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Investigating the role of STAT3 in mouse and rat embryonic stem cell self-renewal and differentiation

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Investigating the role of STAT3 in mouse and rat embryonic stem cell self-renewal and differentiation

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Content INVESTIGATING THE ROLE OF STAT3 IN MOUSE AND RAT EMBRYONIC STEM CELL
 
SELF‐RENEWAL AND DIFFERENTIATION 
 
 
 
 
 
 
 
by 
 
 
Eric Nathaniel Schulze 
 
 
 
 
 
 
A Dissertation Presented to the 
FACULTY OF THE USC GRADUATE SCHOOL 
UNIVERSITY OF SOUTHERN CALIFORNIA 
In Partial Fulfillment of the 
Requirements for the Degree 
DOCTOR OF PHILOSOPHY 
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY) 
 
 
 
 
August 2010 
 
 
 
 
 
 
 
 
 
 
 
 
Copyright 2010 Eric Nathaniel Schulze
ii 
Epigraph 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Somewhere, something incredible is waiting to be known. 
 
‐ Carl Sagan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iii 
Dedication 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
I wish to dedicate this body of work to those Scientists that have both plied their 
essential trade and sought to help others understand its innumerable worth to 
humanity and this cosmos. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
iv 
Acknowledgements 
 
 
 
 
 
 
 
 
I wish to thank my mentor, Dr. Qi‐Long Ying, for his undying passion, creativity, and 
patience in transmitting not just what Science is and how it is performed 
professionally, but what it can be and the utter limitlessness application Science has 
in this world. 
 
I wish to thank my lovely fiancé and partner, Lori, as she has undoubtedly helped 
focus my thinking and sharpened my aspirations in this life with her boundless love, 
care, and intellect. 
 
I wish to thank my family and friends. Without your ability to help me place Science 
into societal context, to realize that seeking observational and reasoned truths 
about this world do matter to non‐Scientists in significant ways, I would have 
missed a key portion of my training. 
 
To my Committee Members, Professors Martin Pera, Gregor Adams, and Carolyn 
Lutzko: Your Scientific acumen and wisdom are forever appreciated. Your belief in 
what I have set out to accomplish is cherished. I hope to make each of you proud and 
also improve humanity’s knowledge base in the process. 
 
To my labmates, past and present, and my colleagues: Biology is nothing if not a 
chance to demonstrate how each individual might un‐weave the tapestry of life. 
Your insights both guide and inspire me to be a better Scientist. 
 
 
 
 
 
 
 
 
 
 
 
 
 
v 
Table of Contents 
 
Epigraph ii 
 
Dedication iii 
 
Acknowledgements iv 
 
List of Figures vii 
 
Abstract xii 
 
Introduction: 1 
 
I.1 ‐ An Overview of the History of Embryonic Stem 
Cell Research 1 
I.2 ‐ Mouse ES Cells Use the LIF/STAT3 Pathway
To Self‐Renew 5 
I.3 ‐ Permissiveness Relates to Metastable Pluripotent
Cell‐States 13 
I.4 ‐ Rationale for This Investigation 19 
I.5 – Conclusions 20 
 
Results: 22 
 
Chapter One ‐ STAT3 Overexpression Enhances Feeder‐ 
Independent Mouse ES Cell Self‐Renewal 22 
Chapter Two ‐ Increased Endogenous STAT3 Activation is
Sufficient to Maintain Feeder‐Independent Mouse ES
Cell Self‐Renewal 53 
Chapter Three ‐ Increased and Sustained STAT3 Activation
Promotes Mouse and Rat ES Cell Self‐Renewal 62 
Chapter Four ‐ Klf4 Overexpression Promotes Mouse and
Rat ES Cell Self‐Renewal 90 
Chapter Five ‐ STAT3 Activation May Promote Tumor
Cell Growth Arrest 101 
 
Discussion: 111 
 
D.1 – STAT3 Is Regulated In a Strain or Species‐Specific
Manner to Promote Rodent ES Cell Self‐Renewal 111 
D.2 ‐ The Role of STAT3 In Mediating Mouse and
Rat ES Cell Self‐Renewal 113 
D.3 ‐ The Role of ERK1/2 In Mouse ES Cell Self‐Renewal 117 
vi 
D.4 ‐ Rodent ES Cell Self‐Renewal: A Stratified Model 118 
D.5 ‐ Future Directions and Implications 123 
D.6 – Conclusions 124 
 
Bibliography 126 
 
Appendices: 140 
 
Appendix A: Materials and Methods 140 
Appendix B: Table of Primer Sets 146 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 vii 
List of Figures 
 
Figure I.1a: Embryonic stem cells permit precise gene‐targeting.  3 
 
Figure I.2a: STAT3 regulates mouse ES cell self‐renewal. LIF
mediates activation of STAT3 through the recruitment of Janus‐ 
kinases (Jaks). 7 
 
Figure I.2b: LIF/Jak/STAT3 signaling regulates mouse ES cell
self‐renewal. 9 
 
Figure I.2c: LIF activates the MAPK/ERK and PI(3)K/Akt
pathways in mouse ES cells. 10 
 
Figure 1.1a: B6 Mouse ES cells enter crisis following feeder depletion.  23 
 
Figure 1.1b: B6 mouse ES cells lose AP positivity in response to 
feeder depletion. 24 
 
Figure 1.2a: Feeder‐independent 46C self‐renew effectively in 
the absence of feeders. 26 
 
Figure 1.2b: Feeder‐independent 46C mouse ES cells increase 
STAT3 expression and activation in response to LIF stimulation 
as compared to B6 mouse ES cells.  27 
 
Figure 1.2c: Densiometry indicates that 46C mouse ES cells
increase STAT3 expression following LIF treatment. 28 
 
Figure 1.2d: Densiometry indicates that 46C mouse ES cells
increase STAT3 phosphorylation following LIF treatment. 28 
 
Figure 1.3a: STAT3 overexpression enhances feeder‐free
self‐renewal of B6 mouse ES cells.  30 
 
Figure 1.3b: STAT3 overexpression prevents crisis and
promotes self‐renewal. 32 
 
Figure 1.3c: Removal of STAT3 transgene results in decreased 
total cell number and decreased AP positive colony formation. 33 
 
Figure 1.3d: Loss of STAT3 overexpression from B6 STAT3
cells results in decreased AP positivity. 34 
 
 viii 
Figure 1.3e: STAT3 overexpressing B6 mouse ES cells remain 
undifferentiated in long‐term feeder‐free culture. 35 
 
Figure 1.4a: 46C mouse ES cells exhibit lower total ERK1/2
expression and activation as compared to B6 ES cells. 37 
 
Figure 1.4b: Densiometry reveals that 46C mouse ES cells
harbor decreased ERK1/2 expression and phosphorylation 
as compared to B6 ES cells.  38 
 
Figure 1.4c: STAT3 overexpression correlates to decreased
ERK1/2 phosphorylation. 39 
 
Figure 1.4d: Densiometry reveals that STAT3 overexpression 
correlates to decreased ERK1/2 phosphorylation in B6 ES cells. 40 
 
Figure 1.4e: Inhibition of MAPK improves STAT3 mediated
feeder‐independent self‐renewal. 42 
 
Figure 1.4f: MAPK inhibition increases feeder‐free self‐ 
renewal when STAT3 is overexpressed. 43 
 
Figure 1.4g: STAT3 overexpression increases RNA expression
of core pluripotency genes.  44 
 
Figure 1.4h: STAT3 overexpression prevents differentiation
in stat3/ mouse ES cells cultured feeder‐free.  46 
 
Figure 1.4i: STAT3 expression is required for LIF‐mediated
self‐renewal feeder‐free. 48 
 
Figure 1.5a: β‐catenin activation does not significantly
enhance feeder‐free B6 self‐renewal in the absence of LIF. 50 
 
Figure 1.5b: β‐catenin Activation Does Not Significantly Increase
Self‐Renewal when despite LIF stimulation and MAPK inhibition. 51 
 
Figure 1.5c: Feeder‐free self‐renewal ability is impaired following
β‐catenin transgene excision. 52 
 
Figure 2.1a: G‐CSF mediated STAT3 activation exerts a dose‐ 
dependent self‐renewal effect. 55 
 
 
ix 
Figure 2.1b: Densiometry reveals that STAT3 phosphorylation
levels decrease in correlation with decreasing G‐CSF concentrations. 56 
 
Figure 2.1c: Jak inhibition decreases STAT3 mediated mouse ES
cell self‐renewal efficiency feeder‐free. 57 
 
Figure 2.2b: STAT3 activation promotes feeder‐independent self‐ 
renewal of BalbC1 mouse ES cells.  59 
 
Figure 2.2c: Feeder‐independent 46C mouse ES cells exhibit a
dose‐dependent self‐renewal response to G‐CSF treatment. 61 
 
Figure 3.1a: G‐CSF mediated STAT3 phosphorylation is increased
and sustained over 24 hours. 63 
 
Figure 3.1b: Densiometry reveals that G‐CSF treated mouse ES
cells retain an increased STAT3 phosphorylation level over 24 hours.  64 
 
Figure 3.1c: Densiometry reveals that G‐CSF treated mouse ES
cells retain a stable STAT3 expression level over 24 hours. 64 
 
Figure 3.1d: G‐CSF activates STAT3 in proportion to STAT3
expression level. Equal total cellular protein from B6 mouse
ES cells was compared via western blot analysis.  66 
 
Figure 3.2a: SOCS3 RNA expression levels do not appear to
significantly differ between LIF or G‐CSF treatments.  68 
 
Figure 3.3a: G‐CSF mediated STAT3 activation correlates
to rapid induction of mouse ES cell differentiation. 70 
 
Figure 3.4a: Feeder‐free maintenance of B6 mouse ES cells
in G‐CSF conditions maintains pluripotent identity. 73 
 
Figure 3.4b: G‐CSF treatment of mouse ES cells regulates a
distinct set of genes as compared to LIF. 74 
 
Figure 3.4c: B6 mouse ES cells maintain pluripotency following
long‐term culture feeder‐free. 76 
 
Figure 3.5a: DAc8 rat ES cells activate STAT3 in response
to LIF stimulation.  80 
 
Figure 3.5b: Densiometry comparing rat and mouse ES
cell STAT3 levels.  80 
x 
 
Figure 3.5c: STAT3 overexpression promotes LIF mediated
self‐renewal of rat ES cells.  82 
 
Figure 3.5d: G‐CSF supports rat ES cell self‐renewal.  83 
 
Figure 3.5e: G‐CSF mediated STAT3 activation increases rat
ES cell self‐renewal ability over 2i.  84 
 
Figure 3.5f: Rat ES cells expressing the chimerical gp130 receptor
activate STAT3 in response to G‐CSF. 85 
 
Figure 3.5g: Densiometry indicates that G‐CSF and LIF
phosphorylate STAT3 comparably in rat ES cells.  86 
 
Figure 3.5h: G‐CSF stimulation sustains STAT3 activation over
time promoting long‐term self‐renewal. 87 
 
Figure 3.5i: Densiometry indicates that G‐CSF increases STAT3
activation over 24 hours in rat ES cells. 88 
 
Figure 3.5j: Densiometry indicates that G‐CSF maintains STAT3
expression over 24 hours in rat ES cells. 88 
 
Figure 3.5k: G‐CSF maintained rat ES cells maintain a pluripotent
identity similar to canonical 2i maintained rat ES cells. 89 
 
Figure 4.1a: Mouse ES cells upregulate Klf4 in response to
G‐CSF treatment feeder‐free. 93 
 
Figure 4.3a: Klf4 overexpression enhances LIF‐mediated feeder‐ 
free B6 ES cell self‐renewal. 95 
 
Figure 4.3b: Klf4 overexpression permits long‐term culture
feeder‐free in a LIF‐dependent manner. 96 
 
Figure 4.3c: Klf4 overexpression enhances BalbC1 mouse ES
cell feeder‐free self‐renewal. 97 
 
Figure 4.3d: Klf4 knock‐down is efficient in mouse ES cells. 98 
 
Figure 4.3e: Klf4 expression is not essential for mouse feeder‐ 
free self‐renewal. 99 
 
 
xi 
Figure 4.3f: Klf4 knock‐down decreases pluripotency gene
expression and increases early differentiation markers.  100 
 
Figure 5.1a: G‐CSF Mediates STAT3 activation and
correlates to inhibited ERK1/2 activation. 103 
 
Figure 5.1b: Densiometry confirms decreased ERK1/2
activation in response to G‐CSF in HT29 tumor cells.  104 
 
Figure 5.1c: Wild‐Type Human Tumor Cell Lines Demonstrate
No Discernible Effect to G‐CSF Treatment. 105 
 
Figure 5.1d: Increased STAT3 Activation Correlates to
Decreased Cell Number in DLD1‐Y118F cells.  106 
 
Figure 5.1e: Increased STAT3 activation correlates to
decreased cell number in HT29‐Y118F cells.  107 
 
Figure 5.2a: G‐CSF stimulation correlates to decreased
cumulative cell number.  109 
 
Figure 5.2b: G‐CSF Prevents Tumor Cell Proliferation at
Low Cell Density and During Exponential Growth. 110 
 
Figure D.2a: STAT3 signaling appears to have a functional
range that effectively promotes ES cell self‐renewal. 112 
 
Figure D.4a: Rodent ES cell self‐renewal is metastable cell‐ 
state within naive pluripotency.  119 
 
Figure D.4b: Self‐renewal represents a finely‐tuned interplay
between STAT3 and MAPK/ERK signaling in rodent ES cells.  121 
 
 
 
 
 
 
 
 
 
 
 
 xii 
Abstract 
 
 
Pluripotent embryonic stem (‘ES’) cells are typically derived and maintained using 
inductive or inhibitory signals that are thought to behave in a binary ‘on/off’ 
manner. Mouse ES cells are maintained in an excess of leukemia inhibitory factor 
(LIF) and rat ES cells in a cocktail of inhibitors that block both GSK3β and MAPK 
signaling in a STAT3‐independent manner. Here, we provide evidence that both 
mouse and rat ES cell self‐renewal is conserved via a STAT3‐dependent mechanism 
that treats STAT3 activation as fluid and dynamic. We observe that increased Klf4 
expression enhances STAT3‐mediated self‐renewal in mouse ES cells but is not 
essential to prevent differentiation. As well, increased STAT3 activation and ERK1/2 
inhibition synergistically enhance self‐renewal. In all, we propose that the self‐
renewal mechanism is a nested metastable cell‐state within naïve pluripotency, and 
that further refinements to the self‐renewal mechanism will allow for direct inter‐
species comparisons and derivation of novel ES and induced pluripotent cell 
population.
1 
 
Introduction 
 
Modern advances in science are predicated upon the unconventional insights of 
previous generations. In order to make clear predictions about the world we 
inhabit, scientists must diligently understand how and why the current path has 
been laid down. 
 
I.1  An Overview of the History of Embryonic Stem Cell Research 
 
Embryonic stem (ES) cells are in vitro cell artifacts that represent a developmentally 
halted tissue type of the pre‐implantation mammalian blastocyst(Evans and 
Kaufman, 1981; Martin, 1981). ES cells have the peculiar, yet useful, properties of 
nearly infinite in vitro self‐renewal and the ability to differentiate into any tissue 
type of the soma and germline, termed ‘pluripotency.’ The concept of harnessing 
pluripotency in vitro arose from early work upon the hematopoietic system. 
Researchers at that time realized that transplanted hematopoietic cells would give 
rise to spleen colonies of hematopoietic cells. In 1964, Till and McCulloch concluded 
that these cells were a progenitor, or ‘stem,’ cell type that, from single cells, 
reconstitute a whole population within a tissue(Till et al.). With the availability of 
mouse ES cells in 1981, researchers hypothesized that precise gene targeting 
experiments could be possible, and therefore specific gene functions might be 
2 
interrogated(Thomas and Capecchi, 1987). For the discovery of mouse ES cells and 
the ability to create gene‐targeted animals via homologous recombination using ES 
cells, Sir Martin Evans , Mario Capecchi and Oliver Smithies were awarded the Nobel 
Prize in medicine in 2007, highlighting the importance of ES cell research and the 
translation of these ideas to in vivo systems. 
 
However Sir Evans’ work was just the culmination of many researchers’ previous 
efforts toiling away on a curious phenomenon observed in germ cell tumors found 
in the 129 strain of mouse. In 1954, Stevens and Little characterized these tumor 
cells from this mouse as a relatively frequent phenomenon, occurring at around 1% 
in the testes(Brook and Gardner, 1997). The tumor types that resulted from these 
cells, termed teratocarcinoma and embryonal carcinoma (‘EC’), were odd in that not 
only did they harbor the characteristic of being able to grow indefinitely in a culture 
dish in an undifferentiated state, a phenomenon referred to as ‘self‐renewal,’ but 
also when single EC cells were transferred from one animal to another, the tumor 
could be reconstituted fully and retain the ability to differentiate into tissue types 
representative of all germ layers of the soma(Kleinsmith and Pierce, 1964). This 
latter characteristic, termed ‘pluripotency,’ is a defining aspect of authentic 
embryonic stem cells. 
 
Maturation, or differentiation, of tumor cell types is not in itself a novel finding, 
however the investigators working on embryonal carcinomas were astute enough to 
note that EC cells share many cell surface antigens and markers of cells of the 
3 
embryonic inner cell mass(Gachelin et al., 1977; Solter and Knowles, 1978). These 
tumors, either by their mechanism of transformation or by their tissue of origin, 
exhibited a novel characteristic that conferred both self‐renewal and multipotent 
differentiation(Strickland and Mahdavi, 1978). 
 
Researchers were aware that, if transplanted to ectopic sites, mouse embryos could 
give rise to teratomas. 
Borrowing from oncology 
studies, researchers routinely 
injected EC cells into the kidney 
capsule as a means of providing 
an environment for their 
pluripotency to be 
observed(Bogden et al., 1979; 
Castro and Cass, 1974). EC cells 
were routinely injected in 
developing mouse blastocyts 
where the tumor cells would 
colonize the developing 
conceptus, eventually forming the mosaically patterned chimeras which form the 
first portion of the assay used to rigorously demonstrate pluripotency: Germline 
transmission(Illmensee and Mintz, 1976; Mintz and Illmensee, 1975). This result 
Figure I.1a: Embryonic stem cells permit precise 
genetargeting.(1) A preimplantation mouse 
blastocyst is subjected to immunosurgery. (2) The 
resulting ES colonies emerge and proliferate. (3) 
Genetargeting by homologous recombination is 
performed. (4) Modified ES cells are injected into a 
host blastocyst and transferred into a donor 
mouse. (5) Coat color contribution determines ES 
penetrance. (6) Mating of chimera results in 
germline transmission of transgene in offspring. 
4 
encouraged researchers because if the rate of transmission could be improved 
somehow, it could become feasible to completely generate a genetically‐modified 
organism by simply altering the gene‐of‐interest in a single pluripotent embryonic 
‘stem’ cell which would then be allowed to re‐enter the normal developmental 
pathway (Figure I.1a).
 
It was Evans and Martin’s insight that led to the discovery of a karyotypically 
normal cell type from an in vivo source that could recapitulate the tumor cell’s 
abilities without the undesirable side effects of using cancer(Martin, 1980). It is 
interesting to note that as of now, the in vivo equivalent of ES cells has yet to be 
definitively determined, although most evidence indicates that the pre‐implantation 
epiblast is the likely equivalent to ES cells(Gardner and Beddington, 1988; Lanza et 
al., 2006). ES cells represent a developmentally frozen cell state that retains nearly 
all of the properties of the pluripotent epiblast. 
 
