contains serum-free N2B27 medium and small molecule inhibitors (3i:
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CHIR99021, PD184352 and SU5402 or 2i: CHIR99021 and PD0325901), resulting in
successful derivation and maintenance of germline-competent rat ESCs (Buehr et al.,
2008; Li et al., 2008; Ying et al., 2008). These rat ESCs can be genetically modified and
robustly propagated in culture, while retaining the ability to contribute to germline-
competent chimeras, as recently demonstrated by the generation of p53 gene knockout
rats by homologous recombination (Tong et al., 2010). The utility of such rat ESC lines
indicated the capability of applying “classical” mouse gene-targeting techniques directly
to the rat, thereby opening a door for a bright future of rat genetic manipulations.

In the meantime, several alternative approaches aiming to circumvent the requirement of
rat ESCs have been developed for rat genetic manipulation. Classical transgenesis via
pronuclear injection has been applied to rats since the early 1990s and contributed a large
variety of transgenic rat disease models, such as hypertension (Popova et al., 2005),
Alzheimer’s disease (Leon et al., 2010), and Huntington’s disease (von Horsten et al.,
2003). The use of N-ethyl-N-nitrosourea (ENU), as well as the mobile DNA technology
of transposons and retrotransposons, has enabled generation of rat mutants in a random
fashion. The first targeted knockout rats were generated in 2009 via Zinc Finger
Nucleases (ZFNs) technology (Geurts et al., 2009), demonstrating an alternative
approach to the classical ESC-based gene-targeting technology. In the next few years,
Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPRs) were also developed to generate gene
knockout rats (Li et al., 2013a; Li et al., 2013b; Ma et al., 2014; Tesson et al., 2011). We
hereby summarize several strategies that have been used in rat genetic manipulations and
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describe the advantages as well as drawbacks of these advances in rat genetics for
ongoing research in the post-genomic era.

ZFN-mediated knockout technology
ZFNs have been invaluable tools for genomic manipulation in many model organisms,
including C. elegans, Drosophila, zebrafish and finally the rat (Geurts and Moreno, 2010;
Urnov et al., 2010). ZFNs are a class of artificial chimeric proteins fused by two
functional domains: a DNA-binding domain consisting of zinc-finger motif repeats, and a
nuclease domain from type II restriction endonuclease FokI. Each ZFN recognizes a
triplet of DNA, and typically three to six individual ZFN “modules” are aligned together
to ensure the targeting of 9-18 specific nucleotides. Because FokI nuclease must work in
pairs to cleave DNA, the targeting specificity is thus doubled up to 36 bases which
isbelieved sufficient enough to achieve single site recognition over the whole genome.
ZFNs take advantage of the DNA repair mechanism in eukaryotic cells to induce gene
disruption (Figure 2.1.1). After examining specificity of ZFNs in cell culture system,
ZFN-expressing RNAs pre-designed to target a particular locus are injected into fertilized
oocytes. A DNA double-strain break (DSB) will be introduced at that locus, which
activates the innate cellular DNA repair system to fix it. The majority of DSBs in cells
are rescued by a mechanism called non-homologous end-joining (NHEJ), which
functions when no homologous DNA template is available in proximity to the DSB (Mao
et al., 2008). NHEJ uses a ligation reaction to eliminate DSBs and restore the physical
integrity of the chromosome, but the imprecise rejoining of the ends results in a loss of
information content, usually a deletion of several base-pairs at the DSB site. And if this
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deletion happens to occur in the coding region of a gene, a frameshift mutation and
knockout genotype will be generated (Geurts et al., 2010).

On the other hand, targeted integration of new sequences can be achieved by stimulating
the homology directed repair (HDR) mechanism (Figure 2.1.1). A donor DNA template
containing the sequence to be inserted, with flanking sequences homologous to genomic
DNA at the ZFN action site, is co-administered with ZFNs. After a DSB is introduced,
cells will preferentially activate the innate HDR to repair the break, leading to precise
integration of the exogenous sequence (or replacement for subtle gene modification) at
the ZFN cleavage site. A number of studies have been performed to induce gene targeting
via ZFN-mediated homologous recombination in cultured cells (Urnov et al., 2005),
ESCs or iPSCs (Hockemeyer et al., 2009; Zou et al., 2009), Drosophila (Bibikova et al.,
2002), plants (Cai et al., 2009), zebrafish (Amacher, 2008) and mouse zygotes (Meyer et
al., 2010). There has also been report testing targeted integration in rat embryos with
ZFN technology (Cui et al., 2011). They were able to knockin an eight base pair NotI
restriction site (6.7-12.5% targeting efficiency), or a 1.5kb human phosphoglycerate
kinase (PGK) promoter-driven GFP cassette (2.4-8.3% targeting efficiency) into the
targeting site. With this success, they have very recently achieved generation of
Cre/LoxP-mediated conditional knockout rats (Brown et al., 2013).