Following fertilization and formation of the zygote, cleavage division begins under 
the supervision of mainly inherited factors and imprinted genes(Surani, 2001). 
Pluripotency markers, such as Oct4, are inherited transcripts that then give way to 
endogenous production as early as the 2‐cell stage (Hansis et al., 2001). Cells then 
begin to partition the embryonic compartment, as indicated by differential, outer 
cell layer expression of cdx2, a marker for trophectodermal lineage, and Oct4 
expression located within the inner cell layer(Suwińska et al., 2008). There is even 
evidence that pluripotency markers are partitioned immediately after fertilization, 
5 
suggesting that the pluripotency compartment is marked and separated early in 
development(Lu et al., 2001; Piotrowska‐Nitsche et al., 2005). 
 
Cell potency refers to the total number of germ layers each cell can form. In addition, 
potency refers to how many differentiated cell types within each tissue layer each 
cell can form. Totipotency indicates the ability for an individual cell to form all 
tissue types of the soma including all extraembryonic tissues(Lanza et al., 2006). 
Dichorionic identical twins are a natural result of totipotent stem cells that separate 
in early cleavage divisions to form separate, genetically identical embryos. 
Pluripotency is generally regarded as a property held by the epiblast alone in 
normal development(Grubb, 2006). 
 
I.2  Mouse ES Cells Use the LIF/STAT3 Pathway To SelfRenew 
 
Embryonic stem cells have two defining characteristics: Self‐renewal and 
pluripotency. While the differentiation potential and characteristics of pluripotency 
have remained a popular area of research, investigating the mechanism as to why ES 
cells decide to divide into two identical daughter cells as opposed to differentiated 
progeny has remained relegated to a small cadre of groups. A thorough 
understanding of ES cell self‐renewal will likely result in derivation of ES cells in 
novel species. In addition, by investigating self‐renewal, clinical applications that 
use ES cells will benefit from the ability to completely terminate and regulate the 
self‐renewal program, a finding that may be applied to both ES‐based therapies and 
6 
cancer studies. And finally, with the recent discovery of induced pluripotent stem 
(iPS) cells, understanding how stem cells decide to self‐renew is tantamount to 
understanding efficient and complete reprogramming(Takahashi and Yamanaka, 
2006). 
 
Self‐renewal is defined as the ability to divide into two identical daughter cells that 
retain the pluripotency properties of the parental cell(Lanza et al., 2006). 
Interestingly, self‐renewal in ES cells is coupled to unlimited proliferative ability. ES 
cells appear to lack a Hayflick limit and never appear to exit the cell cycle, 
demonstrating persistent cyclin D expression, spending the majority of the cell cycle 
in S phase after an extremely abbreviated G1 stage(Burdon et al., 2002; Dalton, 
2009; Singh and Dalton, 2009). Therefore mouse ES cells can be maintained without 
the worry of senescence (Smith, 2001). In addition, telomerase is active in ES cells 
and is required for cellular reprogramming, suggesting that telomerase activity aids 
in proliferation of self‐renewing ES cells(Lee et al., 2005; Niida et al., 2000; 
Takahashi and Yamanaka, 2006).
 
In the mouse, self‐renewal is mediated primarily by the LIF/Jak/STAT3 
pathway(Niwa et al., 1998b) (Figure I.2a). Leukemia Inhibitory Factor (LIF) is an IL‐
6 family cytokine that is secreted by the trophectoderm in vivo to promote diapause: 
Diapause is the phenomenon whereby a suckling mouse triggers a delay in 
blastocyst implantation that can last for several weeks. Estrogen mediates 
trophectodermal secretion of LIF, which in turn induces self‐renewal of the inner 
7 
cell mass (ICM) until implantation occurs and development continues(Mead, 1993; 
Renfree and Shaw, 
2000). 
 
In vitro, LIF is 
secreted by the 
support layer of 
mitotically‐
inactivated cells 
used to anchor and 
maintain mouse ES 
cells, termed 
‘mouse embryonic 
fibroblasts’ 
(‘MEFs’) or ‘feeders’(Rathjen et al., 1990; Smith and Hooper, 1987). LIF secretion 
acts in paracrine to bind and co‐localize the two necessary cell surface receptors: 
LIFRβ and gp130(Chambers et al., 1997; Gearing et al., 1991). LIFRβ and gp130 are 
non‐tyrosine kinase receptors that, in the presence of LIF, heterodimerize(Davis et 
al., 1993). The cytosolic domains then catalyze a series of signal transduction 
pathways that execute self‐renewal for the mouse. 
 
Figure I.2a: STAT3 regulates mouse ES cell selfrenewal. LIF 
mediates activation of STAT3 through the recruitment of 
Januskinases (Jaks). STAT3 activation promotes self
renewal and ‘stemness.’ LIF also activates ERK1/2 which 
aids STAT3 in proliferation, but promotes differentiation as 
well. 
8 
Gp130 is the common IL‐6 family receptor. It frequently heterodimerizes with other 
IL‐6 family members, as well as harboring homodimerization ability(Murakami et 
al., 1993). Gp130‐/‐ mutant embryos fail to develop past implantation and give rise 
to predominantly parietal endoderm(Nichols et al., 2001). It is therefore important 
that gp130 be expressed for both ES maintenance and for normal embryonic 
development. LIFRβ appears to be unnecessary for blastocyst formation and the 
formation of the pluripotent ICM, as LIFRβ‐/‐ embryos develop to term, but die 
perinatally with severe neural defects(Li et al., 1995; Ware et al., 1995). LIF‐/‐ 
embryos are normal into adulthood, but LIF‐/‐ mothers fail to support blastocyst 
diapause and implantation(Stewart et al., 1992). In all, LIF appears to be important 
as a secreted factor by both the mother and trophectoderm to support pluripotency.
 
LIF, once engaged with the receptor complex, recruits a multitude of proteins to the 
cytosolic scaffolding. Most importantly, Janus kinase 2 (Jak2) is recruited to the 
membrane proximal domain(Narazaki et al., 1994). Once docked, Jak2 
phosphorylates Tyk, which in turn recruits inactive STAT3 monomers(Stahl et al., 
1994). While ES cells express several different isoforms, STAT3 is the most common 
species present in mouse ES cells. STAT3 is a highly conserved acute‐phase 
pleiotropic transcription factor, capable of exacting transcriptional regulation upon 
phosphorylation at the cell membrane(Darnell et al., 1994). 
 
9 
STAT3 has several important functional domains important to its role in ES cells. 
Composed of a DNA binding, SH2, coiled‐coil, and transactivation domains, STAT3 
has the ability to both interact directly and as a co‐activator of transcription(Becker 
et al., 1998; Zhong et al., 1994a). Within the transactivation domain lie two distinct 
phosphorylation sites important to ES cells. In the mouse, serine 727 (‘Ser727’ or 
‘S727’) phosphorylation has 
been shown to enhance 
transcriptional activity, likely by 
providing a docking site for 
interaction with other 
proteins(Wen et al., 1995; 
Yokogami et al., 2000). 
Tyrosine 705 (‘Tyr705’ or 
‘Y705’) phosphorylation is 
associated with STAT3 
activation(Zhong et al., 1994b). 
Upon Jak2 docking, STAT3 
monomers are recruited to one of the several STAT3 docking sites distal to the 
membrane(Narazaki et al., 1994). Four distinct YXXQ motifs can bind to STAT3. 
Y265/275 were found to be critical to maintaining the pluripotent state, as mutation 
of these docking sites abolished mouse ES self‐renewal(Niwa et al., 1998a).
 
Figure I.2b: LIF/Jak/STAT3 signaling regulates 
mouse ES cell selfrenewal. (1) LIF ligand binding 
induces heterodimerization of both LIFRβ and 
gp130. (2) Receptor complex formation 
precipitates docking and phosphorylation of Jak2 
and Tyk. (3) Jak2 recruits and phosphorylates 
STAT3 monomers. (4) Active STAT3 dimer 
complexes translocate to the nucleus and effect 
gene transcription. 
 10 
Jak2 phosphorylates STAT3 at Y705, which causes STAT3 to develop affinity for 
other phosphorylated STATs(Hemmann et al., 1996). STAT3 preferentially 
reciprocally binds to other activated STAT3 proteins by phospho‐SH2 
interactions(Heim et al., 1995). This homodimer forms the active complex that then 
immediately translocates to the nucleus whereby transcriptional regulation occurs 
(Figure I.2b).
 
STAT3’s role in mammalian 
cells is broad. In mouse ES cells, 
STAT3 is the central protein 
that maintains ‘stemness’ and 
self‐renewal. However, when 
STAT3 was first discovered in 
tumor cell lines, this protein 
demonstrated it was capable of
responding to a variety of 
cytokines, namely, IFNγ, EGF, 
LIF, and IL‐6(Zhong et al., 
1994a). In both ES and somatic 
tissues, STAT3 activation 
mediates cellular proliferation and anti‐apoptosis(Fukada et al., 1996). In ES cells 
the G1 checkpoint does not appear to exist, suggesting that mouse ES cells employ 
Figure I.2c: LIF activates the MAPK/ERK 
and PI(3)K/Akt pathways in mouse ES cells. 
LIF engagement promotes docking an 
activation of the phosphatase SHP2, which 
in turn activates MAPK signaling. MAPK 
activation results in ERK1/2 activation, 
which regulates proliferative and 
differentiation signals. Concurrently, SHP2 
activates PI(3)K which phosphorylates Akt. 
Akt appears to regulate cell cycle and 
ERK1/2 activation. 
 11 
STAT3 to regulate the cell‐cycle in a novel manner(Savatier et al., 1996). Again in 
both ES and somatic cells, STAT3 promotes pro‐survival signals by regulating 
expression of c‐Myc, Src, and Bcl family genes(Bromberg et al., 1998; Kiuchi et al., 
1999). STAT3 is ubiquitously expressed in mammalian tissues, therefore its 
dysregulation is common in specific cancers(Corvinus et al., 2005). 
 
Once activated by Jak2 in mouse ES cells, STAT3 dimers translocate to the nucleus. 
Concurrent to this activity, Jak2 also phosphorylates Y759 upon the gp130 receptor 
which recruits the protein phosphatase SHP‐2(Symes et al., 1997). SHP‐2 then 
activates the Ras/MAPK/ERK pathway(Boulton et al., 1994). In mouse ES cells 
MAPK activation is associated with decreased cellular proliferation and 
differentiation(Burdon et al., 1999; Kunath et al., 2007). ERK2/ mouse ES cells can 
be derived and maintained with reduced proliferative potential(Yao et al., 2003; 
Ying et al., 2008a). ERK1/ mice are viable suggesting ES cells viable as well. Yet 
ERK1/2 double knock out mouse ES cells have yet to be derived(Selcher et al., 2001). 
Unlike STAT3, ERK activation occurs through a step‐wise regulatory pathway 
culminating in transcriptional control (Figure I.2c). 
 
The third arm of the LIF signaling pathway begins with SHP‐2 phosphorylation by 
Jak2, which activates the phospho‐inositol (3‐OH) kinase (PI(3)K) pathway(Boulton 
et al., 1994). PI(3)K is a highly conserved pathway that regulates many cellular 
processes. In mouse ES cells PI(3)K activates Akt, which in turn acts as a pro‐
survival signal(Sun et al., 1999). There are conflicting reports as to the definitive 
 12 
role of Akt in mouse ES cells. While promotion of Akt signaling has been 
demonstrated to augment mouse ES cell self‐renewal in combination with LIF 
signaling, inhibition of Akt shows no significant deleterious effects upon self‐
renewal(Paling et al., 2004). STAT3 inhibition, either by dominant negative 
mutation, Jak2 inhibition, or siRNA‐mediated knock‐down causes immediate mouse 
ES cell differentiation(Niwa et al., 1998b).
 
Burdon et al. demonstrated clearly that STAT3 is the key effector in mediating 
mouse ES cell self‐renewal(Burdon et al., 1999). By mutating the essential SHP‐2 
docking site at tyrosine 118 they determined that STAT3 alone is sufficient to 
promote self‐renewal by inhibiting LIF‐mediated ERK1/2 and Akt signaling. When 
mutated to a dominant negative form, STAT3 cannot promote self‐renewal in the 
absence of ERK1/2 and Akt signaling despite the presence of LIF/Jak signaling. 
Burdon et al. also treat STAT3 activation as a binary ‘On/Off’ system. In this case LIF 
is either present and active, or absent and off.
 
It is the aim of this work presented here to address the question surrounding STAT3’s 
role in regulating the selfrenewal mechanism. I hypothesize that STAT3 regulates 
self‐renewal not by a binary system, but rather as having a range of activity that 
promotes self‐renewal. Precedent for this idea has been demonstrated by Niwa et al. 
They have demonstrated that Oct4 expression is not binary, but rather tightly 
regulated to fall within a specific range, outside of which results in rapid 
differentiation or death of mouse ES cells(Niwa et al., 2000). This mechanism 
 13 
however applies specifically to pluripotency regulation. Separate from the 
pluripotency mechanism, we propose that STAT3 activation regulates self‐renewal 
in a similar manner. 
 
Also, much is known about STAT3 with regards to mouse ES cells, however little has 
been done to investigate the role STAT3 plays in regulating rat ES cells. Our group 
has reported upon the derivation of germline competent rat ES cells, but to date, 
these cells can only be maintained by inhibiting inductive differentiation cues in a 
STAT3‐independent manner(Li et al., 2008). This ‘ground state,’ permits self‐
renewal by limiting MAPK activation in the absence of STAT3 activation(Ying et al., 
2008b). Also important to promoting ground state self‐renewal is inhibition of 
glycogen synthase kinase 3β (GSK3β) which appears to mimic Wnt/β‐catenin 
signaling(Sato et al., 2004). The role of β‐catenin is likely essential for both mouse 
and rat ES cell self‐renewal but has yet to be determined mechanistically.
 
I.3  Permissiveness Relates to Metastable Pluripotent CellStates 
 
The first mouse ES cell lines derived were isolated from 129 strain mice(Evans and 
Kaufman, 1981; Martin, 1981). Whether by chance or premeditation, the 
investigators chose a strain of mice that have the peculiar ability to be derived and 
maintained either in co‐culture with mitotically‐inactivated mouse embryonic 
fibroblasts (‘MEFs’ or ‘Feeders’) or simply a gelatinized substrate alone(Nichols et 
 14 
al., 1990). Initially, many successful ES derivations made use of conditioned medium 
collected from embryonal carcinoma cells, a transformed pluripotent cell type 
capable of self‐renewal and differentiation. Also presumed at the time was that 
factors within the conditioned medium and/or the feeders were of importance for 
maintenance of the undifferentiated state. However, it was found that the feeders 
could be removed when deriving most 129 strain ES cells as long as the conditioned 
medium was present. No other mouse nor rat strain has been routinely derived in 
the absence of feeders with any significant efficiency and are therefore called 
‘feeder‐dependent’(Gardner and Brook, 1997; Kawase et al., 1994; Li et al., 2008). 
129 mouse ES cells that are able to self‐renew upon a gelatinized substrate alone are 
referred to as ‘feeder‐independent.’ 
 
Recently, evidence has postulated that pluripotency itself is a stratified cell‐state 
based on each strain’s or species’ inherent ‘permissiveness.’ It is proposed that 
mammalian pluripotency consists of at least two metastable cell‐states(Hanna et al., 
2009). These states, termed ‘naïve’ and ‘primed’ pluripotency indicate distinct 
molecular biases towards specific in vitro culture preferences(Nichols and Smith, 
2009).
 
Naïve pluripotency refers to cells that express canonical ES cell transcription factors 
such as Oct4, Nanog, and Sox2. In addition, self‐renewal is maintained by LIF/STAT3 
signaling or in ground‐state culture conditions which are STAT3‐independent. In 
addition naïve pluripotent cells retain two active X chromosomes and can 
 15 
contribute to germline chimeras extensively(Nichols and Smith, 2009). Genetic 
modification produced by homologous recombination in naïve pluripotency is more 
efficient than primed pluripotent cells(Buecker et al., 2010). Mouse and rat 
embryonic stem cells are thought to harbor naïve pluripotency. 
 
Primed pluripotent stem cells can be derived from the post‐implantation murine 
egg‐cylinder of mice at 6.5 dpc. These cells express the same canonical pluripotency 
markers such as Oct4 and Nanog, but also up‐ and downregulate a specific subset of 
genes more indicative of stem cells that have prepared to differentiate(Tesar et al., 
2007). Despite this, primed ES cells exhibit robust in vitro self‐renewal. Rex1, a 
marker of the inner cell mass and ES cells, is absent. FGF5, a canonical marker of 
early differentiation that is not expressed in ES cells, is significantly upregulated in 
primed pluripotent cells(Brons et al., 2007). Most telling, however, is that X‐
chromosome inactivation has occurred, suggesting a unique level of epigenetic 
changes has occurred that may be distinct from naïve pluripotent cells(Nichols and 
Smith, 2009). Primed pluripotent cells becomes refractory to LIF/STAT3‐mediated 
self‐renewal and efficient germline transmission has yet to be demonstrated. 
However, these cells are at least in vitro pluripotent and can form teratomas. Mouse 
primed pluripotent cells, termed ‘EpiSCs,’ self‐renew only in the presence of 
bFGF/Activin signaling, and differentiate in the presence of ground state culture 
conditions(Tesar et al., 2007). Primed EpiSCs can be derived from ES cells via 
bFGF/Activin A induction in culture(Brons et al., 2007). Reprogramming back to 
naïve pluripotency has been demonstrated with ectopic expression of Klf4, 
 16 
suggesting that epigenetic changes to the core pluripotency network are essential in 
the transition between cell‐states(Guo et al., 2009).
 
Permissiveness refers to the idea that each mouse strain or species harbors 
different inherent levels of restrictions that permit switching between naïve and 
primed pluripotency(Hanna et al., 2009). Pertinent to our work, it has been detailed 
that 129 mouse ES cells represent the most permissive ES cell line known currently. 
Determining permissiveness relies upon a combination of criteria such as the 
efficiency of derivation, germline contribution, and reprogramming 
efficiencies(Hanna et al., 2009). Permissive lines also appear to demonstrate feeder‐
independent self‐renewal with no apparent ill‐effects to pluripotency.
 
‘Non‐permissive’ mouse strains are described as historically difficult strains to 
derive germline competent ES cells and their relative recalcitrance to retain self‐
renewal in feeder‐free culture systems(Hanna et al., 2009). Examples of such strains 
form the majority of the ‘feeder‐dependent’ ES cells such as NOD background, 
C57BL/6, and Balb/c. Little work has gone into understanding why such differences 
exist.
 
Currently, the least permissive species is human. Recent work has presented 
evidence that human ES cells currently in use may be more accurately characterized 
as EpiSC‐like based on the current classification system used in rodents(Nichols and 
Smith, 2009). It has been shown that human iPS cells can be reprogrammed back to 
 17 
naïve pluripotency with extensive genetic modification and heavily modified culture 
conditions(Buecker et al., 2010; Hanna et al., 2010). This inefficient process results 
in cells that become LIF/STAT3 sensitive for self‐renewal, can be cultured in 
ground‐state conditions, and express transcriptional markers indicative of mouse 
and rat ES cells. In addition, epigenetic changes are apparent, as the reprogrammed 
human iPS cells restore their X chromosomes to active states. In addition, gene‐
targeting efficiency via homologous recombination is significantly higher in the 
mouse ES‐like human iPS cells as compared to human EpiSC‐like cells. However, 
germline transmission testing is impermissible on human pluripotent cells, 
therefore a full and comprehensive analysis of the identity of these cells is likely 
untenable unless it is shown that ES cell lines can be derived and propagated from 
human blastocysts using the culture conditions and concepts proposed by these 
investigators.
 
Permissive mouse strains are currently limited to the 129. One hallmark feature that 
makes this strain popular for researchers is their unique ability to self‐renew 
feeder‐free. The reason why 129 strain mouse ES cells can be maintained feeder‐
free has remained scientifically understudied. Fundamental advances in the last 
twenty years using 129‐derived mouse ES cells have illuminated several key 
characteristics of ES cell biology: The identification of LIF as the main arbiter of 
mouse ES cell self‐renewal may be the most important thus far. LIF’s isolation and 
characterization by Dr. Austin Smith codified the role of extracellular signals in 
promoting mouse ES cell self‐renewal by demonstrating that the feeder cell layer is 
 18 
the predominant in vitro source that secretes LIF in paracrine to activate STAT3 
within mouse ES cells(Nichols et al., 1990; Smith and Hooper, 1987). The 
conditioned medium as well was later determined to contain LIF. LIF activates 
STAT3 primarily, which in turn regulates the self‐renewal program and helps to 
maintain the undifferentiated state. 
 
Dr. Smith then demonstrated that LIF alone could regulate self‐renewal(Nichols et 
al., 1990). LIF was cloned and recombinant versions were found to be sufficient in 
promoting self‐renewal when supplemented in serum containing non‐conditioned 
medium and feeders. However, it was observed that, again, mainly 129 strain mouse 
ES cells could successfully self‐renew in the absence of the feeder cells. All other 
strains were observed to either die or differentiate. This phenomenon was first 
noticed by Sir Martin Evans, and he appropriately entitled it “ES cell crisis(Nagy et 
al., 2003).” No further progress has been made into determining the mechanism by 
which the feeders augment self‐renewal in non‐129 mouse ES cells.
 
By comparison, 129 strain ES cells are employed frequently for basic research 
because of their convenience and the simplicity of the culture system. Although ES 
strains such as C57BL/6 mice are a popular strain for researchers, their ES cells are 
less so due to their feeder‐dependence, sensitivity to minute culture changes, and 
relatively low derivation efficiencies when compared to 129 strain ES cells(Brook 
and Gardner, 1997).
 
 19 
I was charged with investigating the role the feeders played in promoting self‐
renewal. If the feeders are indeed critical in maintaining the self‐renewal of all but 
129 strain mouse ES cells, could the function of the feeders be dissected? I reasoned 
that 129 mouse ES cells’ ability to selfrenew feederfree was tethered to their 
increased permissiveness as compared to other mouse strains. I hypothesized that 
either the feeders provide a necessary signaling supplement to feeder‐dependent ES 
cells or that 129 strain ES cells have an innate advantage or acquired mutation that 
supplements or replaces the feeders’ function. This advantage seemed unique to 
129 ES cells, and therefore, I reasoned that if we could dissect this mechanism, ‘non‐
permissive’ mouse ES cells could be rendered feeder‐free with no ill‐effects to their 
pluripotency.
 
I.4  Rationale for This Investigation 
 
It has been established that conditioned medium alone is not sufficient to promote 
self‐renewal of feeder‐dependent mouse ES cells(Nichols et al., 1990). It appears 
that feeder‐dependent self‐renewal is indeed a contact mediated effect, although we 
cannot rule out short‐range paracrine cytokine interactions nor a highly unstable 
factor/small molecule. The latter scenario is unlikely however, given the extent of 
work performed to analyze conditioned medium(Lim and Bodnar, 2002). However 
the dearth of knowledge in this area of self‐renewal and the multitude of possible 
factors to assay rendered this line of testing unfeasible. Many cytokines have been 
tested as a possible source of self‐renewal, however, none of these cytokines can 
 20 
replace the function of LIF in vitro. We reasoned that studying the differences 
between both 129 and feeder‐dependent mouse ES cells a more expedient manner 
in which to study the self‐renewal mechanism.
 
In this work, I will employ a metric referred to as the ‘crisis assay.’ The assay’s main 
purpose is to measure self‐renewal ability in feeder‐free culture systems. We 
reasoned that efficient long‐term feeder‐free self‐renewal of mouse ES cells in a 
simplified culture system is an effective manner to compare different mouse ES cell 
strains. 
 
I.5  Conclusions 
 
I conclude that increased STAT3 activation can maintain pluripotent populations of 
both mouse and rat ES cells through a shared STAT3‐dependent mechanism. 
Increased STAT3 activation that is sustained over time mediates both mouse and rat 
ES cell self‐renewal. This effect is synergistically enhanced when the MAPK‐ERK1/2 
pathway is inhibited. I posit that STAT3’s role in rodent ES cell self‐renewal is far 
more sophisticated and refined that once thought, and lack of knowledge of this 
specific level of regulation may have impeded past attempts at deriving rodent ES 
cells. We also conclude that increased Klf4 expression enhances mouse ES cell 
feeder‐free self‐renewal in a LIF‐dependent fashion. Klf4 depletion renders mouse 
ES cells capable of feeder‐free self‐renewal only when STAT3 activation is increased. 
As such we note decreased pluripotency marker expression and increased early 
 21 
differentiation marker expression when Klf4 is depleted, suggesting that Klf4 
expression reinforces naïve pluripotency and that depletion ‘primes’ mouse ES cells 
for differentiation. 
 
We posit that the self‐renewal mechanism, like pluripotency, is itself stratified into 
distinct cell‐states. These cell‐states can transition easily between different ranges 
of STAT3 activation and STAT3‐independent pathways within naïve pluripotency. 
This model predicts that there are different rodent self‐renewal mechanisms which 
are themselves stratified within a specific pluripotent cell‐state. 
 
It is our hope the findings presented here accelerate the derivation and simplify the 
maintenance of naïve pluripotent rodent cell populations. In addition, when 
attempting to derive or maintain naïve pluripotent mammalian cells, one must now 
consider using STAT3 in a species‐specific manner rather than uniformly. Also, one 
must decide whether employing a STAT3 independent mechanism or a STAT3 
dependent system relying upon cytokine induction of self‐renewal is appropriate. 
 
 
 
 
 
 
 22 
Results 
 
Chapter One  STAT3 Overexpression Enhances FeederIndependent Mouse ES 
Cell SelfRenewal 
 
1.1 – B6 Mouse ES Cells Die or Differentiate Upon Feeder Depletion 
 
We first tested if there was a quantifiable difference when C57BL/6, hereto after 
referred to as ‘B6,’ mouse ES cells were maintained upon feeders or feeder‐free. B6 
mouse ES cells were seeded at clonal density, approximately 5x10
3
 cells/cm
2
, either 
upon a gelatinized substrate or in co‐culture with CF‐1 feeder cells and 
supplemented with both serum and recombinant LIF. B6 cells exhibit a significant 
decrease in alkaline phosphatase (AP) positivity after seven days of incubation 
(Figure 1.1a). AP expression is a general marker of undifferentiated, self‐renewing 
ES cell colonies. Both cell populations were passaged and AP positive colonies were 
undetectable when the feeders were absent (Figure 1.1b). This finding suggests that 
LIF is likely insufficient in promoting B6 mouse ES cell self‐renewal feeder‐free. 
 23 
 
 
Figure 1.1a: B6 Mouse ES cells enter crisis following feeder depletion. (A) 
B6 wildtype mouse ES cells seeded at clonal density (5x10
3
 cells/cm
2
) in 
coculture with CF1 feeders in serum medium supplemented with 
10ng/ml LIF. (B) The same cells one day following passage into feeder
free conditions begin to die or differentiate despite serum and LIF. (C) 
Cells from (B) one day following a second passage. Cells appear flattened 
with little to no apparent proliferation observed.
 24 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.1b: B6 mouse ES cells lose AP positivity in response to feeder 
depletion. (A) 10
3
 B6 mouse ES cells were seeded either upon a 
gelatinized substrate or in coculture with CF1 feeders in a 4well plate. 
Each condition was supplemented with serum medium and 10ng/ml LIF. 
Following a seven day incubation, the cells were fixed and stained for 
alkaline phosphatase (AP). (B) Photo demonstrating decreased AP 
stained B6 mouse ES cells cultured upon gelatin (left panel) as compared 
to the same cells cultured with feeders (right). Asterisk indicates pvalue 
<0.05, Error bars indicate SD. 
 25 
1.2 – 129 FeederIndependent Mouse ES Cells Increase STAT3 Expression and 
Activation In Response to LIF Stimulation 
 
We then investigated how 129 derived mouse ES cells avoid crisis and promote 
feeder‐free self‐renewal. We began by directly comparing 46C feeder‐independent 
mouse ES cells to B6 mouse ES cells. 46C mouse ES cells are a derivative cell line 
from the parental 129 strain(Ying et al., 2003). We observe that these cells exhibit a 
significantly higher proportion of AP positive colonies as compared to B6 mouse ES 
cells when cultured in a feeder‐independent manner (Figure 1.2a). Following 
seeding upon a gelatinized substrate at clonal density (5x10
3
 cells/cm
2
), each cell 
type was supplemented with 10ng/ml hLIF for 12 hours, after which the cells were 
serum and cytokine starved for 6 hours. At this point serum medium was re‐applied 
and again 10ng/ml LIF was added to the culture medium. Total protein cell lysates 
were collected at one hour and twenty‐four hours post‐stimulation.
 
Western blotting reveals that the 46C mouse ES cells increase STAT3 expression 
levels in response to LIF stimulation by twenty‐four hours post‐induction as 
compared to B6 mouse ES cells (Figure 1.2b). In addition, 46C cells demonstrate 
increased Tyr705 phosphorylation as compared to B6 mouse ES cells 30 minutes 
post‐LIF stimulation. Densiometric analysis confirms this observation (Figure 1.2c‐
d). This finding suggests that feeder‐independent mouse ES cells may survive 
feeder‐withdrawal by increasing both STAT3 expression and activation in response 
to LIF.
 26 
 
 
 
 
 
 
 
 
 
 
Figure 1.2a: Feederindependent 46C selfrenew effectively in the absence of 
feeders. 5x10
3
 cells/cm
2
 each of either B6 or 46C mouse ES cells were plated 
upon a gelatinized substrate with either 10ng/ml LIF or medium alone 
(‘NT’). Following a seven day incubation, the cells were fixed and AP stained. 
46C mouse ES cells were able to significantly retain AP positive colonies as 
compared to B6 cells when supplemented with LIF. Asterisk indicates p
value <0.05. Error bars indicate SD. 
 27 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.2b: Feederindependent 46C mouse ES cells increase STAT3 
expression and activation in response to LIF stimulation as compared to 
B6 mouse ES cells. (A) 10
5
 cells each of B6 or 46C mouse ES cells were 
seeded upon gelatinized substrates. One day later, each cell type was 
stimulated with 10ng/ml LIF. Western blotting of the resulting cell 
lysates reveals that 46C mouse ES cells increase STAT3 expression by 24 
hrs postLIF stimulation as compared to B6 mouse ES cells. (B) 46C 
mouse ES cells increase STAT3 Tyr705 phosphorylation in response to 
LIF as compared to B6 mouse ES cells at 30 minutes poststimulation. 
 28 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.2c: Densiometry indicates that 46C mouse ES cells increase STAT3 
expression following LIF treatment. Image analysis of Figure 1.2b indicates 
that 46C mouse ES cells increase STAT3 expression almost twofold by 24 
hours posttreatment. Data are normalized to tubulin expression. Scale is 
linear. 
Figure 1.2d: Densiometry indicates that 46C mouse ES cells increase STAT3 
phosphorylation following LIF treatment. Image analysis of Figure 1.2b 
indicates that 46C mouse ES cells increase STAT3 expression nearly twofold 
within one hour of treatment. Data are normalized to tubulin expression. 
 29 
 
 
1.3 – STAT3 Overexpression Prevents FeederDependent Mouse ES Crisis 
 
We observe that 46C feeder‐independent mouse ES cells likely increase their STAT3 
expression and activation in response to LIF treatment in feeder‐free culture. We 
hypothesized that overexpressing STAT3 in feeder‐dependent mouse ES cells may 
recapitulate the effects seen in the 46C mouse ES cells. 
 
To test if increasing STAT3 expression may improve feeder‐free self‐renewal, we 
stably introduced a constitutively active, high expression murine STAT3α vector 
into B6 mouse ES cells. Following drug selection, individual clones were selected 
and tested further. We again seeded equal total cell numbers of both 46C and the 
STAT3 overexpressing B6 ES cells (‘B6‐STAT3’). Both cell types were seeded upon 
feeders or gelatinized substrates and supplemented with 10ng/ml LIF. Seven days 
later, AP staining revealed no significant difference between 46C and B6‐STAT3 
mouse ES cells (Figure 1.3a). Western blotting confirms that, compared to wild‐type 
cells, B6‐STAT3 cells do in fact exhibit increased STAT3 expression, as well as 
significantly increased Tyr705 phosphorylation in response to LIF treatment. 
 30 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3a: STAT3 overexpression enhances feederfree selfrenewal of 
B6 mouse ES cells. (A) 5x10
3
 cells/cm
2
 each of either wildtype B6, 46C, 
or STAT3 overexpressing B6 mouse ES cells were plated upon a 
gelatinized substrate with either 10ng/ml LIF or medium alone (‘NT’). 
Following a sevenday incubation, the cells were fixed and AP stained. B6 
mouse ES cells overexpressing STAT3 significantly increase AP positive 
colony formation as compared to wild type B6 mouse ES cells. 46C and 
B6STAT3 mouse ES cells display a similar AP positive colony number 
when LIF is applied. (B) Western blot indicating that STAT3 expression is 
increased following STAT3α transfection. STAT3 phosphorylation 
increased in response to LIF as well. Error bars indicate SD 
 31 
 
To verify that the effect observed is due to STAT3 overexpression, the transgene 
was excised from the genome via Cre recombinase expression. Complete excision 
results in GFP positive expression as a result of the removal of a premature stop 
codon upstream of GFP (Figure 1.3b). As indicated in Figure 1.3c, when compared to 
wild‐type cells upon gelatinized substrates, GFP positive B6 mouse ES cells 
demonstrate a marked loss of AP positivity after seven days incubation feeder‐free 
despite LIF supplementation. This loss of AP positivity is consistent with wild‐type 
B6 mouse ES cells when cultured feeder‐free, suggesting that the increased AP 
positive colonies observed correlates to the increased STAT3 expression (Figure 
1.3d). STAT3 overexpressing B6 mouse ES cells were able to be maintained in tight, 
dome‐like colonies when cultured with LIF feeder‐free for ten passages and stained 
positive for both Oct4 and Sox2 (Figure 1.3e). These findings suggest that STAT3 
overexpression enhances feeder‐independent self‐renewal of B6 mouse ES cells in a 
LIF‐dependent manner.
 32 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3b: STAT3 overexpression prevents crisis and promotes self
renewal. 5x10
3
 cell/cm
2
 each of wildtype or STAT3 overexpressing B6 
mouse ES cells were seeded upon a gelatinized substrate or in coculture 
with CF1 feeders. Cell were fixed and AP stained on Day 7. STAT3 
overexpression enhances feederfree selfrenewal as indicated by AP positive 
staining as compared to wildtype cells cultured with feeders and LIF. 
Feederdepletion results in the loss of AP positivity. Phase contrast pictures 
demonstrating STAT3 transgene removal following Cremediated excision. 
 33 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3c: Removal of STAT3 transgene results in decreased total cell 
number and decreased AP positive colony formation. (A) Equal total cell 
numbers of either B6STAT3 cells or B6STAT3 cells that were 
transfected with Cre recombinase. Cells were counted every day. STAT3 
overexpressing cells increase their total cell number over five days, while 
Cretransfected initially increase then decrease by day five. (B) Cells from 
(A) were AP stained after five days. Cretransfected cells were observed 
to exhibit decreased AP positive colonies as compared to STAT3 
overexpressing cells. 
 34 
 
 
 
 
 
Figure 1.3d: Loss of STAT3 overexpression from B6 STAT3 cells results 
in decreased AP positivity. 5x10
3
 cells/cm
2
 of either wildtype or Cre
recombinanse transfected B6STAT3 cells were seeded upon 
gelatinized substrates and incubated for seven days. The cells were 
then fixed and AP stained. The removal of the STAT3 transgene by Cre 
results in an inability to sustain selfrenewal feederfree. AP staining 
demonstrates that this loss of selfrenewal is consistent with wild
type cells, suggesting that increased STAT3 expression is critical in 
preventing crisis and promoting selfrenewal feederfree. 
 35 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.3e: STAT3 overexpressing B6 mouse ES cells remain 
undifferentiated in longterm feederfree culture. (A) B6STAT3 cells 
maintained upon gelatinized substrates in serum medium supplemented 
with 10ng/ml LIF for five passages retain pluripotency markers Oct4 and 
Sox2. (B) AP stain comparing entire wells demonstrate STAT3 
overexpression mimics 46C mouse ES cell AP positivity. 
 36 
 
1.4  Inhibition of MAPK and Activation of LIF/STAT3 Have a Synergistic Effect Upon 
Preventing Mouse ES Cell Crisis 
 
It has been established that MAPK/ERK signaling is antagonistic to LIF/STAT3 
signaling for ES cell self‐renewal(Matsuda et al., 1999). Burdon et al. demonstrated 
that ERK1/2 attenuation enhances STAT3 mediated self‐renewal(Burdon et al., 
1999). We hypothesized that ERK1/2 activation may play a significant role in 
regulating feeder‐independent self‐renewal. To test this, we stimulated both 46C 
and B6 mouse ES cells with LIF in feeder‐free conditions for 45 minutes. We 
compared total ERK1/2 and phosphorylated ERK1/2 levels between strains. We 
observe in Figure 1.4a that 46C mouse ES cells exhibit decreased total ERK1/2 
expression as well as decreased ERK1/2 phosphorylation as compared to B6 ES 
cells. Densiometric analysis confirmed this finding and reveals that while 46C ES 
cells phosphorylate ERK1/2 in a similar proportion to total ERK expression as 
compared to B6 cells, the overall decrease in ERK1/2 phosphorylation observed in 
46C ES cells may augment feeder‐free self‐renewal (Figure 1.4b).  
 
We then compared ES cells within the B6 strain to observe if the decreased ERK1/2
signaling was simply a product of strain differences. We compared B6 wild‐type ES 
cells to B6‐STAT3 mouse ES cells to interrogate ERK1/2 expression and activation. 
Equal total cellular protein was compared via western blot. STAT3 overexpressing 
cells demonstrate decreased ERK1/2 phosphorylation regardless of treatment as 
 37 
compared to wild‐type cells (Figure 1.4c). ERK1/2 expression levels remained 
grossly equal between cell types, and did not appear to be affected by any treatment. 
Densiometry analysis of the western blots suggests that of the two ERK isoforms, 
ERK1 is reduced by a factor of approximately five (Figure 1.4d). As well, STAT3 
overexpressing B6 cells demonstrate a slightly increased total ERK expression, yet 
further testing is needed to firmly confirm or deny this observation. We interpret 
this observation to mean that increased STAT3 expression may correlate to 
decreased ERK1/2 phosphorylation, which may improve feeder‐free self‐renewal 
ability.
 