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Figure 2.1.1 ZFN-mediated knockout technology in the rat. ZFN-expressing mRNA is
microinjected into one-cell stage rat embryos to recognize the targeted genomic site and
generate DSB. The DSB is repaired either by NHEJ, which usually bring out-of-frame
deletions, or by HDR, if a donor template is co-administered with ZFN mRNA. The HDR
mechanism achieves a targeted integration of the template DNA. The ZFN-treated
embryos are transferred to a pseudo-pregnant female. The resulting progeny are
genotyped for targeted mutations before breeding to homozygosity (adopted from (Huang
et al., 2011a)).
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TALEN- and CRISPR-mediated knockout technology
TALENs are firstly discovered as important virulence factors from plant pathogenic
Xanthomonas that cause disease symptoms by mimicking eukaryotic transcription factors
in the plant cell nucleus (Boch et al., 2009). TALENs contain a modular DNA binding
domain of tandem repeats; and the number and order of them determine the binding
specificity. In 2009, two independent studies deciphered the code that governs the DNA
binding of TALENs (Boch et al., 2009; Moscou and Bogdanove, 2009). It turns out that
each repeat in the DNA binding domain has a different pair of amino acid residues at the
12th and 13th positions. These two residues (also known as “repeat variable di-residues
(RVD)”) preferentially recognize one or more of different nucleotides (Bogdanove and
Voytas, 2011), and structural studies have shown that RVDs are direct contact with DNA
(Gaj et al., 2013). For example, four of the most common RVD-nucleotide associations
are His-Asp, His-Gly, Asn-Ile and Asn-Asn to C, T, A, and G, respectively (Moscou and
Bogdanove, 2009). Thus, the combination of RVDs residing in a variable number of
repeats determines a specific DNA targeting sequence. Similar to ZFNs, DNA binding
domain of the TALENs can be fused to FokI to create a DSB at a particular genomic site
among a wide range of species, from yeast to humans (Li et al., 2011; Miller et al., 2011).
These engineered TALENs have been successfully applied to disrupt gene function in the
rat through pronuclear injection (Tesson et al., 2011).

However, due to the highly conserved repetitive sequence in tandem repeats, the
assembly of the RVDs is challenging using the regular molecular cloning method. And
chemical synthesis of the entire RVD region is relatively expensive. In 2009, a type II
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restriction enzyme-based DNA cloning method named Golden Gate Shuffling was
reported (Engler et al., 2009). Golden Gate cloning allows a plasmid to be assembled
from 10 separate input plasmids without the introduction of any mutations. This feature
of the technique makes it possible to assemble more than 20 RVDs in just two rounds of
cloning. The first successful assembly of pre-designed TALENs using the Golden Gate
cloning method was recently performed to target a promoter sequence driving GFP
expression in a transgenic plant (Weber et al., 2011). We have also modified the Golden
Gate cloning system and applied it to construct TALENs that can be used to generate
gene-targeted rat ESCs with high-efficiency (Tong et al., 2012). Other methods such as
high-throughput solid-phase assembly and ligation-independent cloning techniques were
also developed to facilitate custom design and construction of TALEN expression vectors
(Briggs et al., 2012; Reyon et al., 2012; Schmid-Burgk et al., 2013).

Apart from ZFN and TALEN technologies that use site-specific endonucleases to induce
specific DNA cleavage, a new system, namely CRISPR/Cas, has recently been developed
as a promising alternative approach for targeted genetic modifications. In prokaryotic
microbes, CRISPR/Cas serves as an adaptive immune mechanism that mediates RNA-
guided site-specific DNA cleavage to destroy exogenous viral nucleic acids (Wiedenheft
et al., 2012). The bacteria acquire shot fragments of foreign DNA that are integrated into
host CRISPR loci through an adaptation process (Barrangou et al., 2007). Then the
foreign DNA-containing CRISPR loci will be transcribed and processed into short
CRISPR RNAs (crRNAs) (Brouns et al., 2008). An additional trans-activating crRNAs
(tracrRNAs) is also required to anneal with crRNAs; and this event leads to the formation
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of effector complexes of crRNAs and Cas proteins, which recognizes target invader DNA
sequence and generate a DSB (Brouns et al., 2008; Gasiunas et al., 2012; Jinek et al.,
2012). In this system, crRNAs contain sequence templates for base-pairing with targeting
DNA sequence and guide Cas protein to the site; while tracrRNAs plays an unspecific yet
essential role in forming the cleavage complex by interacting with crRNAs (Terns and
Terns, 2014). Recent Investigation has shown that the system could be simplified to two
components (RNA and Cas9 protein) by engineering crRNAs and tracrRNAs to a single
guide RNA (sgRNA), which avoids reconstitution of RNA-processing machinery in the
cell (Jinek et al., 2012).

The convenience of designing customized CRISPR/Cas9 vector for gene targeting far
exceeds that of ZFNs and TALENs, and the targeting efficiency is similar, if not greater.
Investigators can simply include selected 19bp target sequence into a U6 promoter driven
sgRNA scaffold construct, which will be used together with a Cas9 expression vector to
achieve targeted genetic modification (Mali et al., 2013). So far it has been demonstrated
that CRISPR/Cas9 system could be used for genome editing in both cultured cells and
whole organisms including rodents, zebrafish, frog, plants, fruit fly, worm, yeast and
bacteria (reviewed in (Terns and Terns, 2014)). The only constraint for the flexibility of
CRISPR-mediated gene-targeting is that the 19-base pair of the targeting sequence must
be followed by a protospacer-adjacent motif (PAM) sequence –NGG. It is expected that
further exploration of Cas9 protein sequence evolution would lead the path toward PAM
independence (Gaj et al., 2013). Importantly, however, concerns have been raised
regarding the specificity of CRISPR/Cas9 because perfect base-pairing match is only
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required for the 3’ end 8 to 12 bases of the targeting sequence and the following PAM
sequence NGG, while single mismatch at the 5’ end can be tolerated by Cas9 recognition
(Jinek et al., 2012). Indeed, a recent study used cell-based reporter assay to evaluate the
potential off-targets of CRISPR/Cas9: they found that Cas9-mediated cleavage could
tolerate up to five mismatches, and many of the off-target sites were cleaved at similar or
higher frequencies than intended targeting sites (Fu et al., 2013). Therefore, substantial
efforts have been made to improve CRISPR specificity, including the use of truncated
guide RNAs (Fu et al., 2014) and double nicking Cas9 proteins (Ran et al., 2013).