 
 
 
 
Figure 1.4a: 46C mouse ES cells exhibit lower total ERK1/2 expression and 
activation as compared to B6 ES cells. Equal total protein from wholecell 
lysates were analyzed via western blot of both wildtype B6 and 46C mouse 
ES cells. Following the addition of 1uM PD0325901, no detectable levels of 
phosphorylated ERK1/2 were observed. 46C mouse ES cells demonstrate a 
significantly lower ERK1/2 expression level as compared to B6 mouse ES 
cells. NT indicates mediumonly treated cells. 
 38 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4b: Densiometry reveals that 46C mouse ES cells harbor decreased 
ERK1/2 expression and phosphorylation as compared to B6 ES cells. Image 
analysis of Figure 1.4a indicates that 46C cells harbor half the total ERK1/2 
expression of B6 ES cells. As well, 46C cells phosphorylate approximately half 
the mount of ERK1/2 as B6 ES cells. The relative proportion of expression: 
phosphorylation within each strain appears equal. Data are normalized to 
tubulin. NT indicates mediumonly treated cells. 
 39 
 
 
 
 
 
 
 
 
 
 
Figure 1.4c: STAT3 overexpression correlates to decreased ERK1/2 
phosphorylation. Total cell lysates were compared via western blot and 
probed for Akt and ERK1/2 phosphorylation following cytokine 
stimulation for 30 minutes. STAT3 overexpressing B6 mouse ES cells 
demonstrate a significant decrease in ERK1/2 phosphorylation as 
compared to wildtype cells. 
 40 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4d: Densiometry reveals that STAT3 overexpression correlates 
to decreased ERK1/2 phosphorylation in B6 ES cells. Image analysis of 
Figure 1.4c reveals that a fivefold increase in STAT3 expression 
correlates to almost a 25 percent reduction in ERK1/2 phosphorylation 
as compared to wildtype cells. Further analysis demonstrates that P
ERK1 is reduced by 75 percent as compared to wildtype cell ERK1 
phosphorylation. Data are normalized to tubulin. 
 41 
 
We then tested if decreasing ERK1/2 phosphorylation would enhance feeder‐free 
self‐renewal. 5x10
3
 cells/cm
2
 B6 wild‐type and B6‐STAT3 mouse ES cells were 
seeded upon gelatinized substrates. One group received LIF only while the other 
received LIF plus the addition of PD0395201, a known specific MAPK inhibitor(Ying 
et al., 2008a). Following seven days of incubation, AP staining revealed that, neither 
LIF nor PD resulted in a significant proportion of AP positive colonies. However, 
when LIF and PD were used in combination, a highly significant increase in AP 
positive colonies resulted (Figure 1.4e). We tested whether STAT3 overexpression 
or 46C mouse ES cells would respond to MAPK inhibition in a similar fashion. STAT3 
overexpression resulted in increased AP positive colonies under LIF stimulation 
alone, and PD also resulted in AP positive colonies. However, again, the combination 
of LIF and PD resulted in a higher proportion of AP positive colonies than either 
condition individually (Figure 1.4f). 46C feeder‐independent mouse ES cells resulted 
in a significant increase in AP positive colonies when compared to B6‐STAT3 mouse 
ES cells upon MAPK inhibition alone. As well, 46C mouse ES cells increase their AP 
positive fraction when both LIF and PD are used in combination, mirroring the B6‐
STAT3 results (Figure 1.4f).
 
Quantitative PCR reveals that STAT3 overexpression increase RNA expression of the 
core pluripotency gene Oct4, Nanog, and Sox2 as compared to wild‐type B6 mouse 
ES cells (Figure 1.4g). Together, these data suggest that MAPK inhibition coupled to 
 42 
LIF stimulation provides a synergistic effect that enhances feeder‐free mouse ES cell 
self‐renewal. 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4e: Inhibition of MAPK improves STAT3 mediated feeder
independent selfrenewal. 5x10
3
 cells/cm
2
 wildtype B6 mouse ES 
cells were seeded upon gelatinized substrates and supplemented 
with LIF, PD0395201, both, or medium alone (‘NT’). Following 
seven days of incubation, the cells were fixed and AP stained. In the 
case of LIF or PD, selfrenewal was impaired. However, when used 
in combination, the selfrenewal was synergistically increased. 
Asterisk indicates pvalue <0.001. Error bars indicate SD. 
 43 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4f: MAPK inhibition increases feederfree selfrenewal when 
STAT3 is overexpressed. Equal total cell amounts (5x10
3
 cells/cm
2
) of 
either B6Y118FSTAT3 or 46CY118F mouse ES cells were seeded upon 
gelatinized substrates and incubated with either 10ng/ml LIF, 1uM 
PD0395201, both or 2pg/ml GCSF for seven days. The cells were then 
fixed and AP stained. Both cell types demonstrate increased AP positive 
staining to either LIF or PD. 46C mouse ES cells significantly increase self
renewal with MAPK inhibition alone in serum medium. Both cell types 
exhibit increased selfrenewal when both LIF and MAPK inhibition are 
used in combination. Asterisks indicate pvalue <0.001. Error bars indicate 
SD. ‘NT’ indicates mediumonly treatment. 
 44 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4g: STAT3 overexpression increases RNA expression of core 
pluripotency genes. Total RNA extracts from passage 15 B6STAT3 
mouse ES cells maintained upon gelatinized substrates supplemented 
with 10ng/ml LIF were converted to cDNA and subjected to 
quantitative PCR analysis to determine expression levels of 
pluripotency genes. Normalized to wildtype B6 mouse ES cells 
maintained in coculture with feeders and supplemented with LIF, 
B6STAT3 cells maintained feederfree demonstrate a robust 
increase in core pluripotency gene expression. A modest 4fold 
increase in STAT3 RNA expression increases Oct4, Nanog, and Sox2 
expression levels over 20fold over wildtype cells, suggesting STAT3 
may regulate these gene’s expression. Rex1, a marker of the 
pluripotent inner cell mass appears unaffected, suggesting it is not 
directly regulated by STAT3 overexpression. Data is normalized to 
wildtype B6 mouse ES cells maintained with feeders, serum, and LIF. 
Data are normalized to GAPDH. 
 45 
 
We then tested if STAT3 expression was required in the context of ERK1/2 
inhibition for enhancing feeder‐free self‐renewal. We employed the use of stat3/ 
mouse ES cells. Stat3/ mouse ES cells are not able to self‐renew via LIF/STAT3 
activation, and can only be maintained as ES cells via chemical inhibition of both 
GSK3β and MAPK(Ying et al., 2008a).
 
To test if STAT3 activation and MAPK inhibition is synergistically promoting mouse 
ES cell self‐renewal, we stably transfected the same constitutively active STAT3α 
vector into these STAT3 null ES cells. Upon drug selection and colony selection to 
isolate individual clones, these stat3/ mouse ES cells overexpressing STAT3 
(‘stat3/(STAT3)’) were seeded at clonal density (5x10
3
 cells/cm
2
) into co‐culture 
with CF‐1 feeders cells. Serum and LIF supplemented medium was applied and, after 
three passages, immunoflourescence analysis revealed positive Oct4 and Sox2 
expression, suggesting these cells are undifferentiated in the presence of the STAT3 
transgene and LIF (Figure 1.4h). Cre‐mediated excision of the STAT3 transgene 
resulted in GFP positive colonies. LIF treatment resulted in immediate 
differentiation, suggesting that STAT3 expression is crucial for preventing 
differentiation. 
 46 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4h: STAT3 overexpression prevents differentiation in stat3/ mouse 
ES cells cultured feederfree. Immunofluorescence images staining for both 
Oct4 and Sox2 in STAT3 overexpressing stat3/ mouse ES cells cultured 
feederfree upon a gelatinized substrate in serum medium supplemented with 
10ng/ml LIF for three passages. 
 47 
 
We then seeded the both stat3/ and stat3/(STAT3) mouse ES cells at clonal 
density upon gelatinized substrates. Either LIF, PD0395201, or both were 
supplemented into the culture medium. Seven days later, AP staining revealed that 
STAT3 overexpressing ES cells exhibited increased AP positive staining after LIF 
treatment. MAPK inhibition did not result in significant AP positive staining by itself 
in either cell type, however the combination of LIF and PD0395201 promoted a 
significantly higher proportion of AP positive colonies than either LIF or 
PD03095201 separately. Lack of STAT3 expression correlated with no positive AP 
staining could be recovered from LIF, PD0325901, nor the combination of the two 
factors (Figure 1.4i). These results suggest STAT3 expression is required for feeder‐
independent self‐renewal, and MAPK inhibition alone is insufficient to promote 
mouse ES cell self‐renewal.
 48 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.4i: STAT3 expression is required for LIFmediated selfrenewal 
feederfree. (A) Stat3/ and Stat3/(STAT3) mouse ES cells were seeded at 
equal density (5x10
3
 cells/cm
2
) upon gelatinized substrates. After various 
cytokine treatments for a seven day period, the cells were fixed and AP 
stained. As indicated, there is no difference between mediumonly (‘NT’) 
treated and LIF treated cells when STAT3 is not expressed. However, upon 
reintroduction of STAT3 via a high expression vector, selfrenewal is 
regained. LIF mediated selfrenewal is enhanced by addition of MAPK 
inhibitor PD0395201, but neither LIF nor MAPK inhibition alone promotes 
selfrenewal. GCSF treatment recapitulates LIF/PD selfrenewal as well. (B) 
AP stain of entire well demonstrates STAT3 expression’s enhances stat3/ 
ES cell selfrenewal. Error bars indicate SD. 
 49 
1.5  βCatenin Activation Enhances Mouse ES Cell SelfRenewal in LIFDependent 
Fashion 
 
β‐catenin likely plays an important role in ES cell self‐renewal. However, most 
reports only examine β‐catenin’s role in isolation(Miyabayashi et al., 2007; Sato et 
al., 2004). To determine if β‐catenin can enhance STAT3‐mediated self‐renewal, We 
hypothesized that increasing nuclear β‐catenin levels would augment STAT3’s 
effects. β‐catenin is canonically activated via the inhibition of GSK3β via the Wnt 
signal pathway(Miller et al., 1999). Since GSK3β inhibition promotes both mouse 
and rat ES cell self‐renewal in the absence of STAT3 signaling, we sought to 
investigate whether it could augment self‐renewal if STAT3 activation was present. 
 
To test this, we employed a mouse ES cell line that contained a floxed, stably 
expressed β‐catenin‐Estrogen receptor fusion protein which, in the presence of the 
chemical 4‐hydroxy tamoxifen (4‐OHT), will cause the nuclear importation of β‐
catenin. This construct was transfected into the B6‐Y118F cell line to take advantage 
of G‐CSF signaling. We then seeded an equal number (10
3
 cells/cm
2
) ES cells at 
clonal density and stimulated this with various permutations of LIF, 4‐OHT, or 
PD184352, a known MAPK inhibitor.
 
 50 
When LIF was used in combination with 4‐OHT, a significant increase in AP positive 
colonies emerged. In the absence of LIF, the combined effects of 4‐OHT and PD18 
did not result in a significant proportion of AP positive colonies (Figure 1.5a). 
 
 
 
 
 
 
 
 
 
 
 
 
 
We observed that despite increasing the proportion of AP positive colonies via the 
combined effects of STAT3 activation and MAPK inhibition, addition of 4‐OHT to this 
condition did not significantly increase the number of AP positive colonies as 
compared to omitting it (Figure 1.5b).
Figure 1.5a: βCatenin activation does not significantly enhance feeder
free B6 selfrenewal in the absence of LIF. B6T2ER ES cells were 
subjected chemical inhibition of MAPK or activation of βcatenin. AP 
positive colonies were quantified at Day 7. βcatenin and MAPK 
inhibition alone did not result in significant AP positive colony 
formation. The combination of LIF and 4OHT increased AP positive 
colony formation only when PD18 was present. Asterisk indicates pvalue 
<0.05. Error bars indicates SD. 
 51 
 
 
 
 
 
 
 
 
 
To demonstrate that the increased self‐renewal observed was due to β‐catenin 
activation via transgene activation, the transgene was excised using Cre 
recombinase. Clones were again selected, tested for transgene excision, and then 
subjected to the previous assay. Figure 1.5c demonstrates that removal of the 
transgene correlates to a loss of AP positive colonies despite LIF and 4‐OHT 
stimulation. These results suggest that β‐catenin may augment LIF‐mediated self‐
renewal, but that this effect is not critical in promoting feeder‐free self‐renewal in 
B6 mouse ES cells.
Figure 1.5b: βcatenin Activation Does Not Significantly Increase SelfRenewal 
when despite LIF stimulation and MAPK inhibition. When LIF plus MAPK 
inhibition was implemented into culture with 4OHT, no significant increase was 
observed in selfrenewal.. Error bars indicate SD. 
 52 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.5c: Feederfree selfrenewal ability is impaired following β
catenin transgene excision. 10
3
 cells/cm
2
 of B6 mouse ES cells expressing 
the T2ER transgene were seeded upon a gelatinized substrate. B6T2ER 
mouse ES cells transfected with Cre recombinase were seeded as well. 
10ng/ml LIF, was added and the cell incubated for seven days. The cells 
were then fixed and AP stained. Cells treated with LIF and 4OHT 
demonstrate selfrenewal feederfree only when the T2ER gene is present. 
Cre transfected cells are unable to selfrenew despite LIF and 4OHT 
stimulation. Asterisk indicates pvalue <0.05. Error bars indicate SD. 
 53 
 
Chapter Two  Increased Endogenous STAT3 Activation is Sufficient to Maintain 
FeederIndependent Mouse ES Cell SelfRenewal 
 
2.1 – Artificial Activation of Endogenous STAT3 Can Functionally Replace LIF in Mouse 
ES Cells 
 
Since STAT3 overexpression is not physiologically relevant, we then tested if 
endogenous STAT3 activation was sufficient to enhance feeder‐independent mouse 
ES cell self‐renewal. We have observed previously that STAT3 overexpression leads 
to significantly increased Tyr705 phosphorylation levels as compared to wild‐type 
cells despite using an equivalent concentration of LIF. We sought to mimic this 
increased STAT3 activation by stable integration and expression of a chimeric 
receptor that has been shown to control STAT3 activation in a dose‐dependent 
fashion.
 
The receptor is constructed as the N‐terminal extracellular domain of the 
granulocyte‐colony stimulating factor (G‐CSF) receptor fused to the cytosolic 
domain of the common IL‐6 receptor gp130(Niwa et al., 1998b). Gp130 has been 
shown to be the key mediator in transducing the LIF signal to STAT3, MAPK, and 
PI(3)K in mouse ES cells. In addition, this chimeric receptor contains a loss‐of‐
function mutation at Tyr118 of gp130 (as calculated from the fusion boundary 
between the GCSF receptor and gp130) that results in the inability to recruit the 
 54 
phosphatase SHP‐2. SHP‐2 has been demonstrated to be the key mediator in 
activating the MAPK/ERK and PI(3)K/AKT pathway in mouse ES cells.
 
The G‐CSF receptor is not expressed in wild‐type mouse ES cells, so we 
hypothesized that addition of G‐CSF to the cell culture would activate the chimeric 
receptor, which would in turn activate STAT3(Burdon et al., 1999).
 
We then stably transfected and expressed the chimeric gp130 receptor in wild‐type 
B6 mouse ES cells. Following drug and colony selection, individual clones were 
tested for their ability to respond to G‐CSF. G‐CSF was applied to transfected clones 
seeded at clonal density upon gelatinized substrates in decreasing two‐fold 
dilutions, beginning with 10ng/ml. AP staining revealed that, after seven days 
incubation, G‐CSF treated cells exhibited a dose‐dependent response in positive 
correlation to the concentration applied. It was found that nominal concentrations, 
~0.2ng/ml, were sufficient to promote feeder‐independent self‐renewal as 
indicated by AP staining (Figure 2.1a). Densiometric analysis revealed that relative 
STAT3 phosphorylation levels decreased by approximately one‐half for every ten‐
fold decrease in G‐CSF for the concentrations tested (Figure 2.1b). Below 0.2pg/ml 
G‐CSF treatment could not prevent crisis.
 55 
 
 
 
 
 
 
 
 
 
 
Figure 2.1a: GCSF mediated STAT3 activation exerts a dosedependent self
renewal effect. (A) 10
3
 cells/cm
2
 B6Y118F mouse ES cells were seeded upon 
a gelatinized substrate in serum medium and supplemented with a 
decreasing concentration of GCSF, 10ng/ml LIF, or medium alone. 
Following a seven day incubation, the cells were fixed and AP stained. AP 
positive staining shows a progressive loss as the GCSF concentration 
decreases. LIF appears to be insufficient to promote feederindependent self
renewal even at high concentration. (B) Western blot comparing the effects 
of decreasing concentrations of GCSF upon STAT3 phosphorylation. As 
indicated, phosphorylated Tyr705 levels decrease in concordance with 
decreasing GCSF concentrations. 
 56 
 
 
 
 
 
 
 
To identify if G‐CSF was exerting the self‐renewal effect through the Jak/STAT3 
pathway, we employed the use of a well‐characterized Jak2 inhibitor, AG490. Again, 
B6‐Y118F mouse ES cells were plated upon gelatinized substrates at clonal density 
and supplemented with 0.2ng/ml G‐CSF, with one well receiving an additional 
supplement of 250nM AG490(Eriksen et al., 2001). AP staining reveals that while G‐
CSF by itself is enough to prevent mouse ES cell crisis, the addition of AG490 
significantly reduced the amount of self‐renewing colonies (Figure 2.1c). This result 
suggests that G‐CSF stimulation of the chimeric receptor is mediating self‐renewal 
through a Jak/STAT3 signal pathway.
Figure 2.1b: Densiometry reveals that STAT3 phosphorylation levels decrease in 
correlation with decreasing GCSF concentrations. Image analysis of Figure 2.1a 
indicate that as GCSF concentration decreases, phosphorylated STAT3 
decreases as well. Data are normalized to tubulin expression. 
 57 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.1c: Jak inhibition decreases STAT3 mediated mouse ES cell self
renewal efficiency feederfree. 5x10
3
 cells/cm
2
 B6Y118F cells were 
seeded upon a gelatinized substrate and incubated with either 10ng/ml 
LIF, 0.2ng/ml GCSF or serum medium alone. One group was 
supplemented further with 250 nM Jak inhibitor AG490 and the other 
DMSO control. Following a seven day incubation, the cells were fixed and 
AP stained. LIF treated cells did selfrenew appreciably with or without 
Jak inhibition, however GCSF treated cell selfrenewal were significantly 
inhibited by Jak inhibition, suggesting GCSF is exerting its effects 
through STAT3. Asterisk indicates pvalue <0.01. Error bars indicate SD. 
 58 
 
2.2 – Increased Endogenous STAT3 Activation Prevents Crisis in Other Feeder
Dependent Mouse ES Cell Lines 
 
We then tested if endogenous STAT3 activation prevents crisis effect was limited to 
the B6 mouse ES cell line. I selected another feeder‐dependent mouse ES cell line, 
Balb/C1. I then stably expressed the same chimeric G‐CSF/gp130 receptor, drug 
selected the transfectants and isolated individual clones. Balb/C1 mouse ES cells 
containing the chimeric receptor (termed ‘Balb/C‐Y118F’) demonstrated AP 
positive colonies in the presence of G‐CSF. Balb/C ES cells were not able to self‐
renew effectively in the presence of LIF when seeded at clonal density upon a 
gelatinized substrate as indicated by alkaline phosphatase staining (Figure 2.2a). 
Our results suggest that the rescue from ES cell crisis is likely not an anomalous 
phenomenon relegated to a specific mouse ES cell strain, but rather a general 
phenomenon . 
 59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.2b: STAT3 Activation promotes feederindependent selfrenewal of BalbC1 
mouse ES cells. (A) 5x10
3
 cells/cm
2
 of either B6Y118F or Balb/c1Y118F mouse ES 
cells were seeded onto a gelatinized substrate in serum medium containing either 
10ng/ml LIF, 10ng/ml GCSF, 1ng/ml GCSF, or medium alone. Balb/c mouse ES 
cells mirror B6 mouse ES cells in that they exhibit morphology consistent with self
renewal in the presence of GCSF but not LIF. (B) AP staining of Balb/cY118F 
mouse ES cells after seven days of culture feederfree in either LIF, GCSF, or 
medium alone. Strong AP positive staining of GCSF treated cells suggest that 
feederfree selfrenewal is enhanced by GCSF treatment Balb/c1 ES cells. 
 60 
 
In addition, I assayed a feeder‐independent mouse ES cell line to ascertain whether 
G‐CSF mediated STAT3 activation could substitute for LIF in this capacity. Using 46C 
mouse ES cells transfected with the chimeric receptor (‘46C‐Y118F’), I added a 
gradient of concentrations of G‐CSF with a range of 200pg/ml – 0.78pg/ml, a range 
of three orders of magnitude. As seen in Figure 2.2c, G‐CSF treatment results in AP 
positive colonies. Peripheral differentiation, as suggested by loss of AP positive 
staining, increases as G‐CSF concentration falls. In addition we observe a general 
flattening of the individual colonies and gradual loss of AP staining. Previously we 
demonstrated that G‐CSF activates STAT3, and that STAT3 activation mediates 
mouse ES cell feeder‐free self‐renewal. It can therefore be inferred that STAT3 
mediates feeder‐free self‐renewal irrespective of mouse ES cell strain.
 