ESC-based gene-targeting technology
Over the years, ESCs have been routinely used to create loss-of-function mutations
(knockout) or gene replacement (knockin) by homologous recombination in mice (Evans
and Kaufman, 1981; Martin, 1981; Thomas and Capecchi, 1990). But numerous attempts
to apply this gene-targeting technique to the rat, until recently, have all failed because of
the imperfect methodology for isolating and manipulating authentic rat ESCs. The 2i/3i
culture system enabled the first successful isolation and propagation of germline
competent rat ESCs. The potential utility of ESC-based gene-targeting technology in rats
was indicated in our recent report on the generation of p53 gene knockout rats in DA rat
ESCs (Tong et al., 2010). Efficient gene targeting by homologous recombination has also
been reported in Fisher344 and Sprague Dawley rat ESCs (Meek et al., 2010). These
results establish a strong foundation for the development of gene-targeted rat models and
the pursuit of rat functional genomics. As one would expect, the strategy for ESC-based
gene-targeting in the rat is similar to the strategy used for the mouse (Figure 2.1.2)
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Figure 2.1.2 ESC-based gene-targeting in the rat. The gene-targeting vector is transfected
into rat ESCs, followed by pick-up, expansion and screening of single colonies for
targeting events. Correctly targeted cells are injected into a host blastocyst and
transferred to foster females. Resulting chimeras are identified and crossed with wildtype
females to generate G1 progeny of heterozygotes (adopted from (Huang et al., 2011a)).
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Comparison of different rat knockout technologies
The development of ZFN, TALEN and CRISPR technologies avoids the requirement of
rat ESCs, and provides much convenience to the whole research community. Compared
with ESC-based knockout technology, ZFN, TALEN and CRISPR create knockout rats
with shorter time course (less than 6 months) by bypassing the chimera stage, and usually
generate gene disruption with higher efficiency. In the meantime, however, the
drawbacks of these methods are also apparent. For example, screening and assembly of
ZFN modules is technically challenging and requires specialized experimental platforms,
while commercialized ZFN modules are expensive. Unlike the construction of ZFN
vectors, TALEN or CRISPR-expressing vector can be rapidly and conveniently
accomplished, yet their off-target effect raises much concern. In addition, these methods
require direct injection of mRNA into one-cell stage embryo, thus founder animals can
only be identified by laborious screening of newborn pups. And in this process, rare
targeting events cannot be enriched, as opposed to various selection strategies performed
in ESC-based targeting. It is thus relatively inefficient to integrate large DNA fragments
into targeted sites, which are usually essential for generating conditional knockout rats.
The insertion of two LoxP sites may have to be handled sequentially and further
optimization for targeting strategy is expected. Therefore, ESC-based gene-targeting still
own unique advantages in terms of sophisticated genetic modifications in the rat. In this
proof-of-principle study, we describe how to perform sophisticated genetic modifications
in rat by homologous recombination in rat ESCs. This strategy has been used routinely in
the mice, and we believe that researchers in many laboratories around the world will take
advantage of it to produce new rat models.
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Experimental design, Results and Discussion
The flowchart in Figure 2.2.1 shows different stages of the whole gene-targeting process,
which requires basic techniques in cell culture, molecular biology, rat embryology and rat
surgery. Some procedures, such as karyotyping, vasectomy, embryo isolation and
manipulation, microinjection and the transfer of embryos to a pseudopregnant recipient,
are provided as a service by animal and transgenic core facilities in many universities and
research institutes. In this study, we chose the POU family transcriptional factor Oct4, a
well-studied pluripotent gene expressed in early embryos, germline cells and
undifferentiated ESCs as the target gene. In vivo expression of Oct4 is essential for the
initial development of pluripotential capacity in the inner cell mass (ICM) and formation
of pluripotent stem cells (Nichols et al., 1998). Oct4 is also one of the four transcription
factors used to create induced pluripotent stem cells (iPSCs), together with Sox2, Klf4
and c-Myc in mouse, rat and human, indicating its capacity to reprogram differentiated
cells into ESC-like state (Hamanaka et al., 2011; Takahashi et al., 2007; Takahashi and
Yamanaka, 2006; Yu et al., 2007). Nevertheless, this makes the investigation on potential
roles of Oct4 in somatic stem cells impossible because of embryonic lethality. Here we
present the experimental design and results on our efforts to generate Oct4-GFP knockin
(and conditional Oct4 knockout) rats. We hope this study will provide a basic platform
for optimization of sophisticated genome engineering in rats. The rapid development of
rat gene-targeting, together with advances in rat genomic resources, will usher the rat
research community into a great future of prosperity.

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Figure 2.2.1 Flowchart outlining how to generate gene knockout rats step by step
(adopted from (Tong et al., 2011)).
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Derivation and propagation of rat ESCs
Using the 2i culture system, we have been able to derive ESC lines at a relatively high
efficiency from all the rat strains we have tested, including Dark Agouti (DA), Fischer
344 (F344), Sprague-Dawley, Brown Norway and Long-Evans (Buehr et al., 2008; Li et
al., 2008; Ying et al., 2008). Other groups have reported the derivation of ESCs from
other strains of rats (summarized in (Tong et al., 2011)). It is generally believed that
ESCs can be established from most if not all strains of rats by using the 2i condition. The
derivation procedure is relatively simple. After the removal of the zona pellucida,
blastocysts are transferred to a 4-well plate pre-coated with feeders and cultured in 2i
medium. ESC lines can be established from about 50% of the rat blastocysts plated. Rat
ESC derivation efficiency can be further increased if the ICM is isolated by
immunosurgery after the removal of the zona pellucida (Mia Buehr, personal
communication). If a particular rat strain has a low efficiency in deriving ESCs, isolation
of the ICM will most likely increase the chance of success. Feeder cells are essential for
the maintenance of pluripotent rat ESCs in the 2i condition. Embryonic fibroblasts
derived from both mouse and rat embryos work well for the culture of rat ESCs. Prior to
use as feeders, embryonic fibroblasts must be mitotically-inactivated either by γ-
irradiation or by mitomycin C treatment (Nagy et al., 2003). As shown in Figure 2.2.2a,
Rat ESCs grow as loosely attached or floating aggregates in the 2i condition, so extra
care must be taken to avoid washing away the cells when changing the medium or
passaging the cells. For routine passaging, we detach rat ESCs by pipetting and harvest
them by centrifugation, after which trypsin is added to dissociate the cell aggregates into
single cells. This method avoids the carryover of feeders that adversely affects rat ESC
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growth. Healthy rat ESCs grow as compact, round, and dome-shaped colonies; they are
uniformly alkaline phosphatase (AP) staining positive, and express all pluripotency
factors including Oct4, Sox2 and Nanog (Figure 2.2.2b-e).

Figure 2.2.2 Rat ESCs derived and maintained in the 2i condition. (a) DA rat ESCs
cultured on feeders in 2i. (b) DA rat ESCs stained with alkaline phosphatase. (c-e) DA rat
ESCs stained for pluripotency markers Oct4 (c), Sox2 (d), and Nanog (e). Scale bar = 50
μm.

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Design of the targeting vector
Gene-targeting by homologous recombination in ESCs has provided a powerful means to
elucidate gene function and create gene knockout animal models (Capecchi, 2005). The
design of the gene-targeting vector is the first critical step (Wu et al., 2008). Key
components of a targeting vector include two homology arms with the same DNA
sequences as the genomic DNA fragments flanking the region to be modified, and a
positive and a negative selection marker. Construction of the targeting vectors has been
described in detail in various protocols (Cotta-de-Almeida et al., 2003; Liu et al., 2003;
Nagy et al., 2003; Sharan et al., 2009; Wu et al., 2008). Although the basic principle for
designing a gene-targeting vector is the same in the mouse and the rat, we found that
several modifications to the classic strategy are required. The first of these is the choice
of promoter used to drive the expression of the positive selection marker. In mouse gene-
targeting vectors, phosphoglycerate kinase (PGK) promoter is most often used to control
the selection marker gene expression. Rat ESCs are very sensitive to drug selection and
the activity of the PGK promoter is too weak for the efficient isolation of drug-resistant
colonies of rat ESCs. The CMV early enhancer/chicken-actin (CAG) promoter has a
much stronger activity than that of the PGK promoter (Figure 2.2.3). The use of the CAG
promoter to drive the expression of the positive selection marker has been proven to be
very effective for isolating drug-resistant colonies in rat ESCs (Tong et al., 2010).

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Figure 2.2.3 Luciferase assay showing that the PGK promoter activity is much lower than
that of the CAG promoter in rat ESCs.

The second modification to the strategy is the choice of the negative selection marker,
which is the gene used to eliminate cells with targeting vectors integrated at non-
homologous recombination sites. We use the diphtheria toxin-A chain (DTA) gene as a
negative selection marker instead of the thymidine kinase (TK) gene. Cells with random
integration of targeting vectors containing the DTA negative selection gene will produce
DTA that kills the cells. As a result, correctly targeted cells can be enriched without the
addition of any selection drugs. We found that gancyclovir at the working concentration
(2.5 μM) is toxic to all rat ESCs lines we have tested, including ESC lines derived from
DA, F344 and Sprague-Dawley rats. Gancyclovir also promotes the differentiation of rat
ESCs maintained in the 2i condition. Consequently, the TK gene is not suitable for use as
a negative selection marker in the rat gene-targeting vector.