 
 61 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.2c: Feederindependent 46C mouse ES cells exhibit a dosedependent 
selfrenewal response to GCSF treatment. 10
3
 cells/cm
2
 46CY118F mouse ES 
cells were seeded upon gelatinized substrates and supplemented with 
decreasing concentrations of GCSF, 10ng/ml LIF, or medium alone (‘NT’). 
Following seven days of incubation, the cells were fixed and AP stained. GCSF 
appear to exert a similar effect as B6 mouse ES cells in response to GCSF 
mediated STAT3 activation. AP positive staining decreases with decreasing G
CSF concentration. LIF stimulation produces AP positive colonies, but overt 
marginal differentiation is apparent.
 62 
 
Chapter Three  Increased and Sustained STAT3 Activation Promotes Mouse and 
Rat ES Cell SelfRenewal 
 
3.1  GCSF Stimulation Promotes Increased STAT3 Activation Over 24 Hours 
 
LIF/STAT3 signaling reaches a peak activation level approximately thirty minutes 
after induction(Niwa, 2001; Niwa et al., 1998a). At which point, negative feedback 
loops such as SOCS3 are upregulated by active STAT3 which attenuate STAT3 
signaling by binding Jak2 and preventing it from phosphorylating STAT3(Li et al., 
2005; Sasaki et al., 1999). We sought to compare G‐CSF stimulation to LIF induction 
to investigate if G‐CSF reached a similar peak of activation. Following stimulation of 
B6‐Y118F mouse ES cells upon gelatinized substrates, total protein lysates were 
collected at thirty minutes, sixteen hours, and twenty‐four hours post‐induction 
with both cytokines. Western blotting revealed that G‐CSF activated an increased 
amount of STAT3 at thirty minutes as compared to LIF, but also this activation level 
is sustained over twenty‐four hours (Figure 3.1a). Densiometric analysis indicates 
that G‐CSF treatment increased STAT3 phosphorylation levels approximately two‐
fold as compared to LIF. This increase degraded slightly over time, but remained at 
least two‐fold higher than LIF treated cells (Figure 3.1b). LIF, on the other hand, 
shows a return to basal activation levels by sixteen hours, with a maximum at thirty 
minutes. STAT3 expression levels remain relatively unchanged during either 
 63 
treatment (Figure 3.1c). These data suggest that increasing STAT3 activation over 
time may enhance mouse ES cell feeder‐free self‐renewal.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.1a: GCSF mediated STAT3 phosphorylation is increased and 
sustained over 24 hours. Total cell lysates extracted from B6Y118F mouse 
ES cells cultured feederfree were compared for Tyr705 phosphorylation. 
Cells stimulated with 10ng/ml LIF demonstrate peak phosphorylation at 30 
minutes poststimulation with a return to basal levels by 16 hours. GCSF 
treated cells demonstrate increased phosphorylation at 30 minutes and 
relatively stable maintenance of this phosphorylation over 24 hours. Both 
cell types appear to modestly increase STAT3 expression levels over time as 
well.
 64 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.1b: Densiometry reveals that GCSF treated mouse ES cells retain 
an increased STAT3 phosphorylation level over 24 hours. Image analysis of 
Figure 3.1a indicates that GCSF stimulation of B6 mouse ES cells increases 
STAT3 phosphorylation levels with minimal signal degradation over 24 
hours as compared to LIF treated cells. Data are normalized to tubulin 
expression. 
Figure 3.1c: Densiometry reveals that GCSF treated mouse ES cells retain a 
stable STAT3 expression level over 24 hours. Image analysis of Figure 3.1a 
indicates that GCSF stimulation of B6 mouse ES cells causes both LIF 
treated and GCSF treated cells to retain relatively similar STAT3 expression 
levels over 24 hours. Data are normalized to tubulin expression. 
 65 
To compare STAT3 activation level‐profiles between cell lines, we then expressed 
the chimeric gp130 receptor in STAT3 overexpressing B6 mouse ES cell lines using 
the methods previously outlined, a line now termed ‘B6‐Y118F‐STAT3’. We then 
transfected Cre recombinase into the B6‐Y118F‐STAT3 cell line to remove the 
STAT3 transgene. Following selection of colonies, we expanded both cell lines to 
compare their ability to activate STAT3 in response to G‐CSF. After stimulating each 
cell type with either 10ng/ml LIF, 0.2ng/ml G‐CSF or serum medium alone, we 
compared each cell line via western blot. When compared to both wild‐type cells or 
those only expressing the chimeric receptor alone (B6‐Y118F), B6‐Y118F‐STAT3 
mouse ES cells demonstrate a significant increase in Tyr705 phosphorylation in 
response to both LIF and G‐CSF. Following removal of the STAT3 transgene, STAT3 
expression and activation levels return to wild‐type levels, suggesting that 
increasing STAT3 expression allows for increased STAT3 phosphorylation in 
response to LIF or G‐CSF (Figure 3.1d). 
 66 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.1d: GCSF activates STAT3 in proportion to STAT3 expression level. 
Equal total cellular protein from B6 mouse ES cells was compared via 
western blot analysis. Wildtype B6 mouse ES cells respond to 10ng/ml LIF 
but not GCSF, while all remaining cell lines stably expressing the chimeric 
gp130 receptor activate STAT3 in response to 0.2ng/ml GCSF treatment. 
STAT3 overexpression results in a significant increase in STAT3 Tyr705 
phosphorylation by both 10ng/ml LIF and 0.2ng/ml GCSF. Cremediated 
excision of the STAT3 transgene results in a return to wildtype STAT3 
expression levels. 
 67 
3.2 – GCSF Stimulation Correlates to Increased Socs3 RNA Expression 
 
We then investigated whether G‐CSF treatment targets similar LIF/STAT3 gene 
targets. We chose Socs3 as it is well characterized primary target of STAT3 signaling 
in mouse ES cells(Duval et al., 2000; Heinrich et al., 2003; Li et al., 2005). We 
decided to ascertain the kinetics of RNA expression in G‐CSF treated mouse ES cells 
over time.
 
Quantitative PCR revealed that G‐CSF treated mouse ES cells increase Socs3 RNA 
expression rapidly by one hour and this RNA level decreased after 24 hours of 
stimulation (Figure 3.2a). LIF treatment demonstrated a similar expression pattern 
in Socs3 RNA expression level as well. Indeed this finding is corroborated by 
microarray data that indicates that Socs3 is upregulated in response to G‐CSF and 
LIF treatment (Section 3.3). These data suggest G‐CSF treatment may be targeting 
similar genes as LIF, suggesting that G‐CSF regulates mouse ES cell self‐renewal by 
targeting a similar gene set as LIF, yet possibly regulating this set in a distinct 
manner from LIF. 
 
 68 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.2a: SOCS3 RNA expression levels do not appear to 
significantly differ between LIF or GCSF treatments. Total RNA 
extracts from B6Y118F mouse ES cells stimulated with either 
0.2ng/ml GCSF, 10n/ml LIF, or medium alone over 24 hours. 
Both GCSF and LIF treated cells exhibit similar expression 
patterns over time. Data normalized to mediumonly treatment. 
Error bars indicate SEM. 
 69 
 
3.3 – STAT3 Activation Induces Differentiation When STAT3 is Overexpressed 
 
The duration of the STAT3 activation signal appears to regulate mouse and rat ES 
cell self‐renewal feeder‐free. We sought to understand if STAT3 expression has a 
functional ceiling. A functional ceiling is defined as increasing either STAT3 
expression or activation levels so that the protein no longer increases self‐renewal 
ability or possibly begins to harbor a deleterious effect upon self‐renewal.
 
We then applied 0.2ng/ml G‐CSF, the ideal concentration determined for B6 mouse 
ES cells with wild‐type STAT3 expression levels, to B6‐Y118F‐STAT3 mouse ES cells. 
Two days later, we observed B6‐Y118F‐STAT3 mouse ES cells were no longer 
arranged in colonies, but rather as individual flattened cells that could not be 
passaged. RT‐PCR analysis suggests these cells are predominantly primitive 
endoderm following G‐CSF treatment (Figure 3.3a). This result suggests that 
increasing STAT3 activation in the context of overexpression may induce ES cell 
differentiation.
 70 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.3a: GCSF mediated STAT3 activation correlates to rapid 
induction of mouse ES cell differentiation. (A) B6Y118FSTAT3 mouse 
ES cells maintained in coculture with CF1 feeders and serum medium 
supplemented with 10ng/ml LIF. (B) B6Y118FSTAT3 mouse ES cells 
passaged into feederfree conditions and supplemented with 0.2ng/ml G
CSF immediately differentiate within 3 days of stimulation. (C) 5x10
3
 
cells/cm
2
 B6Y118FSTAT3 mouse ES cells were seeded upon a 
gelatinized substrate in serum medium supplemented with 0.2ng/ml. 
Reversetranscription PCR analysis reveals that GCSF mediated 
differentiation results in Sparc positive cell types. 
 71 
3.4  Non129 Mouse ES Cells Maintained FeederFree Exhibit Robust In Vitro 
Pluripotency 
 
To verify that feeder‐free self‐renewal does not impinge upon pluripotency, B6‐
Y118F mouse ES cells were seeded at clonal density upon a gelatinized substrate. 
Serum‐containing medium was supplemented with 0.2ng/ml G‐CSF and the cells 
were allowed to proliferate. Following fifteen passages in this condition, the cells 
were subjected to in vitro pluripotency assays to verify ES identity:
 
Embryoid body (EB) formation assays were performed by seeding 5x10
6
 B6‐Y118F
mouse ES cells onto a uncoated, bacterial grade petri dish in serum medium. 
Medium was changed every other day as the ES cells aggregate and form embryoid 
bodies that proceed with differentiation. Following maturation by approximately 
Day 12‐14, roughly four or five mature EB’s were seeded upon a gelatinized four‐
well substrate. The cells were allowed to continue differentiating until 
spontaneously beating areas were observed by approximately Day 7, post‐plating. 
At this point the cells were stained for markers of differentiation. 
 
 As indicated in Figure 3.4a, B6‐Y118F mouse ES cells cultured feeder‐free for fifteen 
passages demonstrate the ability to form tissue types of the three somatic germ 
layers. This finding was confirmed by RT‐PCR and quantitative PCR analysis of both 
conventionally LIF/feeder maintained B6 mouse ES cells and the G‐CSF maintained 
versions. 
 72 
 
In addition, we interrogated the B6‐Y118F mouse ES cells for their global RNA 
expression patterns to determine if G‐CSF treatment significantly overlaps with LIF 
treatment. Since G‐CSF can support feeder‐independent self‐renewal and LIF 
cannot, it can be hypothesized that the gene targets may be different for the two 
conditions. We subjected B6‐Y118F mouse ES cells to stimulation with either 
0.2ng/ml G‐CSF, 10ng/ml LIF, or medium alone for 45 minutes. The total RNA was 
analyzed and globally compared. G‐CSF increases expression of a small subset of 
genes and downregulates the remainder, as compared to medium‐treated only cells. 
LIF treatment presents a gene expression pattern similar to medium‐only treated 
cells. This results suggests that LIF’s gene targets are consistent with a 
differentiating phenotype when feeder‐dependent mouse ES cells are placed into a 
feeder‐free environment (Figure 3.4b). Upon further examination of the 
LIF/Jak/STAT3 pathway, G‐CSF treatment reveals that it significantly 
downregulates SHP‐2, Ras, and Akt pathway RNA. This result confirms that G‐CSF 
mediated STAT3 activation is likely the critical effector of feeder‐independent self‐
renewal, and that inhibition of MAPK likely enhances this effect. 
 73 
 
 
 
 
 
 
 
 
Figure 3.4a: Feederfree maintenance of B6 mouse ES cells in GCSF 
conditions maintains pluripotent identity. (A) Passage 15 B6Y118F mouse 
ES cells maintained in 0.2ng/ml GCSF upon gelatinized substrates in the 
absence of feeders were subjected to embryoid body formation. The resulting 
differentiated cell types were probed for markers of individual germ layers. 
The cells demonstrate positive staining for all three germ layers  βIII
tubulin (ectoderm), gata4 (endoderm), and muscle sarcomere MF
20(mesoderm) – suggesting these cells are likely pluripotent. (B) RTPCR 
analysis further suggests no significant difference in differentiation ability 
exist between feederfree and feederdependent populations. 
 74 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.4b: GCSF treatment of mouse ES cells regulates a distinct set of 
genes as compared to LIF. (A) Microarray analysis of B6Y118F mouse ES 
cells maintained feederfree in either 0.2ng/ml GCSF, 10ng/ml LIF, or 
medium alone (‘NT’) reveals that GCSF gene patterning is significantly 
distinct from LIF. GCSF upregulated only a small set of genes, while 
generally downregulating the remainder as compared to LIF or medium 
alone. (B) Specific genes of the LIF/Jak/STAT3 pathway were analyzed in 
detail and reveal that GCSF downregulates MAPK and Akt family member 
genes, while LIF appears to increase expression of these genes. (C) Table 
comparing the total significantly up or downregulated genes for direct 
treatment comparisons. Heatmap colors indicate relative foldchange in 
expression. Red equals increased expression, while green indicates decreased 
expression, and black indicates no change. 
 75 
We interrogated the G‐CSF maintained B6‐Y118F ES cells for pluripotency markers. 
Immunofluorescence and western blotting reveal positive staining for Oct4, Nanog, 
and Sox2 at passage fifteen. Quantitative PCR was performed to directly compare 
pluripotency gene RNA expression levels of G‐CSF B6‐Y118F mouse ES cells 
maintained feeder‐free to wild‐type B6 mouse ES cells maintained upon feeders and 
supplemented with LIF. qPCR results revealed similar expression patterns between 
both cell types (Figure 3.4c). This result indicates that G‐CSF/STAT3 maintained 
cells are likely pluripotent populations of ES cells as compared to their feeder‐
maintained counterparts. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 76 
Figure 3.5c: B6 mouse ES cells maintain pluripotency following long‐term culture 
feeder‐free. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 77 
Figure 3.5c continued 
 
 
 
 
 
 
 
 
 
 
 
 78 
Figure 3.5c continued 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.4c: B6 mouse ES cells maintain pluripotency following longterm 
culture feederfree. (A) B6Y118F mouse ES cells were maintained upon 
gelatinized substrates in serum medium containing 0.2ng/ml GCSF and 
passaged at regular intervals. (B) RTPCR analysis of passage 15 GCSF 
maintained B6 mouse ES cells. Feederfree maintenance does not appear to 
adversely affect core pluripotency gene expression as compared to wildtype 
B6 mouse ES cells maintained with feeders and LIF. (C) Western blot 
demonstrating protein expression of Oct4 and Nanog is comparable between 
both feederfree and canonical culture conditions. (D,E) 
Immunofluorescence demonstrating feederfree maintained mouse ES cells 
express Oct4 and Sox2. (F) Quantitative PCR analysis of B6Y118F cells 
maintained in 0.2ng/ml GCSF feederfree as compared to cells in 10ng/ml 
LIF with feeders or medium alone. Feederfree maintenance results in a 
comparable gene expression pattern over 24 hrs as compared to LIF and 
feeders. FGF5, a marker of early differentiation is also downregulated in 
response to GCSF treatment. Error bars indicate SEM. 
 79 
 
3.5  Sustaining Rat ES Cell SelfRenewal Through Manipulating STAT3 Signaling 
 
Our group has reported the derivation and characterization of rat embryonic stem 
cells(Li et al., 2008). This pluripotent cell type relies upon derivation and 
maintenance upon mouse embryonic fibroblasts, serum‐free defined medium, and 
the combined inhibition of both GSK3β and MAPK. In direct contrast with their 
murine counterparts, rat ES cells rely upon shielding from inductive differentiation 
cues in a STAT3‐independent manner. This cell state has been described as the 
‘ground state’ of embryonic stem cells, and has been shown to be applicable in the 
derivation and maintenance of mouse ES cells as well. 
 
However, we observed in our work that rat ES cells, despite their ability to self‐
renew in a STAT3 independent manner, demonstrated an acute sensitivity to LIF 
stimulation. Upon addition of LIF to the culture medium, DAc2 rat ES cells robustly 
phosphorylate Tyr705 upon STAT3 in a manner consistent with mouse ES cells. In 
addition rat ES cells phosphorylate Ser727 in concordance with mouse ES cells 
(Figure 3.5a). Rat ES cells express slightly less STAT3 than mouse ES cells, yet 
activate a comparable fraction of when stimulated with LIF (Figure 3.5b). 
 80 
 
 
 
 
 
 
 
 
 
Figure 3.5a: DAc8 rat ES cells activate STAT3 in response to LIF 
stimulation. Total protein lystates were directly compared via 
western blot to ascertain if LIF causes phosphorylation of STAT3. As 
compared to mouse ES cells, rat ES cell do appear to phosphorylate 
Tyr705 robustly and demonstrate comparable STAT3 expression 
levels to mouse. 
Figure 3.5b: Densiometry comparing rat and mouse ES cell STAT3 levels. 
Image analysis of Figure 3.5a demonstrates that rat ES cells express 
slightly less STAT3, yet activate slightly more as compared to mouse ES 
cells. Cells were stimulated with LIF for 30 minutes.
 81 
 
Previously, we have established that sustained STAT3 activation can promote 
robust feeder‐independent mouse ES cell self‐renewal in non‐129 mouse strains. 
We hypothesized that rat ES cells may respond similarly to STAT3 activation as long 
as STAT3 is regulated appropriately.
 
It was demonstrated that rat ES cells overexpressing STAT3 are able to self‐renew 
effectively in the presence of LIF and feeders. The STAT3 transgene was stably 
expressed in DAc2 rat ES cells. Western blotting reveals that STAT3 expression is 
increased and responsive to LIF stimulation. STAT3 overexpressing rat ES cells 
remain Oct4 and Sox2 positive in long‐term culture maintained in LIF and serum 
supplemented medium (Figure 3.5c). This result suggests that rat ES cells are 
sensitive to STAT3 expression and activation levels, akin to mouse ES cells. We then 
hypothesized that specific activation and regulation of endogenous STAT3 would 
recapitulate the positive effects observed upon self‐renewal observed in the mouse. 
 
To test this hypothesis, we began by stably introducing the chimeric gp130 receptor 
into DAc8 rat ES cells. Following drug selection and clone isolation, we tested 
individual clones for their ability to self‐renew feeder‐free. Under no condition 
(laminin, fibronectin, or matrigel‐coated substrates) could we identify a G‐CSF 
concentration that would promote feeder‐independent self‐renewal. This is 
unsurprising, as extracellular matrix (ECM) components that have been 
demonstrated to promote either mouse or human ES cell self‐renewal in lieu of 
 82 
feeder cells are well‐documented, but in rat ES cell culture, no such condition 
faithfully reproduces the effects of feeders. 
 