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The third strategic modification involves the source of homology arms. Currently there is
no DA rat bacterial artificial chromosome (BAC) end sequence information available,
thus homology arms can only be amplified from DA rat genomic DNA by using PCR-
based methods with high fidelity DNA polymerases. Importantly, the published rat
genomic sequences are from the Brown Norway strain of rats (Gibbs et al., 2004). If there
are any sequence differences between the amplified DNA and the published data, it is
important to determine whether these differences are polymorphisms specific to DA rat
genomic DNA or mutations created by PCR. After retrieving the whole sequence of the
gene of interest and ~15kb of upstream and ~15kb of downstream sequences from UCSC
genome browser, the sequence is imported into sequence analysis software for primer
design of amplifying homology arms. Decision on the location and size of short and long
homology arms is based on the structure and function of the gene, information of exons
and introns, percentages of repetitive sequences, and the restriction enzyme sites. The
minimal length of the homology arms required for successful gene-targeting in rat ESCs
has not been determined yet. From our experience, ~6 kb of the long homology arm and
~1.5 kb of the short homology arm are sufficient for gene targeting by homologous
recombination in rat ESCs. The 5’ and 3’ homology arms can be amplified together or
separately from genomic DNA extracted from isogenic rat tissues depending on different
gene-targeting strategies. The homology arms are then sub-cloned into a pUC vector for
further replication (and introduction of restriction enzymes sites on both ends), using
Clontech’s infusion method as illustrated in Figure 2.2.4a. The targeting vector is
constructed by inserting the homology arms and the PGK-DTA-polyA cassette into the
pCAG-EGFP-IRES-Pac plasmid (Figure 2.2.4b).
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Figure 2.2.4 Schematic diagram showing the strategy for constructing a rat gene-targeting
vector. (a) Amplification and construction of homology arms. pUC backbone vector is
amplified by PCR, using primers 1 and 2 containing restriction enzyme sites SalI and
SpeI, respectively. Primers 3 and 4 are designed to amplify the homology arms from DA
rat genomic DNA. Primers 1 and 2 contain 15 bp extensions (highlighted in red)
complementary to primers 3 and 4. The homology arms generated by PCR are subcloned
into the pUC vector, using Clontech’s infusion method. (b) Construction of the targeting
vector by sequentially inserting the homology arms and the PGK-DTA negative selection
cassette into the pCAG-EGFP-IRES-Pac plasmid (adopted from (Tong et al., 2011)).
107

We designed primers to amplify the whole genomic sequence of rat Oct4 and attempted
to subclone it into the pUC vector (Figure 2.2.5a). In two of the four primer pairs used,
we successfully obtained the ~10 kb PCR product of rat Oct4 genomic sequence and
confirmed pUC vector incorporation (Figure 2.2.5b).

Figure 2.2.5 Amplification of genomic sequence of rat Oct4. (a) Molecular features
(exons and major enzyme sites) of genomic sequence of rat Oct4. (b) Left: PCR product
of rat Oct4; Middle: Incorporation of rat Oct4 sequence into pUC vector; Right: EcoRV
digestion confirming the incorporation. Black arrow: ~10 kb sequence of rat Oct4.

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Next, to construct the gene-targeting vector (Figure 2.2.6), the Oct4 sequence was first
transferred to a PL611 targeting vector containing PGK-DTA-polyA negative selection
cassette. The IRES-EGFP-CAG-Pac fragment was inserted at immediate downstream of
Oct4 coding sequence. This strategy allows the EGFP gene under Oct4 endogenous
promoter control, serving as reporter for targeting events in ESCs and also adult animals.
Importantly, IRES-EGFP-CAG-Pac was engineered in advance to incorporate two Frt
sites in the same orientation flanking CAG-Pac positive selection cassette. In this way,
selection cassette can be excised to create a “clean” modification manner. Two LoxP sites
were also introduced ahead of exon 2, and after CAG-Pac sequence, to induce the
removal of exon 2-5 of Oct4 gene under conditional Cre expression in vivo.

Figure 2.2.6 Schematic diagram showing the gene-targeting strategy of generating Oct4-
GFP knockin and conditional knockout rats. The five exons of rat Oct4 gene with
flanking genomic sequence are shown. The targeting vector contains the full length Oct4
gene. The IRES-EGFP-Frt-CAG-Pac-Frt-LoxP cassette is inserted into the downstream
of Oct4 gene locus at BclI restriction site. Another loxP site is also introduced between
exon 1 and 2 (Adopted from (Huang et al., 2011b)).

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Introduction of gene-targeting vectors into rat ESCs.
Several methods are available for introducing foreign DNA into ESCs, including
electroporation, nucleofection and chemical-based transfection. We have evaluated these
methods for introducing targeting vectors into rat ESCs, and our results suggest that
electroporation is still the most effective means of generating correctly gene-targeted rat
ESC clones (unpublished data). The quality of the mouse ESC line used for the in vitro
manipulation is often the major stumbling block in developing the mouse model
containing the modified gene locus (Liu et al., 1997). This is also likely to be the case in
the rat. The major factors that affect the quality of ESCs and their ability to generate
germline chimeras include culture medium, quality of feeder cells, methods of passaging
and the number of passages (Nagy et al., 2003). Under sub-optimal culture conditions,
ESCs progressively acquire chromosomal abnormalities as their number of passages
increases, and this is one of the major reasons ESCs might fail to contribute to the
germline. The quality of rat ESCs should be regularly monitored by examining their
karyotypes, in vitro differentiation potential, expression of pluripotency markers, and by
determining how efficiently they produce germline chimeras, which still remains the
gold-standard test. Rat ESCs grow as compact dome-shaped colonies in the 2i condition
and as the number of passages increases, some rat ESCs start attaching to the feeders and
forming flat colonies. Those rat ESCs with flat colony morphology should not be used for
gene-targeting because they are most likely karyotypically abnormal (Tong et al., 2010).

110

Isolation of gene-targeted rat ESC colonies
Rat ESCs cultured in the 2i condition are very sensitive to drug selection. We add the
selection drug at half of the normal concentration used for mouse ESCs and we also
apply a pulse selection scheme to increase the selection efficiency (Tong et al., 2010).
This pulse selection scheme works well for puromycin, G418 and hygromycin that we
have tested on ESCs derived from DA, F344 and Sprague-Dawley rats. However, it is
possible that this selection scheme will not work well for other drugs or a particular rat
ESC line. If this is the case, optimization of the selection condition by determining the
killing curve of the selection drug on the rat ESC line is expected. As previously
mentioned, most of the rat ESC colonies are either floating or loosely attached to the
feeders, and extra care should be taken when changing the medium so that ESC colonies
are not washed away. We developed a simple and efficient method for picking rat ESC
colonies after drug selection (Figure 2.2.7). Rat ESC colonies are detached by pipetting
and pooled together in a sterile tube. Each colony is then distributed into one drop (10 μl)
of trypsin/EDTA to dissociate the colony into single cells. In this way, up to 500 colonies
can be picked, dissociated and seeded in less than two hours. The dissociated cells are
then transferred into duplicate 96-well plates, using a multi-channel pipette.
111

Figure 2.2.7 Flow chart of a modified method to pick up rat ESC colonies after
electroporation and drug selection (adopted from (Tong et al., 2011)).

Freezing and thawing of the rat ESCs cultured in the 96-well plate results in a poor
recovery rate. To circumvent this problem, we split each rat ESC colony into two 96-well
plates with one plate having three times more cells than the other. The plate with more
cells is used for the initial screening by PCR, which takes 1-2 days to complete, while
cells in the other plate are kept in culture. Colonies that test positive in PCR-screening are
then expanded from the duplicate plate for further confirmation by southern blot and/or
sequencing analysis. The strategies for southern blot confirmation of the targeting events
in rat ESCs are essentially the same as in mouse ESCs.

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Characterization of gene-targeted rat ESC colonies
The design of PCR primers and optimization of PCR conditions are critical for the initial
screening. One of the PCR primers should be located on the genomic sequence beyond
the 3’ short arm and the other located on the drug resistance cassette. If mouse fibroblast
cells are used as feeder layers, the PCR screening primer sequences should be BLASTed
against mouse genomic DNA sequences to rule out potential non-specific amplifications.
We recommend constructing a control vector containing the 3’ short homology arm with
a 300bp to 500bp extension in which one of the PCR primer pair is located. Rat ESCs
transfected with the control plasmid mimic the targeting event; therefore they can be used
to optimize the PCR screening conditions. The genomic DNA extracted from these rat
ESCs can also be run in parallel with PCR primers as a positive control for the screening.

We linearized the Oct4-GFP gene-targeting vector, transfected it into DA rat ESCs via
electroporation and selected with puromycin. Resistant ESC colonies were picked and
expanded individually. We then performed PCR screening, and obtained 23 positive hits
from ~200 picked colonies (Figure 2.2.8). These positive colonies were expanded, and
those that are GFP-positive and exhibit the typical rat ESC morphology (Figure 2.2.9)
were subject to southern blot. We next PCR amplified the long and short homology arms
from the selected colonies (Figure 2.2.10), and sequenced the junction points between the
homology arm and the genomic locus, as well as the LoxP integration site, to confirm the
correct targeting of those rat ESCs (Figure 2.2.11).

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Figure 2.2.8 PCR screening of gene-targeted rat ESC colonies after electroporation and
drug selection. Samples that generated a single bright band of PCR product were
considered potential positive ones

Figure 2.2.9 A GFP-positive colony of gene-targeted rat ESCs selected from PCR
screening. Images from left to right: Light, Fluorescence, Merge.
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Figure 2.2.10 PCR amplification of the long homology arm (~10 kb, primer P1 and P2)
and the short homology arm (~2 kb, primer P3 and P4) from the selected Oct4-GFP
targeted rat ESC colony. These primers were designed to amplify sequences containing
the junction points between the homology arm and the genomic locus. Plox primer was
designed to examine the introduction of the LoxP site ahead of Oct4 exon 2.

115

Figure 2.2.11 Sequence analysis of the selected Oct4-GFP targeted rat ESC colony. (a)
Sequencing results using primer P3 and P4 were aligned with the predicted sequence of
targeted locus to confirm correct integration of the short homology arm. (b) The
introduction of the LoxP site ahead of Oct4 exon 2 was confirmed by sequencing analysis
using primer Plox.

116

The above results together suggested we have obtained correctly targeted rat ESCs. We
next attempted to test the proposed function of this gene-targeting vector: Oct4-GFP
knockin and Cre-mediated Oct4 deletion. After transfection of Cre-expression vector in
targeted rat ESCs, we found that, as expected, the cells rapidly differentiated and EGFP
signal also turned off (Figure 2.2.12).

Figure 2.2.12 Oct4-GFP targeted rat ESCs differentiate and lose GFP fluorescence after
Cre-mediated Oct4 deletion.

Karyotyping and subcloning of correctly targeted rat ESCs
Low-efficiency contribution of rat ESCs to the germline is often caused by chromosomal
abnormalities in the contributing rat ESCs. We have frequently observed a high
percentage of cells with an abnormal karyotype in gene-targeted rat ESC colonies,
despite their parental cell lines having normal karyotypes. Rat ESCs with a normal
117

karyotype can be isolated by subcloning, which may improve the germline competency
of gene-targeted rat ESCs. Subcloning is performed by plating rat ESCs at single cell
density (1000 cells in a 10 cm tissue culture dish) and culturing them for one week under
2i condition. Then colonies with compact dome-shaped morphology are isolated under a
dissection microscope (Figure 2.2.13). These colonies are to be expanded for karyotyping
analysis as preciously described (Tong et al., 2011). Karyotypically normal rat ESC
subclones should be used in subsequent experiments. In our experience, one rat ESC
population in which >80% of the cells have a normal karyotype is suitable for proceeding
to the next step, that is, production of chimeras by blastocyst injection.