 
 
 
 
 
 
 
Figure 3.5c: STAT3 overexpression promotes LIF mediated selfrenewal of rat ES 
cells. (A) Total protein lysates were compared via western blot following stable 
overexpression of STAT3 in DAc2 rat ES cells. As is demonstrated, Tyr705 
phosphorylation is increased in response to LIF following STAT3 overexpression. 
Following gene excision via Cre recombinase, STAT3 phosphorylation levels 
return to wildtype levels. (B) STAT3 overexpression results in LIF mediated self
renewal of rat ES cells longterm, as indicated by Oct4 and Sox2 expression. (C) 
Cremediated excision of the transgene results in the loss of exogenous STAT3 
expression and loss of Oct4 and Sox2 expression despite LIF stimulation. 
 83 
We therefore performed our assays upon feeder cell layers. Previous research has 
revealed that only the combined use of specific GSK3β and MAPK inhibition (‘2i’) 
prevents rat ES cell differentiation. As a point of comparison, we employed both LIF 
and 2i treated wild‐type DAc8 rat ES cells to compare self‐renewal efficiency. To test 
if G‐CSF could prevent rat ES cell differentiation, we seeded DAc8 rat ES cells 
expressing the chimeric gp130 receptor (termed ‘DAc8‐Y118F’) at clonal density 
upon a gelatinized substrate pre‐coated with CF‐1 feeder cells. We determined that 
0.02ng/ml G‐CSF was optimal for promoting rat ES cell self‐renewal. As seen in 
Figure 3.5d, both 2i and 0.02ng/ml G‐CSF promoted rat ES cell self‐renewal, yet 
10ng/ml LIF stimulation results in rapid differentiation. 
 
 
 
 
 
 
Figure 3.5d: GCSF supports rat ES cell selfrenewal. (A) Phase contrast 
photo displaying DAc8 rat ES cells maintained in the canonical 2i culture 
system in serumfree N2B27 medium. (B) 10ng/ml LIF application to DAc8 
rat ES cells results in differentiation within two passages. (C) 0.02ng/ml G
CSF application to DAc8Y118F rat ES cells are able to selfrenew efficiently 
over fifteen passages with no overt differentiation. (D) GCSF maintained 
DAc8Y118F rat ES cell AP stain at passage 15.
 84 
We then seeded equal total Dac8‐Y118F rat ES cells into co‐culture with CF‐1 
feeders and stimulated them with either LIF, G‐CSF, 2i, or medium alone. G‐CSF 
treated cells demonstrate significantly increased AP positive colonies as compared 
to either 2i or LIF. We observed no AP positive colonies in the LIF‐treated condition 
(Figure 3.5e). Thus, we hypothesized that the chimeric receptor may be functioning 
as in mouse ES cells to promote self‐renewal. 
 
 
 
 
 
 
To verify the chimeric receptor’s function, I seeded both wild‐type DAc8 and DA‐
Y118F rat ES cells at clonal density, approximately 5x10
3
 cells/cm
2
, upon a layer of 
feeder cells. Both cell types were stimulated with either 10ng/ml LIF or 0.02ng/ml 
Figure 3.5e: GCSF mediated STAT3 activation increases rat ES cell self
renewal ability over 2i. 5x10
3
 cells/cm
2
 DAc8Y118F rat ES cells were seeded 
in coculture with CF1 feeders in serumfree N2B27 medium. Either 10ng/ml 
LIF, 3uM CHIR99201 plus 1uM PD0395201 (‘2i’), or 0.02ng/ml GCSF were 
added and allowed to incubate for seven days. The cells were then fixed and 
AP stained. GCSF significantly promotes selfrenewing rat ES cell colonies as 
compared to 2i or no treatment. LIFtreatment reveals almost no self
renewing colonies. Asterisk indicates pvalue <0.05. Error bars indicate SD. 
 85 
G‐CSF and total cell lysates were compared for protein expression. Western blotting 
revealed that both LIF and G‐CSF demonstrate robust Tyr705 phosphorylation in 
DA‐Y118F rat ES cells (Figure 3.5f). Densiometric analysis confirms that DAc8‐
Y118F rat ES cells phosphorylate STAT3 comparably to wild‐type cells when either 
LIF or G‐CSF is employed (Figure 3.5g). Wild‐type cells did not demonstrate 
detectable STAT3 phosphorylation in response to G‐CSF stimulation. This result 
suggests that the chimeric receptor functions to activate STAT3 through G‐CSF 
solely in rat ES cells. 
 
 
 
 
 
Figure 3.5f: Rat ES cells expressing the chimerical gp130 receptor 
activate STAT3 in response to GCSF. Total protein lysates were collected 
from either wildtype or rat ES cells expressing the chimeric gp130 
receptor following a 30 minute stimulation with either 10ng/ml LIF or 
0.02ng/ml GCSF. Only cells that express the chimeric receptor 
phosphorylate Tyr705 in response to GCSF stimulation.
 86 
 
 
 
 
 
 
 
We then hypothesized that, like mouse ES cells, STAT3 activation likely exerts its 
effects in a temporal fashion. To test this, we stimulated DAc8‐Y118F rat ES cells 
with either LIF or G‐CSF and collected cell lysates at thirty minutes, sixteen hours, 
and 24 hours post‐induction. As indicated in Figure 3.5h, G‐CSF treated rat ES cells 
demonstrate increased STAT3 Tyr705 phosphorylation that is sustained over 
twenty‐four hours. LIF‐treated cells reveal maximal Tyr705 phosphorylation at 
thirty minutes with a return to basal levels by sixteen hours. Densitometry confirms 
Figure 3.5g: Densiometry indicates that GCSF and LIF phosphorylate 
STAT3 comparably in rat ES cells. Image analysis of Figure 3.5f suggests 
that GCSF and LIF stimulation result in STAT3 phosphorylation. Data 
are normalized to tubulin expression.
 87 
this finding. G‐CSF stimulation maintains almost two‐fold increase in STAT3 
activation as compared to LIF over 24 hours (Figure 3.5i‐j). Quantitative PCR 
analysis performed upon DA‐Y118F cells maintained in 0.02ng/ml G‐CSF for fifteen 
passages suggests that these cells are undifferentiated (Figure 3.5k). Together, these 
results suggest that, like feeder‐dependent mouse ES cells, increased and sustained 
STAT3 activation promotes rat ES self‐renewal. 
 
 
 
 
 
 
 
Figure 3.5h: GCSF stimulation sustains STAT3 activation over time 
promoting longterm selfrenewal. Total protein lysates were compared 
via western blot. (A) DAc8Y118F rat ES cells were stimulated with 
either 0.02ng/ml GCSF to 10ng/ml LIF for 30 minutes. Analysis reveals 
that GCSF induces Tyr705 phosphorylation robustly and sustains the 
majority of this activity over 24 hours. LIF rapidly phosphorylates and 
returns to basal levels by 16 hours. (B) AP stain demonstrating 
undifferentiated cells after fifteen passages stimulated with GCSF (C) 
identical cells maintained in 2i after fifteen passages. 
 88 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.5i: Densiometry indicates that GCSF increases STAT3 activation 
over 24 hours in rat ES cells. Image analysis of Figure 3.5h(A) indicates 
that GCSF stimulation increases STAT3 phosphorylation almost twofold 
over LIF treated rat ES cells. Both signals degraded over time, yet GCSF 
maintains twice as much active STAT3 as LIF at any given moment over 
24 hours. Data are normalized to tubulin expression. 
Figure 3.5j: Densiometry indicates that GCSF maintains STAT3 
expression over 24 hours in rat ES cells. Image analysis of Figure 3.5h(A) 
indicates that GCSF and LIF stimulation maintain constant STAT3 
expression over 24 hours. Data are normalized to tubulin expression. 
 89 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.5k: GCSF maintained rat ES cells maintain a pluripotent identity 
similar to canonical 2i maintained rat ES cells. Quantitative PCR analysis of 
DAc8Y118F rat ES cells stimulated with either 0.02ng/ml GCSF, 10ng/ml 
LIF, 2i (3uM CHIR99201, 1uM PD0395201), or serumfree N2B27 medium 
alone (‘NT’) reveals that GCSF treated cells mirror 2i treated cells in their 
pluripotency gene expression pattern. LIF treated cells, although expressing 
pluripotencyrelated RNA, demonstrates a more aberrant pattern similar to 
mediumonly treatedcells, suggesting dysregulation of the core 
pluripotency program. Data are normalized to mediumtreated cells. Error 
bars indicate SEM. 
 90 
Chapter Four  Klf4 Overexpression Promotes Mouse and Rat ES Cell Self
Renewal 
 
There are many proteins that may regulate self‐renewal downstream of STAT3. We 
then wished to understand if any STAT3 downstream effectors may recapitulate the 
effects of increased STAT3 expression or activation in promoting mouse and rat ES 
cell self‐renewal.
 
4.1  Introduction and Rationale 
 
Our initial choice was to investigate Klf family proteins: Krüppel‐Like Factor (Klf) 
family genes are a highly conserved set of transcriptional factors that were 
originally discovered in Drosophila studies(Kaczynski et al., 2003; Marin et al., 
1997; Schöck et al., 1999). Klfs are notable for their conserved zinc‐finger motif and 
their tendency to regulate GC‐rich promoter regions(Marin et al., 1997). Klfs 
regulate specific aspects of embryonic patterning, such as erythroid lineage 
commitments (Klf2), gut tissue patterning (Klf4), and epithelial lineage patterning 
(Klf5)(Kuo et al., 1997). Klf2, 4, and 5 are expressed in mouse ES cells and appear to 
harbor significant functions that promote self‐renewal(Jiang et al., 2008). 
 
Klfs have been known to be important in self‐renewing mouse ES cells for several 
years, however, with the discovery of induced pluripotent (iPS) cells, Klf4 was found 
to be one of the four core factors needed mediate this process(Takahashi and 
 91 
Yamanaka, 2006). It was determined that four genes, Oct4, Sox2, cMyc, and Klf4, 
were needed to reprogram fibroblasts into iPS cells. Oct4 and Sox2 were known to 
directly regulate pluripotency in mouse and human ES cells. CMyc was found to be 
essential in expediting the remodeling of the chromatin and is a potent mitogen. It 
has been determined that Klf4 is critical in remodeling the epigenetic structure of 
reprogramming cells and for also regulating the core pluripoteny network(Jiang et 
al., 2008).
 
Recently, it has been established that Klf4 is the necessary factor to fully, and finally 
reprogram pluripotent epiblast stem cells back into an ES‐like pluripotent state(Guo 
et al., 2009). Since then, Dr. Hitoshi Niwa’s group has determined that Klf4 is likely 
an important target of STAT3 upon LIF activation of gp130(Niwa et al., 2009). 
Adding to this model, Dr. Austin Smith and colleagues conclude that Klf4 is regulated 
by STAT3, with Klf5 as secondary target of regulation as well(Hall et al., 2009). Klf2 
has been found to be a direct target of Oct4 in response to LIF/STAT3 signaling in 
mouse ES cells. In sum, Klf2, 4, and 5 therefore make attractive targets immediately 
downstream of STAT3 as effectors of the self‐renewal program. Therefore, I sought 
to investigate the role of Klfs in regulating the self‐renewal of mouse ES cells. 
 
4.2 – Klf4 Is Upregulated in Response to STAT3 Activation in Mouse ES Cells 
 
We hypothesized that since STAT3 activation is sustained over time, it is likely true 
that STAT3’s putative downstream targets are up‐regulated and maintained in a 
 92 
sustained fashion. To test this, we began by seeding high density (~10
4
 cells/cm
2
) 
B6‐Y118F ES cells in co‐culture with CF‐1 feeders. Following serum‐starvation, the 
mouse ES cells were stimulated with either 10ng/ml LIF, 0.2ng/ml G‐CSF, or 
medium only. Total RNA was extracted at one hour and twenty‐four hours post‐
stimulation. 
 
Quantitative PCR was performed to ascertain relative amounts of mRNA for Klf2,4, 
and 5 (Figure 4.2a). As expected, Klf4 mRNA levels increase significantly by one hour 
post‐LIF stimulation, and by twenty‐four hours have only increased modestly over 
one hour. However, G‐CSF treated cells demonstrate only a slight increase in Klf4 
expression by one hour. By twenty‐four hours, G‐CSF treated cells increase Klf4 
expression five‐fold above controls. Despite a lagging increase as compared to LIF, 
G‐CSF treatment increases expression of Klf4 higher than LIF induction does at 
twenty‐four hours post‐induction. This finding suggests that increased and 
sustained STAT3 activation may increase the expression of Klf4 RNA which remains 
increased over twenty‐four hours .
 93 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.2a: Mouse ES cells upregulate Klf4 in response to GCSF 
treatment feederfree. B6Y118F mouse ES cells were treated with 
either 0.2ng/ml GCSF, 10ng/ml LIF, 10ng/ml LIF plus 3uM 
CHIR99201, or serum medium alone (‘NT’). Quantitative PCR analysis 
reveals that LIF induces a significant increase in Klf4 RNA expression 
by one hour as compared to GCSF or LIF/CHIR. By 24 hrs, however, 
GCSF and LIF/CHIR significantly increase Klf4 RNA expression over 
LIF. Both Klf2 and Klf5 show modest increases in response to GCSF. 
Data is normalized to nontreated cells. Error bars indicate SEM. 
 94 
4.3 – Klf4 Overexpression Prevents Crisis in Mouse ES Cells But Is Not Essential For 
Maintaining SelfRenewal 
 
To determine if overexpression of specific Klf proteins could enhance STAT3 
mediated self‐renewal, we transfected wild‐type B6 mouse ES cells with retroviral 
constructs containing either Klf2, 4, or 5 isoforms. After viral Klf RNA production 
was verified (Figure 4.3a(A)), each cell type was then seeded upon a gelatinized 
substrate containing serum medium and LIF. Only cells transfected with Klf4 
displayed colonies morphologically consistent with self‐renewing ES cells that could 
be passaged. Removal of LIF from Klf4 transfected cells also resulted in cellular 
morphology consistent with differentiation (Figure 4.3a(B)). Klf4 overexpression 
was confirmed by western blotting and densiometry (Figure 4.3a(C)). After five 
passages feeder‐free, Klf4 overexpressing B6 mouse ES cells stain positive for Oct4 
(Figure 4.3b). 
 95 
 
 
 
 
 
 
 
 
 
Figure 4.3a: Klf4 overexpression enhances LIFmediated feederfree B6 ES cell self
renewal. (A) Wildtype B6 mouse ES cells were retroviral transfected with Klf2, 4, or 
5 isoforms. RTPCR confirms viral RNA production. (B) Clones infected with each Klf 
were seeded onto gelatinized substrates and supplemented with LIF. Only Klf4 
transfected cells demonstrated colony morphology consistent with selfrenewal. LIF 
was then removed from the Klf4 transfected cells and immediate differentiation was 
observed. (C) Western blot indicating Klf4 overexpression following transfection. 
(D) Densiometric analysis of (C) that demonstrates that Klf4 is expressed almost 
twofold higher than over wildtype. Data are normalized to βactin. 
 96 
 
 
 
 
 
 
We then overexpressed Klf4 in BalbC1‐Y118F mouse ES cells. As indicated in Figure 
4.3c, wild‐type BalbC1 ES cells fail to form tight, compacted, and dome‐shaped 
colonies when seeded into feeder‐free environment despite supplementation with 
LIF. However, when transfected with Klf4, LIF supplementation results in colonies 
consistent with undifferentiated, self‐renewing ES cells despite the absence of 
feeders. These data suggest that Klf4 overexpression can enhance feeder‐free self‐
Figure 4.3b: Klf4 overexpression permits longterm culture feederfree in a LIF
dependent manner. Wildtype B6 mouse ES cell overexpressing Klf4 are able 
stain Oct4 positive after five passages in feederfree conditions stimulated by 
LIF.
 97 
renewal in a LIF‐dependent fashion in more than one non‐permissive mouse ES cell 
line. Klf4 overexpression plus LIF supplementation results in cells morphologically 
consistent with self‐renewing ES cells expressing the chimeric gp130 receptor and 
stimulated with G‐CSF. These results suggest that Klf4 overexpression augments 
STAT3 mediated self‐renewal feeder‐free. 
 
 
 
 
 
 
 
Figure 4.3c: Klf4 overexpression enhances BalbC1 mouse ES cell feederfree 
selfrenewal. (A) Wildtype BalbC1 mouse ES cells seeded feederfree cease 
proliferating and appear to differentiate despite LIF supplementation. (B) 
Klf4 transfection into wildtype BalbC1 mouse ES cells renders them 
consistent with undifferentiated ES cells in feederfree culture. (C) BalbC1 ES 
cells expressing the chimeric gp130 receptor fail to selfrenew in the 
presence of LIF feederfree. (D) GCSF stimulation renders the same cells 
able to selfrenew. 
 98 
To determine if Klf4 expression was essential for STAT3 mediated self‐renewal, we 
transfected B6‐Y118F mouse ES cells with shRNA constructs targeted against Klf4 
(‘B6‐Y118F‐K4KD’). Quantitative PCR and western blotting verify significant knock‐
down of Klf4 RNA and protein levels as compared to untransfected cells (Figure 
4.3d). We then seeded Klf4‐depleted B6‐Y118F and mock‐infected B6‐Y118F mouse 
ES cells onto gelatinized substrates supplemented with 0.2ng/ml G‐CSF. Despite 
Klf4 knock‐down, B6‐Y118F cells were able to self‐renew comparably to cells with 
normal Klf4 expression levels (Figure 4.3e(A)). Immunostaining reveals Oct4 
positive cells after five passages feeder‐free in G‐CSF (Figure 4.3e(B)). These data 
suggest that Klf4 expression may not be essential in promoting STAT3‐mediated 
mouse ES cell self‐renewal. 
 
 
 
 
Figure 4.3d: Klf4 knockdown is efficient in mouse ES cells. (A) Western blot 
demonstrating endogenous Klf4 in B6Y118F mouse ES cells is depleted as 
compared to scrambled shRNA control. (B) Densiometry indicates that both 
shRNA constructs deplete Klf4 protein by at least 50 percent or more. Data 
normalized to βactin expression (C) qPCR indicates that Klf4 RNA levels are 
depleted as well following shRNA transfection. Data normalized to mock
infected. Error bars indicate SD. 
 99 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.3e: Klf4 expression is not essential for mouse feederfree self
renewal. (A) B6Y118F mouse ES cells infected with Klf4 shRNA and 
stimulated with GCSF in a feederfree environment selfrenew efficiently. 
(B)Immunostaining of B6Y118F transfected with Klf4 shRNA after five 
passages in GCSF in feederfree conditions. Both cell lines stain positive for 
Oct4. 
100 
 
Despite the ability to be passaged with no apparent differentiation, quantitative PCR 
analysis reveals that Oct4, Nanog, and Rex‐1 RNA expression levels are significantly 
reduced in Klf4 depleted ES cells (Figure 4.3f(A)). In addition, early markers of 
differentiation are upregulated as a result of Klf4 knock‐down, yet as observed 
earlier, self‐renewal appears to not be adversely affected (Figure 4.3f(B)). These 
data suggest that Klf4 expression is likely accessory to, but not critical for 
maintaining STAT3‐mediated mouse ES cell self‐renewal. 
 
 
 
 
 
Figure 4.3f: Klf4 knockdown decreases pluripotency gene expression and 
increases early differentiation markers. (A) qPCR analysis demonstrates that 
Klf4 knockdown results in decreased RNA levels of Oct4, Nanog, and Rex1. 
(B) qPCR analysis upon the same cell lines indicate that Klf4 knockdown 
results in increased early differentiation markers. Data are normalized to 
untransfected B6Y118F cells. Error bars indicate SD. 
101 
Chapter Five  STAT3 Activation May Promote Tumor Cell Growth Arrest
 
5.1 – Introduction and Rationale 
 
STAT3 as an oncogene is well‐characterized in the literature(Corvinus et al., 2005; 
Kusaba et al., 2005). The most common transformation event related to STAT3 is 
constitutive activation. Aberrant STAT3 regulation is often found in specific subsets 
of breast, leukemic, and colon cancers. Dysregulated STAT3 has been correlated to 
enhanced tumor survival and poor prognosis(Ling and Arlinghaus, 2005). It is 
worthwhile then to investigate if specific aspects of STAT3 regulation in ES cells can 
translate to tumor cells with known STAT3 dysregulation. Many tumors, like ES 
cells, have at least a small population of cells that self‐renew and resist therapeutic 
treatment(Villalva et al., 2010; Welte et al., 2010). Is STAT3 integral in mediating 
this process? ES cells provide an isolated environment to study STAT3 without the 
limitations of other or more numerous mutations found in cancer cell lines(Foster et 
al., 2000; Yang et al., 2005).
 
As previously shown, in specific instances, STAT3 activation can induce 
differentiation in mouse ES cells. It is well‐characterized that many cancers 
demonstrate aberrant gene behavior or protein production. Of the many oncogenes 
activated or mutated, STAT3 is found to be either constitutively activated or exhibit 
slightly higher than normal expression levels(Bromberg et al., 1998; Corvinus et al., 
2005; Kusaba et al., 2005; Ling and Arlinghaus, 2005; Ling et al., 2007). We 
102 
hypothesized that increasing STAT3 activation may have the effect observed in 
mouse ES cells, whereby increasing STAT3 activation may actually promote 
differentiation in tumor cells. 
 
To test if increasing endogenous STAT3 activation in human tumor cells is able to 
induce differentiation, we selected tumor cell lines that had well‐characterized 
STAT3 mutations. Two cell lines, DLD‐1 and HT‐29 were selected for their aberrant 
STAT3 activity(Corvinus et al., 2005; Kusaba et al., 2005). DLD‐1 and HT‐29 are both 
human colon cancer cell lines of epithelial origin(Fogh and Trempe, 1975; Trainer et 
al., 1988).
 
Both cell lines were expanded, transfected stably with the chimeric gp130 receptor, 
and drug selected for individual clones. Western blotting revealed a functional 
chimeric receptor was able to robustly activate STAT3 in human tumor cells (Figure 
5.1a). Western blotting also revealed a significant decrease in ERK1/2 activation 
upon high G‐CSF concentration (2.5ng/ml) stimulation. Upon stimulation by G‐CSF, 
several clones demonstrated little cellular attachment to the tissue culture dish, 
suggesting the cells may be dying. Densiometry confirms that ERK1/2 
phosphorylation is decreased in response to high G‐CSF concentrations (Figure 
5.1b). To rule out off‐target effects of G‐CSF, wild‐type HT‐29 and DLD‐1 cells were 
exposed to 10ng/ml G‐CSF for seven days. No apparent change in growth was 
observed as compared to untreated cells (Figure 5.1c). 
103 
 
 
 
 
 
 
Figure 5.1a: GCSF Mediates STAT3 activation and correlates to 
inhibited ERK1/2 activation. HT29Y118F cells were treated with 
a gradient of GCSF for 30 minutes. Western blotting reveals a 
decrease in STAT3 activation as the GCSF concentration is 
decreased. ERK1/2 activation is inhibited to background levels 
under the highest GCSF concentration. STAT3 expression appears 
to decrease in correlation with STAT3 activation. 
104 
 
 
 
 
 
 
Figure 5.1b: Densiometry confirms decreased ERK1/2 activation in response 
to GCSF in HT29 tumor cells. Image analysis of Figure 5.1a demonstrates 
that high STAT3 phosphorylation correlates to decreased ERK1/2 
phosphorylation at 2.5 ng/ml GCSF. STAT3 expression also decreases with 
GCSF concentration. ERK1/2 activation remains constant below 2.5 ng/ml 
GCSF. Data normalized to tubulin. 
105 
 
 
 
 
 
To quantify the effects of G‐CSF in tumor cells, we plated an equal number of HT‐29 
and DLD‐1 human tumor cells cells, ~5x10
3
 cells/cm
2
 into separate wells, and 
stimulated them with either 1ng/ml G‐CSF or medium alone. Compared to wild‐type 
cells, the clones expressing the chimeric receptor exposed to G‐CSF demonstrated 
almost a total reduction in cell number by Day 7 (Figure 5.1d‐e). This result suggests 
that STAT3 activation may be correlated to decreased total cell number. 
Figure 5.1c: WildType Human Tumor Cell Lines Demonstrate No Discernible 
Effect to GCSF Treatment. (A) Wildtype HT29 human colon cancer cells 
were subjected to high doses of GCSF for 5 days. No appreciable effect was 
observed. (B) Wildtype DLD1 human colon cancer cells were subjected to 
high doses of GCSF for 5 days. Again, no appreciable effect was observed. 
106 
 
 
 
Figure 5.1d: Increased STAT3 Activation Correlates to Decreased Cell 
Number in HT29Y118F cells. Top: (A) Nontreated HT29Y118F cells, 
day 7. (B) Western blot demonstrating GCSF mediated STAT3 
phosphorylation (C) 100 pg/ml GCSF stimulation demonstrating 
detached cells, Day 7 (D) 1ng/ml GCSF stimulation demonstrating 
complete detachment of cells to substrate, Day 7. 
 
 
 
107 
 
 
 
 
STAT3 has been well‐characterized as a positive regulator of cell cycle progression 
in somatic tissues, namely by facilitating the G1 to S phase transition(Fukada et al., 
1998). This is accomplished via gp130 activation of STAT3 and the associated 
upregulation of cyclins D and A and cdc25a. STAT3 activation is also a potent 
inhibitor of the cell‐cycle inhibitor p21. STAT3’s effect upon DLD‐1 and HT‐29 
human tumor cells may be through cell‐cycle regulation, therefore it must be 
established that proliferation is inhibited first. 
Figure 5.1e: Increased STAT3 activation correlates to decreased cell number 
in DLD1Y118F cells. (A) Nontreated DLD1Y118F cells, day 7. (B) 1ng/ml 
GCSF treatment, Day 7. (C) Western blot demonstrating GCSF meditated 
STAT3 phosphorylation. 
 
108 
5.2 – STAT3 Activation Correlates to Decreased Tumor Cell Proliferation 
 
To further investigate STAT3’s effects upon tumor cells, we plated 10
5
 DLD‐1 or HT‐
29 clones containing the chimeric receptor. The cells were passaged every two days. 
10
5
 cells of each type were passaged for six passages, or until there were less than 
10
5
 cell available to passage. Each cell type was stimulated with a gradient of G‐CSF 
concentrations or medium only. Cells stimulated with G‐CSF showed a stagnant cell 
number that decreased with each passage when compared to no treatment (Figure 
5.2a). Non‐treated cells exhibit an initial decrease in total cell number, but recover 
by passage five. Supplementation with 1ng/ml G‐CSF correlated to an inability for 
the cells to recover, as no cells could be subcultured by passage four. These data 
indicate that high G‐CSF concentrations applied to human tumor cells expressing the 
chimeric gp130 receptor may increase cellular death, inhibit proliferation, or both 
may be occurring . 
109 
 
 
 
 
 
We assayed for cumulative cell number changes in HT29‐Y118F cells seeded at 
clonal density (250 cells/cm
2
) supplemented with a gradient of G‐CSF (1ng/ml – 
0.01ng/ml G‐CSF) in serum medium for six days. Indeed, when given a multitude of 
concentrations, the lower cell density appears to affect total cell number in an 
inversely proportional manner as compared to the G‐CSF concentration present 
(Figure 5.2b). We observed no adherent cells by Day 4 when 1ng/ml or 0.1ng/ml G‐
CSF was employed. These observations indicate that G‐CSF treatment of chimeric 
Figure 5.2a: GCSF stimulation correlates to decreased cumulative 
cell number. HT29Y118F cells were seeded at 10
4
 cells/cm
2
. GCSF 
was added and the cells were counted every other day at passage. 
1ng/ml GCSF resulted in complete ablation by passage 4, while all 
other treatments did not significantly affect cell number. 
110 
receptor‐harboring human tumor cells may be adversely affecting cell survival 
and/or proliferation.
 
 
 
 
 
 
 
 
 
 
Figure 5.2b: GCSF Prevents Tumor Cell Proliferation at Low Cell Density and 
During Exponential Growth. Top: 250 HT29Y118F cells/cm
2
 were seeded 
and GCSF was applied in a gradient fashion. Cells were counted everyday 
for six days. All treatments prevented significant growth, while 1ng/ml and 
0.1ng/ml GCSF concentrations resulted in no surviving cells to count.
111 
Discussion 
 
D.1 – STAT3 Is Regulated In a Strain or SpeciesSpecific Manner to Promote 
Rodent ES Cell SelfRenewal 
 
Previously, mouse and rat ES cells have shared only a STAT3‐independent self‐
renewal pathway, however this mechanism is poorly understood (Ying et al., 
2008a). Since mouse ES cells can self‐renew in both a STAT3‐dependent and 
independent fashion, we hypothesized that rat ES cells must share both features as 
well. Our work has presented evidence that rat ES cell do in fact share both a STAT3‐
dependent as well as independent self‐renewal mechanism with the mouse.
 
To elucidate this claim, we had to discard with the idea that STAT3 activation 
functions as a binary ‘on/off’ system. Rather, modulating STAT3 activation in a 
strain or species‐specific manner has the effect of enhancing both mouse and rat ES 
cell self‐renewal ability. One obstacle that likely prevented previous derivations of 
rat ES cells is that rat ES cell STAT3 activation requirements are different from the 
mouse, as mouse ES cells require an approximately ten‐fold higher G‐CSF 
concentration than rat to maintain self‐renewal. This increased G‐CSF concentration 
likely correlates to a significant increase in STAT3 activation. Receptor expression 
levels are likely important in regulating STAT3 activation levels as well. In addition, 
LIF stimulation has been shown to be refractory to rat ES cell derivation. This 
112 
apparent discrepancy in response to LIF may not be because LIF does not support 
rat ES cell self‐renewal, but rather because STAT3 is regulated differently upon LIF 
induction within each species. This evidence suggests that additional species‐
specific levels of STAT3 regulation are likely necessary to promote rodent ES cell 
self‐renewal. 
 
 
We have presented evidence that attempts to determine why specific ES cell lines 
are able to self‐renew feeder‐independently. This work demonstrates that STAT3 
activation does not function as a binary ‘on/off’ system, but rather more in line with 
a rheostat‐like effect composed of intermediate cell‐states aggregately referred to as 
‘self‐renewal.’ This finding not only refines our understanding of the role of STAT3 
in mouse ES cell self‐renewal, but also provides a signaling connection that explains 
how 129 mouse ES cell strains, but not B6 mouse ES cells, manage feeder‐
independent self‐renewal.
 
Our results suggest that STAT3 expression and activation levels are critically 
important in permitting feeder‐independent mouse ES cell self‐renewal. This finding 
argues that the differences in feeder‐free self‐renewal ability between mouse ES cell 
strains are related to each strain or species’ inherent permissiveness(Hanna et al., 
2009). Our data suggests that feeder‐independent 46C mouse ES cells harbor an 
increased STAT3 expression level in response to LIF. This increased expression level 
permits an increased proportion of activated STAT3, which correlates to the ability 
113 
to self‐renew feeder‐free. B6 mouse ES cells, when engineered to either overexpress 
STAT3 or artificially increase endogenous STAT3 activation, are also rendered able 
to self‐renew feeder‐free, thus enhancing their self‐renewal ability. This result also 
implies that the STAT3‐dependent self‐renewal mechanism is more accurately 
described as the aggregate of many intermediate STAT3‐phosphorylation levels 
which can be finely tuned by the cell or the investigator given the specific culture 
milieu. 
 
D.2  The Role of STAT3 In Mediating Mouse and Rat ES Cell SelfRenewal 
 
Interestingly, STAT3 overexpression correlates to increased core pluripotency gene 
transcript expression and decreased ERK1/2 phosphorylation. Together, this result 
suggests that increasing STAT3 expression levels has a net positive effect in 
promoting ES cell self‐renewal feeder‐free. However, in contrast to this finding, we 
report that overexpressing STAT3, then hyperactivating such a large pool results in 
acute differentiation of mouse ES cells, and may reduce proliferation of specific 
tumor cell populations. If STAT3 activation is found to have a differentiation‐
inducing ability as well in specific circumstances, then novel approaches to tumor 
suppression may be developed. Instead of complete inhibition of STAT3 activation, 
hyperactivation of STAT3 may exacerbate proliferation inhibition. Within ES cells 
however, our observation that STAT3 may induce differentiation when 
hyperactivated reaffirms that STAT3 likely functions within a self‐renewal range 
with a functional ceiling on self‐renewal (Figure D.2a). 
114 
 
 
 
Building upon this premise, we provide evidence that rat ES cells are able to self‐
renew in a STAT3‐dependent manner. Prior to this work the rat ES cell self‐renewal 
had been suggested to be STAT3‐independent(Buehr et al., 2008; Li et al., 2008). 
However, STAT3 overexpression or artificial STAT3 activation results in robust self‐
renewal for both mouse and rat ES cells. What remains to be determined is if feeder‐
independent rat ES cell self‐renewal is possible. Currently, rat ES cells cannot self‐
renew in the absence of feeders. Determining an appropriate feeder‐independent 
Figure D.2a: STAT3 signaling appears to have a functional range that 
effectively promotes ES cell selfrenewal. Based on the work of others and 
myself, mouse and rat ES cells harbor a speciesspecific range for STAT3 
expression and activation that fundamentally outlines the selfrenewal 
program for each celltype. Finding the functional range by modulating 
specific aspects of the selfrenewal machinery for each species is tantamount 
to understanding how to derive novel mammalian ES cell species, or at least 
in the shortterm novel rodent ES cell species. 
115 
rat ES cell culture system will allow for more accurate direct comparisons of mouse 
and rat ES cells in vitro. 
 
We also report on putative downstream targets of STAT. Since it has been reported 
that Klf genes are likely critical targets of STAT3 activation, we sought to understand 
the relationship specific Klf genes may have with STAT3(Hall et al., 2009; Niwa et al., 
2009). We note that only Klf4 demonstrates a significant LIF‐mediated increase in 
RNA expression over time in mouse ES cells. G‐CSF stimulation further bolstered 
this finding by demonstrating that increasing STAT3 activation correlates to an 
increase in Klf4 RNA expression, suggesting Klf4 expression is potentially regulated 
by active STAT3. 
 
Klf4 overexpression in B6 feeder‐dependent mouse ES cells renders the cells able to 
self‐renew feeder‐free in a STAT3 dependent manner. This result is in contradiction 
to the finding by Hall et al. that forced Klf4 expression promotes LIF‐independent 
self‐renewal(Hall et al., 2009). We note that Hall et al. indicate that self‐renewal was 
severely impaired in the absence of LIF, and that self‐renewal was improved when 
LIF was included. We speculate that ES strain differences or transfection efficiency 
may account for the discrepancy that our cells require LIF to self‐renew feeder‐free. 
In addition there is evidence that Klf4 overexpression itself has cytostatic or toxic 
effects upon self‐renewing mouse ES cells(Niwa et al., 2009). We however, confirm 
also the specific finding by Hall et al. that STAT3 expression is necessary to support 
self‐renewal despite Klf4 overexpression. This evidence supports the notion that 
116 
Klf’s are but one aspect of a critical cadre of downstream targets of STAT3 necessary 
to execute self‐renewal(Jiang et al., 2008). It has been also demonstrated that either 
Klf2 or 4 forced expression is sufficient to reprogram fibroblasts(Nakagawa et al., 
2008). Evidence suggests that unlike STAT3, however, Klf4’s role is a gate that 
promotes naïve pluripotency while preventing the transition to the EpiSC 
state(Feng et al., 2009). Given that Klf4 augments self‐renewal, but does not appear 
to be essential in promoting it, this view likely true. Therefore, further 
investigations into Klf4 as it pertains to self‐renewal may be unnecessary, as Klf4 
appears to be a reprogramming regulator and developmental gate, but not directly a 
self‐renewal regulator. 
 
It has been demonstrated that, of the Klfs expressed in mouse ES cells, only Klf2 
overexpression can promote LIF‐independent mouse ES cell self‐renewal(Hall et al., 
2009). However, no one particular Klf expressed in mouse ES cells appears to be 
critical for maintenance of the naïve pluripotent state, suggesting that Klf proteins 
are but one aspect of the cloud of signal networks mediating self‐renewal (Jiang et 
al., 2008). 
 
D.3  The Role of ERK1/2 In Mouse ES Cell SelfRenewal 
 
We confirm the finding that ERK1/2 activation is detrimental to STAT3‐mediated 
mouse ES cell self‐renewal(Burdon et al., 1999; Kunath et al., 2007). However we 
reveal to what extent ERK1/2 signaling potently inhibits STAT3‐mediated feeder‐
117 
independent self‐renewal: Neither LIF nor ERK1/2 inhibition alone increased 
feeder‐free self‐renewal capability, yet in combination feeder‐free self‐renewal was 
significantly increased. This suggests a synergy between activation and inhibition, 
and not simply STAT3 activation, supports naïve pluripotent self‐renewal. This idea 
is firmly in line with other reports that MAPK/ERK signaling is directly in opposition 
to STAT3 signaling in mouse ES cells (Greber et al., 2010).
 
Intriguingly, STAT3 overexpression correlates to a significant decrease in ERK1/2 
activation, suggesting increased STAT3 expression may indirectly inhibit ERK1/2, 
thus reinforcing naïve pluripotency. More work is needed to verify if in fact STAT3 
might be regulating ERK1/2 activation. However, if true, our results may shed light 
upon the mechanism behind murine diapause. It is known that LIF is secreted by the 
trophectoderm which signals to the ICM to self‐renew. However, cell‐cycling in the 
diapaused epiblast is slowed and proliferation is halted, but evidence exists that 129 
strain epiblasts have a significantly higher proportion of cycling cells as compared 
to other mouse strains(Batlle‐Morera et al., 2008). Increased STAT3 expression in 
the epiblast in utero may inhibit ERK1/2 activation, decreasing proliferation, which 
would promote self‐renewal.
 
Also, recent evidence suggests that ERK signaling is needed to stabilize the primed 
pluripotent state by inhibiting Klf4 expression and therefore acts as a direct 
impediment in reprogramming from EpiSC to ES cell pluripotency(Greber et al., 
2010). If true, this suggests that increased STAT3 activation may further increase 
118 
Klf4 and Klf2 expression, which has been shown to stabilize naïve pluripotency by 
inhibiting ERK1/2 activation(Greber et al., 2010). Finally, ERK expression is likely 
unnecessary for naïve pluripotency, as erk2 null mouse ES cells appear to be normal 
in their capacity to self‐renew and erk1 null mice are viable(Pagès et al., 1999; Yao 
et al., 2003). 
 
D.4  Rodent ES Cell SelfRenewal: A Stratified Model 
 
STAT3 also likely plays a critical role in regulating the naïve pluripotent state. 
Recent evidence has emerged that indicates that pluripotency itself is stratified into 
different stable states. Hanna et al. demonstrate that STAT3 harbors a critical role in 
creating human iPS cell populations that mirror mouse ES cell naïve pluripotency by 
using the combination of STAT3 activation, the combined inhibition of GSK3β and 
MAPK, and the combined ectopic expression of Klf2 and 4 (Hanna et al., 2010). 
Indeed, mouse self‐renewal can be maintained using mechanisms that are STAT3‐
dependent (LIF), independent (2i), or both (LIF/2i) (Guo et al., 2009). We propose 
that the naïve pluripotent state’s self‐renewal mechanism is itself a stratified, 
metastable cell‐state composed of at least two distinct mechanisms that are shared 
between mouse and rat (Figure D.4a). Now, due to the conserved nature of STAT3 in 
mouse, rat, and human ES cells, direct in vitro comparisons can now be made that 
address the significance of downstream self‐renewal targets.
119 
 
 
 
The ground state of ES cells fits into this model as well, suggesting that the ground 
state can be interconverted to employ the more traditional inductive, or STAT3‐
dependent, self‐renewal mechanism by induction with LIF. Differentiation in this 
model may be induced by increasing or decreasing either STAT3 expression, 
Figure D.4a: Rodent ES cell selfrenewal is metastable cellstate within naive 
pluripotency. The ground state of mouse ES cells is defined as a STAT3 independent 
mechanism that relies upon inhibited MAPK/ERK and GSK3β to promote self
renewal. These two selfrenewal mechanisms can be switched depending on the 
culture conditions. By increasing STAT3 expression or activation acutely, and by 
inhibiting MAPK/ERK signaling slightly less, more traditional inductivebased ES 
cells can be cultured. Further increases in MAPK signaling or a decrease in STAT3 
activation will result in primed ES cell transition or overt differentiation. 
120 
activation, or increasing ERK1/2 activation (Figure D.4b). Differentiation may be 
averted by chemical or genetic modification to ‘re‐set’ the balance between 
competing signal cascades.
 