Figure 2.2.13 Karyotyping and subcloning of correctly targeted rat ESCs. (a) Rat ESC
colonies emerged from the tissue culture dish. (b) A round dome-shaped colony with a
normal karyotype. (c) A flat colony with an abnormal karyotype. The colony in (b) is
suitable for proceeding to blastocyst injection. Scale bar, 50 μm.

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Production of knockout rats
Male DA rat ESC lines (as determined by karyotyping or PCR) are used for the
production of rat chimeras. On the day of injection, cells are collected as single cell
suspension in N2B27 medium. At the same time, blastocysts are isolated from timed-
pregnant F344 rats at 4.5 d.p.c, and incubated to allow the expansion of blasocyst cavity.
Usually 12-15 rat ESCs are injected into each blastocyst cavity (Figure 2.2.14a) and the
blastocysts are transferred to pseudo-pregnant surrogate mothers. The produced male
chimeras are bred with Sprague-Dawley female rats to test germline transmission of the
ESC genome (Figure 2.2.14b, c). DA rat ESC has an agouti (A/A) coat color genetic
background, which is dominant to albino (c/c) of the F344 and Sprague-Dawley rats. The
genetically determined coat color distinctions provide a convenient indicator of
chimerism and germline transmission of the DA rat ESC genome. Potential heterozygote
rats are identified by PCR genotyping and southern blot analysis on tail biopsies using
standard methods. And homozygote mutant rats can be generated by intercross male and
female heterozygote rats.

Figure 2.2.14 Production of gene-targeted knockout rats. (a) Isolated rat blastcysts ready
for microinjection. (b) Oct4-GFP chimera rats. (c). Oc4-GFP germline heterozygote rats.
119

Strain combination
Different strains of rats have different suitabilities as models for particular human
diseases. For instance, the Long-Evans rat is a good model for diet-induced obesity and
the spontaneous hypertensive rat (SHR) has been widely used for the study of
hypertension. The availability of ESCs from different strains of rats provides
investigators the option of making gene-targeted rat models based on a relevant genetic
background. For the production of gene-targeted rats by ESC-based technologies,
however, it is essential that rats ESCs used are able to contribute to chimera formation
and re-enter the germline. Therefore, before performing gene-targeting experiments on a
rat ESC line, it is important to determine whether the line is germline-competent and to
identify the ideal strain combination that leads to its efficient germline transmission.

In the mouse, it has been shown that the genetic background of host embryos chosen for
the production of ESC-mouse chimeras is critical for the efficient transmission of the
ESC genome to their progeny. One of the early findings during the development of gene-
targeting in 129 mouse ESCs was that the proportion of chimeras that gave germline
transmission was controlled by the genetic background of the host blastocyst.
Schwartzberg et al used outbred MF-1, outbred CD-1, and inbred C57BL/6 mouse
blastocysts to produce chimeras with CCE 129 mouse ESCs (Schwartzberg et al., 1989).
All three blastocyst donor mouse strains successfully produced ESC-mouse chimeras.
However, only C57BL/6 host blastocysts produced chimeras that transmitted the
genetically modified ESC genome through the germline. The same is true for the mouse
C57BL/6 ESC lines. The efficiency of germline transmission is increased when the host
120

blastocyst used to make ESC chimeras with C57BL/6 ESCs is derived from C57BL/6-
Tyrc-2J/J mice instead of BALB/c, SWR, FVB/N, C57BL/6-Tyrcbrd, or 129/Sv host
blastocysts (Auerbach et al., 2000; Ledermann and Burki, 1991; Lemckert et al., 1997;
Schuster-Gossler et al., 2001; Seong et al., 2004).

The strain combination is also critical for the germline transmission of rat ESCs. Our
results suggest that DA rat ESCs can transmit through the germline when they are
injected into F344 rat blastocysts but not when they are injected into Sprague-Dawley rat
blastocysts (Li et al., 2008). ESCs derived from Sprague-Dawley and Wistar rats have
also been shown to be germline-competent (Buehr et al., 2008; Hirabayashi et al., 2010;
Kawamata and Ochiya, 2010). Another factor that needs to be considered when choosing
the strain combination is the coat color genetic background. The combination of strains
with distinct coat colors will facilitate the identification of chimerism and germline
transmission. Identification of the ideal strain combination will facilitate the broad
application of ESC-based technology for the production of gene-targeted rat models.

Generation of Cre-expressing rat lines
Conditional knockout mice are generated by intercrossing two transgenic mouse lines,
one line bearing Cre recombinase transgene under control of a promoter directing tissue-
and/or time-specific expression, or estrogen receptor T2(ERT2) allowing inducible Cre
excision, and another mouse line with homozygosity of “floxed” (flanked by loxP sites)
alleles derived from gene-targeted ESCs. There have been reports demonstrating the
feasibility of Cre-loxP system in Cre-activated reporter rat line by either crossing to Cre-
121

expressing transgenic rats (Ueda et al., 2006) or somatic delivery of Cre-expressing
viruses (Sato et al., 2004). Since methods such as pronuclear injection to generate Cre
transgenic rats are similar enough to be transferrable, we are now attempting to generate
a transgenic PGK-Cre rat strain that can be crossed with Oct4-GFP gene-targeted rat
strain to achieve inducible gene knockout. On the other hand, we are also working on
generating a Rosa26 Cre-ERT2 knockin rat strain as an alternative approach (Figure
2.2.15). The Rosa26 locus was originally cloned and characterized in gene-trap studies on
mouse ESCs (Friedrich and Soriano, 1991). It was believed to encode nuclear RNA of
unknown function and gene-targeting mice harboring homozygous disruption of Rosa26
locus are viable and phenotypically normal (Zambrowicz et al., 1997). Rosa26 locus is
convenient and amenable to genetic modification as it can be targeted with high
efficiency and is expressed universally in mice. Various kinds of Rosa26 knockin mouse
strains have been produced so far and have become useful tools for biomedical research
(Mao et al., 2001; Mao et al., 1999; Soriano, 1999). Therefore, such strains in the rat will
be also of great help for the rat research community.

Figure 2.2.15 Gene-targeting design of the Rosa26 Cre-ERT2 knockin rat.
122

Concluding remarks and future directions
Although the lack of efficient tools to manipulate the rat genome has made mouse the
leading rodent for genetic research, recent advances in transgenic technology have
explored new paths for rats returning back to the stage. Starting from the Rat Genome
Project launched in 1995 by National Institutes of Health (NIH), rat genomic resources
have been expanding unprecedentedly, including the landmark sequence data of Brown
Norway rat genome (Gibbs et al., 2004), with more and more other strains being added
(Abbott, 2009). The rapid development of reference database has greatly facilitated
establishment of rat models. Genetically modified rats are precious platforms for the
study of human physiology and disease. For example, as compared with transgenic mice,
transgenic rat models of Huntington’s disease not only present a more typical adult
patient pathology, but are also more suitable for in vivo metabolic and structural imaging
(von Horsten et al., 2003). Also, a triple transgenic rat model of Alzheimer’s disease can
successfully mimic human disease phenotype of amyloid deposition (Leon et al., 2010).
With the availability of genome sequences of commonly used rat strains, genetic
modifications in rats will rise up to a new level with exquisite specificity and fidelity.

Loss-of-function models are critical to investigate gene function in vivo. And as research
advances, an increasing number of scientists are hoping to study gene function in a
conditional manner, either in a defined temporal window, or in a specific type of cells or
tissue. For example, since the rat is the choice of species in pre-clinical studies of
diabetes, a pancreas-specific conditional gene knockout rat model would be important for
drug discovery and pharmacological experiments. For neuro-scientists, the rat is now
123

playing a new role in optogenetics - an emerging field that is revolutionizing this area of
research by combining genetic approaches with optics to (in)activate a particular class of
neurons using laser light with the specificity conventional electrophysiology does not
allow (Boyden et al., 2005). In 2009, the first rat optogenetic study reported manipulation
of different circuit elements in Parkinson’s disease rat models with the purpose of
identifying cells responsible for Deep Brain Stimulation (DBS) (Gradinaru et al., 2009).

Inadequate animal models have been believed by pharmaceutical industry as one of the
major hurdles to drug discovery, especially drugs for the central nerve system (CNS). A
more sophisticated genetic modification will allow scientists to establish reliable rat
models of psychiatric and neural disorders. Interesting topics should include conditional
mutagenesis in dopaminergic and glutamatergic neural systems and generation of
“humanized” rats after cell type-specific depletion. Progress in such fields will greatly
promote elucidation of patho-physiologic mechanisms and drug discovery.

Over the years, many strategies for manipulating rat genes were adapted from the mouse
genetic toolbox, including conventional transgenesis by pronuclear injection (Cozzi et al.,
2009), RNA interference (Dann et al., 2006), ENU mutagenesis (van Boxtel et al., 2010;
Zan et al., 2003) and transposon mutagenesis (Izsvak et al., 2010; Kitada et al., 2007;
Kitada et al., 2009). Most importantly, ZFN, TALEN and CRISPR technology and ESC-
based gene-targeting have enabled the species of rat to step into a true “knockout” era.
These methods are highly complementary and each of them has its unique advantages
that assist investigators in achieving their specific objectives: ZFN, TALEN and CRISPR
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can generate knockout rats in a short time with high targeting efficiency, whereas ESC
based gene-targeting gives better precision and offers convenience for sophisticated
genetic modifications as shown in our Oct4-GFP knockin (and inducible/conditional
knockout) rats. In summary, the development of these rat genome engineering
approaches will contribute tremendously to the deciphering of gene functions and the
establishment of rat disease models.

125

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Abstract (if available)

Abstract STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residues. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK‐mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule‐based system to post‐translationally modulate the quantity of transgenic STAT3 in STAT3-/- mESCs. We demonstrated that pY705 is absolutely required for STAT3‐mediated mESC self‐renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency by enhancing STAT3 transcription activity. S727 phosphorylation is regulated directly by FGF/Erk signaling, a differentiation cue in mESCs, and is crucial in the transition of cells from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neural differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to naïve pluripotency, suggesting the opposing functions of STAT3 pY705 and pS727 in reprogramming. Together, these data demonstrated a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate.

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Creator Huang, Guanyi (author)

Core Title Role of STAT3 phosphorylation in mouse embryonic stem cell self-renewal and differentiation

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag differentiation,embryonic stem cell,OAI-PMH Harvest,phosphorylation,self‐renewal,Stat3

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ying, Qi-Long (committee chair), Adams, Gregor B. (committee member), Ichida, Justin (committee member), Kobielak, Krzysztof (committee member), Maxson, Robert E., Jr. (committee member)

Creator Email guanyihu@usc.edu,kylin1000a@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-464664

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