We posit that the self‐renewal mechanism is itself stratified into distinct cell‐states. 
These cell‐states can transition between different ranges of STAT3 activation and 
STAT3‐independent pathways within naïve pluripotency depending on the 
permissiveness of the species or strain. This model predicts that there are different 
rodent self‐renewal mechanisms which are themselves stratified within a specific 
pluripotent cell‐state. Both the STAT3‐dependent and ground‐state rodent self‐
renewal mechanisms are relegated to describing naïve pluripotency (Nichols and 
Smith, 2009). 
 
 
 
 
 
 
 
121 
 
 
 
 
 
 
 
 
 
Figure D.4b: Selfrenewal represents a finelytuned interplay between STAT3 
and MAPK/ERK signaling in rodent ES cells. Selfrenewal can be defined as any 
cellstate that maintains a significantly high proportion of expressed and active 
STAT3 as compared to active MAPK/ERK signaling. Either increasing ERK or 
decreasing STAT3 activation disrupts this balance and results in ES cell 
differentiation. In this scenario, no signal protein exerts all the selfrenewal 
effect, as some beneficial mitogenic effects are likely important from ERK 
signaling.
122 
The idea of a multiplicity of self‐renewal mechanisms within naïve pluripotency is 
likely transferable to other pluripotent stem cell populations. This model may be 
transferable to other species, and may help explain why LIF is insufficient in 
deriving and maintaining porcine or bovine ES cells(Alberio et al., 2010; Ezashi et 
al., 2009; Gong et al., 2010). In addition, we have shown that STAT3 is regulated 
temporally. As well, STAT3‐independent mechanisms may prove to be temporally 
regulated as well. Likely, this temporal regulation coupled to conserved 
downstream targets may explain why the observed self‐renewal mechanisms 
appear separate, but in reality may be highly interconnected. In addition, this model 
predicts the existence of additional self‐renewal mechanisms within the primed 
pluripotent cell‐state in addition to the bFGF/MAPK pathways already 
characterized. There may be a ‘ground‐state’ of primed pluripotency, which is yet to 
be observed, but likely exists.
 
We add that this idea of conserved, metastable self‐renewal mechanisms will also 
aid in the derivation and maintenance of novel mammalian ES cell species, and will 
underpin future direct comparisons of mouse and rat ES cells. A more thorough 
understanding of the self‐renewal mechanisms and their conserved aspects will 
likely expedite the derivation of mammals that may less permissive than humans.
 
 
 
 
123 
D.5  Future Directions and Implications 
 
As a general future direction, it is worth noting that currently two distinct ES culture 
systems exist: STAT3‐dependent or STAT3‐independent. What is of fundamental 
importance is how these two vastly different signaling systems result in the same 
outcome: Self‐renewal. If and where these distinct mechanisms converge upon the 
self‐renewal program is of great interest to our group, as this point likely represents 
the conserved aspect of self‐renewal necessary to facilitate a wider variety of 
mammalian ES cell species to be derived. Conversely, these two pathways may 
represent two truly independent self‐renewal pathways, acting in a compensatory 
fashion when the other is impaired or removed. Therefore transcriptional and 
genomic analysis of the downstream targets of both mechanisms in both mouse and 
rat ES cells is necessary. It is also worth determining if the suggestion by Niwa et al. 
is true that a STAT3‐independent mechanism is running in parallel to the STAT3‐
dependent mechanism, as the ground state model has been extensively studies 
within STAT3‐devoid systems(Niwa et al., 2009). 
 
With regards to tumor biology, it seems more than tenuous that the inherent 
proclivity to develop pluripotent tumor types in the 129 strain of mouse is not 
correlated to the fact that the ES cell lines yielded are capable of feeder‐free 
derivation. Since ES cells share many characteristics with tumor cells, it seems 
reasonable to posit that 129 ES cells, in their ability to adhere and proliferate 
efficiently without the aid of any supporting tissue, likely by their ability to increase 
124 
STAT3 expression and activation, is behavior consistent with metastasizing tumor 
cells. Strains of mice that yield only feeder‐dependent mouse ES cells not only lack 
this ability to quickly modify their STAT3 expression levels to retain feeder‐
independence, they lack the significant occurrence of spontaneous pluripotent 
tumor cell types. It is worth investigating the connection between the relative 
incidence of cancer as compared to efficiency of ES cell derivation, specifically 
feeder‐free derivation between different rat and mouse strains. Indeed, evidence 
exists that the attempts to connect the 129 strains relatively high tumor 
susceptibility rate to its high permissiveness is genetically determined(Anderson et 
al., 2009).The ability to derive ES cells feeder‐free may indicate a reasonably 
significant relationship between overall susceptibility to germ cell tumors and ease 
of ES cell derivation. Whether the rate of ES cell derivation significantly correlates to 
other tumor type susceptibilities is less certain, but would be of interest as well. Gail 
Martin herself speculated upon this issue tangentially in her 1981 manuscript 
outlining the derivation process, hinting that the connection between ES cells and 
tumor cells may be indicative of a more fundamental relationship than once thought. 
 
D.6  Conclusions 
 
In rodent ES cell biology, Stat3 has begun to reveal its true pleiotropy. Further 
investigations will no doubt uncover the downstream targets, their likely intimate 
relationship with STAT3, and the ultimate effect upon the stem cell phenotype. In 
all, a more thorough comprehension of STAT3 and the ES cell self‐renewal 
125 
mechanism will likely aid in the successful derivation of ES cells in novel species. To 
the extent of rodent ES cells, our work has laid the foundation for accurate in vitro 
inter‐species comparisons, which will likely aid in further refinements to the 
fundamental mechanism of ES cell self‐renewal. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
126 
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140 
Appendix A 
Materials and Methods 
 
Cell Culture 
 
 
C57BL/6, 46C, and Balb/c1 mouse ES cells were routinely cultured in GMEM 
(Sigma) supplemented with 10% FBS (HyClone) and 10ng/ml human LIF (Sigma) 
on gelatinized culture flasks. In the case of the C57BL/6 and Balb/c1 mouse ES cells, 
the flasks were first pre‐coated with gamma‐irradiated mitotically‐inactive CF‐1 
mouse embryonic fibroblasts (MEFs) at a density of 2.1 x 10
4
 cells/cm
2
. DAc8 rat ES 
cells were routinely cultured in serum‐free N2B27 medium (previously described in 
Nichols and Ying, 2006) supplemented with 3 uM CHIR99201 and 1 uM PD0325901 
(both from Stem Cell Sciences). Rat ES cell culture flasks were gelatinized and pre‐
coated with gamma‐irradiated CF‐1 MEFs at a density of 2.1 x 10
4
 cells/cm
2
. Stat3 
null mouse ES cells were derived and maintained as described previously in Ying et. 
al., 2008.
 
Plasmid Construction 
 
To over express Klfs genes in ES cells, retrovirus based gene expression vector, 
pMIG, was used. The coding region of KLF4 gene was amplified by RT‐PCR from 
cDNA of ES cells. Klf2 and Klf5 were kindly gifted by Shinya Yamanaka. The 5’BglII 
141 
and 3’XhoI sites were introduced into the PCR product and then subcloned into 
BglII/XhoI site in pMIG.
 
For RNA interference, a lentivirus mediated system was used according to the 
standard protocol. Briefly, two pair of sense and antisense oligonucleotides of 
shRNA were synthesized. After annealing, the duplexes were cloned into AgeI/EcoRI 
sites of pLKO.1. The target sequences are as follows: klf4‐shRNA1: 5’ ‐
GCGCTACAATCATGGTCAAGT;
klf4‐shRNA2: 5’ ‐ GTCAGCTTGTGAATGGATAAT. 
 
Virus production and infection of ES cells 
 
Retroviruses or lentiviruses were produced by transfection of the transducing 
vector into 293T cells with packaging vectors through Lipofectamine 
2000(Invitrogen), The supernatant containing secreted viral particles was 
harvested at 48 and 72 hours after transfection and filtered through 0.45um low‐
protein binding syringe filter, and used to infect ES cells.
 
For virus transduction, ES cells were trypsinized and re‐suspended in growth media 
at concentration 5 x 10
5
/ml. Then 1.5 ml cells were mixed with 0.5 ml viral 
supernatants and plated onto gelatinized 3.5 cm dishes. Polybrene was added at a 
final concentration 8 ug/ml. Cells were incubated with virus overnight and then fed 
with fresh media. Two days after infection, drug selection with puromycin (2 
142 
ug/ml,Sigma) was performed for 1 week. Puromycin‐resistant ES cell pools were 
collected and maintained under drug‐selection conditions. 
 
Transfection of Mouse and Rat ES Cells 
 
STAT3 overexpression clones were created by inserting the full‐length stat3 
transgene, acquired from Addgene, into the pPyFloxedMTIPgfp vector (previously 
described in Ying et al., 2008). 20 ug of linearized pPyFloxedMTSTAT3IPgfp 
plasmid was electroporated into 2 x 10
6
 mouse or rat ES cells using 500 uF and 280 
V on a BioRad GenePulser XCell apparatus. The cells were then cultured with DR4 
MEFs for two days before the transfectants were selected using 1 ug/ml puromycin 
for 5‐7 days. Individual stably transfected colonies were picked and expanded. 
STAT3 transgene excision was mediated by transient Lipofectamine LTX 
(Invitrogen) transfection of pCAGCreIP plasmids into STAT3 overexpressing 
C57BL/6 mouse ES cells. The resulting GFP positive colonies were picked, 
dissociated, re‐picked, and expanded to obtain a pure population of cells. Stat3 null 
mouse ES cells were transfected with the STAT3 expression vector as above, but 
instead the cells were cultured in serum‐free N2B27 medium supplemented with 3 
uM CHIR99201 and 1 uM PD0325901 in co‐culture with DR4 MEFs prior and 
following transfection. Creation of Y118F clones were obtained in a similar manner, 
except transfection of the GCSFRgp130Y118F expression vector was linearized and 
stably introduced instead. Drug selection was enforced using 20 ug/ml Zeocin 
(Invitrogen) for 7‐10 days.
143 
In Vitro Differentiation 
 
Mouse ES cells were differentiated by induction of embryoid body formation. Mouse 
ES cells were plated onto uncoated bacterial grade petri dishes in MEF medium 
(GMEM, 10% FBS, 1 mM L‐Glutamine). Cells would form aggregates in suspension 
which would expand into embryoid bodies. The embryoid bodies would then be 
plated intact upon a gelatinzed cell culture dish in GMEM/10% FBS medium after 7‐
10 days. The embryoid bodies were allowed to continue differentiation for another 
5‐7 days before subsequent analysis was performed. 
 
Immunostaining and AP Staining 
 
Immunostaining was performed using conventional methods. Primary antibodies 
were used in the following concentrations: Oct4 (C‐10, Santa Cruz, 1:500), Sox2 (Y‐
17, Santa Cruz, 1:1000), Gata‐4 (G‐4, Santa Cruz, 1:200), βIII‐Tubulin (Sigma, 1:200), 
and muscle sarcomere (Developmental Studies Hybridoma Bank, MF‐20, 1:3). 
Secondary antibodies were all purchased from Alexa Fluor (Invitrogen) and 
employed at a 1:2000 dilution. Nuclei were visualized via DAPI staining. Alkaline 
phosphatase staining was performed as per manufacturer’s instructions (Sigma).
 
 
 
144 
RTPCR and Quantitative PCR 
 
Total RNA was collected and purified using RNA Easy Mini Kit (Qiagen). cDNA was 
prepared by reverse transcribing 2 ug total RNA using the Cloned AMV First‐Strand 
cDNA Synthesis Kit (Invitrogen) using oligo dT primers and following the 
manufacturer’s instructions. PCR reactions were prepared using 1/20 of the reverse 
transcription reaction as template and amplified using Taq polymerase (Invitrogen). 
qPCR was performed by using SYBR Green PCR Master Mix (Applied Biosystems) 
according to the manufacturer’s instructions. Amplicons were detected by the 
ABI7900HT Real‐Time PCR System (Applied Biosystems) and analyzed using RQ 
Manager v1.2 (Applied Biosystems). Relative expression was determined by the 
ΔΔCt method and normalized to the GAPDH expression. Unless otherwise noted, all 
reactions were performed in technical triplicate and representative reactions from 
biological duplicates were presented. 
 
Western Blotting 
 
Western blotting was performed using conventional methods. In general, cells were 
serum starved for 6‐12 hours prior to stimulation. Cells were then stimulated with 
either 10 ng/ml human LIF (Sigma) or 0.2 ng/ml human G‐CSF (Peprotech) in the 
presence in serum‐free basal medium (GMEM, 1 mM L‐Glutamine) for the amount of 
time specified in the assay. PD0325901 was used at 1 uM concentration. Cells were 
145 
lysed in RIPA buffer. Total cell lysates were compared under denaturing conditions 
and probed using the following primary antibodies: Anti‐STAT3 (BD, 1:1000), anti‐
phosphoY705‐STAT3 (Cell Signaling, 1:1000), anti‐ERK1/2 (K‐23, Santa Cruz, 
1:1000), anti‐phosphoY204‐ERK1/2 (E‐4, Santa Cruz 1:1000), and anti‐α‐tubulin 
(Sigma, 1:2000), anti‐Klf4 (R&D, AF3158, 1:1000), anti‐β‐actin (Sigma, A5441, 
1:2000). 
 
Densiometry 
 
Digitized western blot films were analyzed using ImageJ software available through 
the National Institutes of Health website. All images were quantified by normalizing 
the density of the signal to tubulin expression unless otherwise noted. Data was 
compared to a reference signal to derive the relative intensities reported as 
Densiometric Units. Scale is linear unless otherwise noted.
 
 
 
 
 
 
 
 
146 
Appendix B: 
Primer Set List 
Primer Name Sequence (5' - 3')
Species
Recognized
Klf4 Forward CTGAACAGCAGGGACTGTCA Mouse, Rat
Klf4 Reverse GTGTGGGTGGCTGTTCTTTT Mouse, Rat
Klf4 Forward qPCR CGGCTGTGGGTGGAAATTC Rat
Klf4 Reverse qPCR CCGGTGTGTTTGCGGTAGT Rat
Klf2 Forward qPCR CAACTGCGGCAAGACCTACA Rat
Klf2 Reverse qPCR CAATGATAAGGCTTCTCACCTGTGT Rat
Klf5 Forward qPCR CCACCTACTTTCCCCCATCA Rat
Klf5 Reverse qPCR CTGGAGCATCTCAGCTTGTCTATC Rat
Tbx3 Forward qPCR TCCGGCTCAGTGTCCTTGTC Rat
Tbx3 Reverse qPCR TGCTCTGCAGTTCGCTGGTA Rat
STAT3 Forward qPCR CCTTACTGGGCCTAGGGTCAAC Rat
STAT3 Reverse qPCR GGACATGGGAAGGAGACATACC Rat
FGF4 Forward GGGAGGCTACAGACAGCAAG Mouse
FGF4 Reverse CTGTGAGCCACCAGACAGAA Mouse
FGF5 Forward GCGACGTTTTCTTCGTCTTC Mouse
FGF5 Reverse ACAATCCCCTGAGACACAGC Mouse
HPRT Forward CTCGAAGTGTTGGATACAGG Mouse, Rat
HPRT Reverse TGGCCTATAGGCTCATAGTG Mouse, Rat
Acta2 Forward GGGAGTAATGGTTGGAAT Mouse
Acta2 Reverse TCAAACATAATCTGGGTCA Mouse
AFP Forward CTGGCGATGGGTGTTTAG Mouse
AFP Reverse CCTGGAGGTTTCGGGATT Mouse
Brachyury Forward CTTTCTTGCTGGACTTCG Mouse
Brachyury Reverse TTACATCTTTGTGGTCGTTT Mouse
Cdx2 Forward CTTCCTGCCAGCAACGAC Mouse
Cdx2 Reverse CAGACATACATTCCGCCTACA Mouse
Gata4 Forward CCGAGGGTGAGCCTGTAT Mouse
Gata4 Reverse GCCTGCGATGTCTGAGTG Mouse
Hand1 Forward GGCGAGAAGAGGATTAAAGG Mouse
Hand1 Reverse CGAGAAGGCATCAGGGTAC Mouse
Nestin Forward GTCTGATGGGTTTGCTGA Mouse
Nestin Reverse AATCGCTTGACCTTCCTC Mouse
Plat Forward TCTGCCAGTGCCCTGATG Mouse
Plat Reverse TGGGTGCCACGGTAAGTC Mouse
Sparc Forward GGTGGTTTGGAGTTAGGC Mouse
Sparc Reverse GTAGAAGGTTTCAAGTGGC Mouse
Tuj1 Forward ACGCATCTCGGAGCAGTT Mouse
Tuj1 Reverse CGATTCCTCGTCATCATCTTC Mouse
Oct4 Forward GAAGCAGAAGAGGATCACCTTG Mouse
Oct4 Reverse TTCTTAAGGCTGAGCTGCAAG Mouse

Abstract (if available)

Abstract Pluripotent embryonic stem (‘ES’) cells are typically derived and maintained using inductive or inhibitory signals that are thought to behave in a binary ‘on/off’ manner. Mouse ES cells are maintained in an excess of leukemia inhibitory factor (LIF) and rat ES cells in a cocktail of inhibitors that block both GSK3β and MAPK signaling in a STAT3-independent manner. Here, we provide evidence that both mouse and rat ES cell self-renewal is conserved via a STAT3-dependent mechanism that treats STAT3 activation as fluid and dynamic. We observe that increased Klf4 expression enhances STAT3-mediated self-renewal in mouse ES cells but is not essential to prevent differentiation. As well, increased STAT3 activation and ERK1/2 inhibition synergistically enhance self-renewal. In all, we propose that the self-renewal mechanism is a nested metastable cell-state within naïve pluripotency, and that further refinements to the self-renewal mechanism will allow for direct inter-species comparisons and derivation of novel ES and induced pluripotent cell population.

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Asset Metadata

Creator Schulze, Eric Nathaniel (author)

Core Title Investigating the role of STAT3 in mouse and rat embryonic stem cell self-renewal and differentiation

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 08/11/2010

Defense Date 05/24/2010

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

Tag cell,Development,differentiation,embryonic,embryonic stem cell,ERK1/2,ground state pluripotency,GSK3,human,Jak,Klf4,Krüppel-like factor 4,MAPK,metastable pluripotency,mouse,naive pluripotency,OAI-PMH Harvest,pluripotency,pluripotent,rat,self-renewal,Stat3,STEM,stem cell

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ying, Qi-Long (committee chair), Adams, Gregor B. (committee member), Lutzko, Carolyn (committee member), Pera, Martin F. (committee member)

Creator Email eschulze@gmail.com,eschulze@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3349

Unique identifier UC153735

Identifier etd-Schulze-3501 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-377882 (legacy record id),usctheses-m3349 (legacy record id)

Legacy Identifier etd-Schulze-3501.pdf

Dmrecord 377882

Document Type Dissertation

Rights Schulze, Eric Nathaniel

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu