‫عنوان البحث باللغة اإلنجليزية‬

‫‪Research Title: The interesting reality of‬‬

‫‪stress granules formed in regulating stem‬‬
‫‪cell fate: a step towards successful‬‬
‫‪personalized medicine‬‬

‫عنوان البحث باللغة العربية‬

‫الواقع المثير للحبيبات السيتوبالزمية الناتجة عن اإلجهاد في‬
‫تحديد مصير الخاليا الجذعية‪ :‬خطوة ناجحة في اتجاه تصنيع‬
‫األدوية الشخصية‪.‬‬

‫‪A‬‬
Tables of contents
Abstract………………………………………………………………………………………………C
Introduction…………………………………………………………………………………..……1-2
Research Problem…………………………………………………………………………………...3
Research Questions…………………………………………………………………………………3
Research Hypothesis…………………………………………………………………………….….3
Research Objectives……………………………………………………………………..……….3-4
Research Significance………………………………………………………………………………4
Research Obstacles……………………………………………………………………………….4-5
Research Terminology……………………………………………………………………………....5
Theoretical perspective & literature review……………………………………………………6
Stem Cells………………………………………………………………………………………..…6-7
Embryonic Stem Cells (ESCs) ………………………………………………………………..…7-8
Induced Pluripotent Stem Cells (iPSCs) ……………………………………………………..…8-9
Stress Granules…………………………………………………………………………………9-10
Stress regulation and pluripotent stem cells ………………………………………………….…10
Research Methodology……………………………………………………………………….….11
Methodology…………………………………………………………………………………..…….11
Research community………………………………………………………………………….……11
Research sample…………………………………………………………………………………...11
Data collection and analysis tools………………………………………………………………...11
Research variables…………………………………………………………………………………12
Materials & Methods………………………………………………………………………………..12
I. Tissue Culture…………………………………………………………………………………12-13
II. Cancer cells MEM……………………………………………………………………………….14
III. Sodium chloride Treatment…………………………………………………………………….14
IV. Immunocytochemistry ………………………………………………………………….….14-15
V. SG formation quantification…………………………………………………………………….16
Efficiency of Methodology………………………………………………………………………….16
Research Results (Display and Analyze data and Information) ………………………….16
I. Hyperosmotic stress induces SGs assembly in IPSCs…………………………………..16-17
II. The morphology of induced pluripotent stem cells are not altered under hyperosmotic
stress……………………………………………………………………………………….…….17-18
III. Hyperosmotic stress induced SGs have a specific pattern of assembly in IPSCs……....18
IV. NaCl stress negatively affect cell morphology and capacity of MDA-MB-231 BCCs.18-19
Conclusions (Research Discussion) ……………………………………………………...19-20
Research Recommendations (future applications)……………………………………..….20
Acknowledgments…………………………………………………………………………….….20
Resources and References………………………………………………………………….21-23
Appendices……………………………………………………………………………………….….I
IPSCs background……………………………………………………………………………......….I
Research Results figures………………………………………………………………..………II-IV
Students work pictures…………………………………………………………………………VI-VII

B
Abstract
We have previously shown the formation of SGs in Embryonic stem cells
(ESCs) and induced pluripotent stem cells (iPSCs) subjected to heat and
oxidative stresses (sodium arsenite treatment). In this study have examined
the effect of other stresses (hyperosmolarity) on SG formation in iPSCs by
using NaCl to induce hyperosmolarity. Our results showed that hyperosmotic
stress induces SGs formation in iPSCs. SGs is initiated within this normal
range of NaCl and reached its maximum (100%) in a concentration little
beyond the normal range (200 and 300 mM). However, when the
concentrations increase to reach 400 mM, no SG formation was observed. In
addition, under the hyperosmotic stress conditions ranging from 50mM-400mM
no alteration of the shape and morphology of iPSCs was observed. The time
course of 200mM NaCl treatment showed SG formation that lasts up to 2 hrs,
however with high concentrations of 400mM iPSCs are resistance to SGs
formation was observed at any time of treatment. In conclusion, SG formation
occur under hyperosmotic stress but under specific concentrations and for an
extended period of time, indicating its role in regulating stem cell fate. In the
future we aim to test this effect on cancer stem cells.

C
Introduction

Pluripotent stem cells (PSCs), Embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs), are presently recognized jointly of the foremost
potential sources for cell based medical care, attributable to their distinctive
ability to self-renew or differentiate into numerous varieties of cells (1 & 2). The
power of ESCs to settle on between self-renewal and differentiation depends,
primarily, on changes on ESC micro-environmental conditions (3). Within the
early stages of embryonic development, ESCs reside in hypoxic surroundings,
wherever the cells manufacture terribly low amounts of ATP, whereas
throughout cell differentiation ATP production will increase through
mitochondrial biological process (oxidative phosphorylation), that in-turn
produces Reactive gas Species (ROS) like O2- and H2O2 changes in
mitochondrial biogenesis and antioxidant enzymes during the spontaneous
differentiation of human embryonic stem cells (4). High amounts of ROS
subject the cell to oxidative stress, that enforces the cell to enter their old state
or results in cell necrobiosis. Though many studies self-addressed the result of
oxidative stress on ESCs propagation and differentiation, our epigenetic data
of how ESCs start and off its gene expression in these adverse environmental
conditions is extremely very little.

Oxidative stress and other different varieties of environmental stresses

induces rapid response among the cell that results in a timely adaptation of
various restrictive processes like transcriptional regulation and translational
management that maximize the power of cells to survive beneath these
adverse environmental conditions (5). Throughout these adverse surroundings,
protein-producing cell arrest has been discovered (6, 7, 8, 9, 10, & 11). Stop of
cell macromolecule synthesis is caused by translation initiation inhibition that
results in speedy polysome dismantlement and is related to the activation of
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restrictive stress-response programs. These restrictive programs take care of
stress conditions by decreasing the expression of common work genes and
increasing the expression of genes that repair stress-induced disorder (12).
One part of the stress response program is the formation of a particular
varieties of granules called stress granules (SGs). SGs are non- membranous
structures of 100–200 nm in size and are related to the ER (Endoplasmic
Reticulum). These granules isolate elected ribonucleic acid transcripts and
proteins and store them throughout stress conditions (13 & 14). Once cells are
mitigated from the stress condition, hold on transcripts are the discharged for
either translation or degradation (after recovery).These dynamic granules are
thought to play a vital role in stress-induced reprogramming that regulate cell
translation and promotes cell survival.
It has been known that LIN28A, the master regulator of development in
C. elegans, is sequestered to SGs (15). Additionally, Palangi et al previously
observed the formation of SGs in ESCs subjected to stress conditions.
Thermal conditions and oxidative stresses (sodium arsenite treatment), but not
H2O2, are the selected stresses to form SGs (16). These granules sequestered
the pluripotent markers (LIN28A, L1TD1, and DPPA5). Altogether, allow us to
expect that those granules have the power to play a role in the managing the
switch between self-renewal and differentiation in iPSCs. (see the fig. IPSCs in
the appendices).
Since the formation of SGs in pluripotent stem cells is stress type
selective, so in this proposal we aim to test the effect of another type of
stress, hyperosmotic stress, on SG formation in pluripotent stem cells.

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Research Problem
Although, iPSCs have been widely used in disease modeling, to some
extent in drug screening, and starts its application in clinical trials, we still have
limited knowledge of how the fate of pluripotent stem cells is determined. The
limited knowledge of how these cells decide for self-renewal or differentiation
hinder us from producing high quality iPSCs and induces some obstacles in
maintaining them in culture. Thus create a problem in producing efficient and
safe cells that can be used in cell replacement therapy. Therefore, it is crucial
to understand the mechanisms of how these stem cell fate is controlled to
overcome this problem.
Research Questions
1- Would hyperosmotic stress induce SG formation in pluripotent stem cells
(iPSCs) like sodium arsenite and heat shock?
2- Would hyperosmotic stress change the morphology of pluripotent stem cells
(iPSCs)?

Research Hypothesis
Our central hypothesis is that beneath oxidative stress and thermal
conditions, SGs, a widely known part of the stress response program, are
formed in ESCs. Thus it's highly possible that hyperosmotic stress could
stimulate SGs in pluripotent stem cells (iPSCs) to sequester pluripotent RNA or
proteins to regulate stem cell fate.
Research Objectives
The overall objective of this project is to grasp the result of hyperosmotic
stress on SGs formation in pluripotent stem cells. To approach this question
we'll use iPSCs as our model and treat it with NaCl to stimulate hyperosmotic
stress in these cells. iPSCs are well-established cell lines that are
straightforward to govern and have outlined matter, so provide a singular

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technical tool to totally study queries of iPSCs reprogramming under stress
conditions.
Our analysis focused on the subsequent specific aims:
I. Determine whether iPSCs induce SG formation under hyperosmotic
stress.
II. Determine the minimum and maximum concentration(s) that induce SGs.
III. Determine the time course of SG induction.
IV. Determine the effect of hyperosmotic stress on iPSC morphology.
Research Significance
o Success during this project will pave the way to perceive extra roles in
epigenetics in control somatic cell fate that may in-turn enhance our
ability to understand the mechanism of stem cell fate regulation.
Understanding this mechanism is crucial in producing high quality iPSCs
that can be used efficiently and safely in:
1. Cell therapy for the treatment of many serious diseases, such as

diabetes and Autism, which are of significance importance among

Qatari population.
2. Testing the safety and quality of drugs (drug screening) to obtain

specific properties of the cells targeted by the drug.

3. Modeling of different diseases to enhance our understanding of their

pathogenesis.
o Altogether will help in using iPSCs in personalized medicine, which is
one of the main goals of Qatar National Strategy.
Research Obstacles
Among the most prominent difficulties and challenges that we faced:
Financial issues: unavailability of induced pluripotent stem cells, which
are necessary to carry out this research due to its high cost, and that
problem was overcome with the help of Dr. Mohamed Emara from the
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 College of Medicine at Qatar University, who provided us with these
cells.
Laboratory equipment: the difficulty of having laboratories equipped to
carry out this research, this problem was overcome by using the
laboratories of the College of Medicine at Qatar University.
Time trouble: the time of carrying out the research experiments, this
problem was overcome in coordination between the school
administration and the laboratories of Qatar University.
Research Terminology
❖ Stem cells are Non-specialized cells that can differentiate into various
types of cells, and can also divide in self-renewal to produce more of the
same type of stem cells (17).
❖ Embryonic stem cells (ESCs) are pluripotent stem cells derived from the
inner cell mass of a blastocyst, its distinguished by their ability to
differentiate and to self-renew (19).
❖ Induced pluripotent stem cells (iPSCs): adult cells that have been
genetically reprogrammed to an embryonic stem cell–like state by being
forced to express genes & factors essential for maintaining the defining
properties of ESCs (1 & 2).
❖ Pluripotent stem cells (PSC) are cells that have the capacity to self-
renew by dividing and to develop into all type of cells of the adult body,
but not extra-embryonic tissues, like placenta (18).
❖ Stress conditions are changes in the environment in which stem cells live
(3).
❖ Stress granules (SGs) are non- membranous structures of 100–200 nm
in size and are associated with the ER. These granules sequester
selected mRNA transcripts as well as proteins and store them during
stress conditions (5,13 & 14).
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Theoretical perspective & literature review
Stem Cells
The concept of stem cells has been defined as undifferentiated cells that
are characterized by their capability to proliferate, self-renew and give rise to
different cell types (17). Stem cells can be classified into five categories based
on their differentiation potential namely as totipotent, pluripotent, multipotent,
oligopotent and unipotent cells (18). The zygote that is produced after ovum
fertilization is termed as totipotent cell as it has the potential to develop into
both extra-embryonic cells (trophoblast cells that are later differentiated to form
the placenta) and embryonic cells (which form the embryo) (19). The second
category of stem cells are known as pluripotent stem cells (PSCs), which have
the ability to differentiate into all cell types that are derived from the three germ
layers (ectoderm, mesoderm and endoderm) from which all cells and tissues
are developed. Another type of stem cells that has lower potency and their
differentiation is restricted to cells from a single germ layer are called
multipotent stem cells (MSCs). Finally, those cells that differentiate into two or
more progeny within a specific tissue are called oligopotent stem cells; while
those that are able to only differentiate into a single and specific cell type are
referred to as unipotent stem cells (18).

The concept of stem cells has been divided into classical and evolving
views. In the traditional view of stem cells, a one-way direction of differentiation
is considered; where a specific mature cell is a product of a series of stem cell
differentiation (totipotent → PSCs→ MSCs→ oligopotent → unipotent stem
cells), which finally develop and differentiate to generate the specific mature
cell. During this developmental series, both differentiation potentiality and self-
renewal ability are decreased (17, 19, 20). On the other hand, the evolving
concept of stem cells has emerged based on several experimental data that
gave a state of plasticity in stem cell function. These observations have
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evidenced that tissue-specific stem cells have the ability to migrate and
contribute in producing other cell types when exposed to specific
environmental influences. The function of those migrated stem cells can be
determined by simultaneous effects of both microenvironment and growth and
differentiation factors that are present in their new environment. Moreover,
other studies have even suggested the ability of tissue specific mature cells to
reverse their differentiation and contribute to the stem cell pool (17, 19). As
reviewed, the emerged somatic cell reprogramming technologies have
supported the evolving concept of stem cells and reversed the linear hierarchy
of stem cell development (20).

Embryonic Stem Cells (ESCs)

ESCs are in vitro isolated cells from the inner cell mass of a blastocyst,
which is formed during the preimplantation stage of a fertilized ovum. These
ESCs are mainly characterized by their pluripotency and high expression levels
of telomerase activity. These telomerases are involved in maintaining cells’
telomere length which is an important tool in cells’ replicative life-span. In
addition, it has been reported that these cells have high nucleus to cytoplasm
ratio, prominent nucleoli and express several surface markers including
alkaline phosphatase, TRA-l-60, TRA-1-81, stage-specific embryonic antigen
(SSEA)–3 and SSEA-4 (20). Moreover, the molecular profile of ESCs showed
upregulated self-renewal genes along with pluripotent genes including OCT4
(octamer-binding transcription factor 4), SOX2 (sex determining region Y box
2), LIN28A (lin-28 homolog A), NR0B1 (nuclear receptor subfamily 0 group B
member 1) and TCF3 (transcription factor 3); whereas, developmental
differentiation genes have been observed to be downregulated such as CDX2
(caudal type homeobox 2), GATA6 (encodes GATA binding protein 6) and
HOXD8 (homeobox D8) (21, 22). Additionally, alteration at the level of

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epigenetic regulators of ESCs has also been reported such as the elevated
levels of HDAC5 (histone deacetylase 5) and DNMT3A (DNA
methyltransferase 3A) (22).

The unique characteristics of pluripotent ESCs made them a useful tool

in human research field mainly in studying human developmental processes,
developing and screening of new drugs, and offering a potentiality for
regenerative therapies (19, 21). Despite all these advantages, there are
several challenges that limit the use of ESCs as a research and therapeutic
tool. These limitations can be summarized in two main points: (i) ethical issues
related to embryo destruction during the process of ESCs extraction from the
inner cell mass, and (ii) problems associated with immune rejection that can
emerge when ESCs are used as regenerative medicine (18, 21).

Induced Pluripotent Stem Cells (iPSCs)

The first attempt of somatic cell reprogramming into pluripotency state
was reported in 1962 by nuclear transfer. The concept of this reprogramming is
based on inserting a somatic cell nucleus into an enucleated egg which has
factors that can induce somatic nuclear reprogramming (23, 24). Later in 2001,
Tada et al. reported the ability of ESCs to induce somatic cell reprogramming
through obscure factors (25). Collectively, these findings encouraged
researchers to identify the combination of factors that is able to induce somatic
cell reprogramming and obtain all the information and conditions needed for
pluripotent cell culture. Indeed, these identified factors and conditions were
used to generate induced pluripotent stem cells (iPSCs) (1, 2).

Takahashi & Yamanaka were the first researches to generate iPSCs

using mouse fibroblast cells in 2006. These mouse fibroblast cells were
reprogrammed into pluripotent state by retrovirally introduction of four
transcription factor genes known as OSKM or called later the Yamanaka
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factors (26). OSKM factors refer to OCT3/4, SOX2, KLF4 (Krüppel-like factor
4) and c-MYC (which is an oncogene). The ability of these four genes also to
reprogram human fibroblast cells (27). Importantly, the generated iPSCs
derived from both mouse and human fibroblasts showed indistinguishable
characteristics from ESCs in terms of morphology, proliferation, telomerase
activity, teratoma formation, surface markers, gene expression and epigenetic
state of pluripotent genes (26, 27). Therefore, a great interest in iPSC research
has boomed significantly.

The use of iPSC technology in research has created a new era in stem
cell research that carry several advantages. First, it overcomes the limitations
and challenges associated with ESCs usage. Second, iPSC technology
allowed the generation of human-derived cellular models of human diseases
that were difficult to be recapitulated using other types of models, such as
animal models. This places iPSCs in the forefront as a real human-derived cell
model that could help in understanding the molecular mechanisms underlying
the pathogenesis of a disease. Third, generating patient specific iPSCs in vitro
create a great advantage to screen for drugs and thus help in drug discovery
that is specific for each patient, which is a step towards personalized medicine.
Moreover, using iPSCs that are genetically identical to the patient as a
replacement therapy for some diseases is a long-term advantage for this
technology, which would avoid immune rejection problems associated with
using ESCs in regenerative medicine. Collectively, using iPSCs can be a
promising approach for applying the concept of personalized medicine (28, 29).

Stress Granules
Stress granules: when cells are under stress, condensed accumulations
in the cytoplasm composed of RNA and proteins that appear when the cells
undergo stress.

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 SGs are sites where messenger ribonucleoproteins (mRNPs) aggregates
under adverse environmental (stress) conditions. Those granules are selective
in their mRNA sequestration. They store mRNA that encodes housekeeping
genes, but others encoding stress-induced genes. Also, they are able to
aggregate a vast types of RNA binding proteins (RBPs) that are involved in
many different metabolic and signaling pathways. Given this, SGs are
implicated in different stress-induced signaling events within the cell such as
stress-induced apoptotic signaling. Once stress is removed, those granules are
disassembled and the sequestered RNAs become available back for
translation (13, 14).
Stress regulation and pluripotent stem cells
A variety of internal and external factors regulate the fate of PSCs self-
renewal and differentiation (30). The alteration in the micro-environmental
conditions is considered one of these factors that control such important cell
fate decision (31). Although, several studies shed the light on the possibility
that stress conditions could regulate pluripotency, there are several questions
that need to be answered of how (PSCs) respond to stress conditions to
regulate stem cell fate. In line with this, the presence of SGs had been
previously reported in PSCs subjected to stress conditions (15). These
granules sequestered the pluripotent markers (LIN28A, L1TD1, and DPPA5).
One of the possibilities of how PSCs regulate stem cell fate is through the
formation of SGs that can sequester differentiation genes/proteins to allow self-
renewal and switch this mechanism during differentiation. There are still more
to learn about the different stresses that induce SGs and whether other
stresses such as hyperosmotic stress form SGs or not.

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Research Methodology
Methodology
In this proposal, we used the experimental method.

Research community
Studying the effect of stress factors on pluripotent stem cells fate (PSCs).
Research sample
Studying the effect of stress factors (ex. hyperosmolarity, Sodium chloride) on
induced pluripotent stem cells fate (iPSCs). We also examined this effect on
breast cancer cell
Data collection and analysis tools
• Data: Quantitative & Quality data.
• Sources: Materials & methods.
• Collecting tools: Experiments & observation.

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Research variables
Table (1): Research variables, Control variables, Independent variables, &
Dependent variables.
Testable ScientificControl Variables Independent Dependent
Question Variable Variable

1- Would hyperosmotic Petri dishes, Different SG

stress induce SG media, NaCl formation
formation in pluripotent PSCs, Pipette, concentrations. and cell
stem cells like sodium tubes, Chemical Different time morphology
arsenite? reagents, of incubations. and
2- Would hyperosmotic microscopes, integrity.
stress change the incubator,
morphology of pluripotent centrifuge.
stem cells?

Table (2): Comparison between the control & Experimental sample in term
of stress granules formation.
Control Sample Experimental sample Formation of SGs
- ve control = iPSCs, iPSCs, treat with - ve control = No SGs
no treat with NaCl. different NaCl formation.
+ ve control = iPSCs, concentrations & + ve control = SGs
treat with Sodium Different time of formation.
arsenite. incubations.
Materials & Methods
I. Tissue Culture
Induced Pluripotent stem cells (hiPSCs) purchased from WiCell were
cultured in feeder-free system to grow cells exploitation Matrigel (Corning) for
cell adherent. Human somatic cell colonies were full-grown and maintained in
̊ in an exceedingly 5%
StemFlex media (Thermo Fisher Technologies) at 37 C
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CO2 incubator. Cells were fed each different day and subject to passage once
cell confluency was around eighty percent (from 4 to 5 days once a passage).
To organize for cell passage, 35 mm dishes were coated with Matrigel for ½
hrs, 2 hrs or nightlong before the passage. At the time of passage, colonies
were washed with 1ml DPBS before being unconnected to the suitable size of
cell colonies (usually aggregates are close to 100μm in size) (32). Then, cells
were detached from the dish by using 500μl of non-enzymatic chemical agent
(EDTA; STEMCELL technologies). Once the addition of EDTA, cells were
̊ in a 5% CO2 incubator; then, EDTA were
incubated for 45 seconds at 37 C
aspirated from the dish that was reintubated for two minutes. Afterward,
StemFlex media was added to collect the colonies in an exceedingly 15 ml
cone-like tube. This step were recurrent with another 1 cm3 of StemFlex media
to clean the dish and collect nearly most of the detached colonies. later, cells
were centrifuged for 4 mins at 800 RPM and 22̊ C. Supernatant was then
aspirated and also the cell pellet was resuspended with 1cm3 media. Then,
70μl of cells were side to the dish that was antecedently coated with Matrigel
that was aspirated and replaced with 2 cm3 StemFlex media. Lastly, even
distribution of the little cell aggregates among the dish are going to be ensured
by north , south & east, west motion. (see the fig.I)

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II. Cancer cells MEM
MDA-MB-231 breast cancer cells were cultured in Dulbecco’s Minimum
̊ in an exceedingly 5% CO2
Essential Media (DMEM; Life Technologies) at 37 C
incubator. When cells became 80% confluent cells were treated with different
concentration of the sodium arsenite or sodium chloride for different time
courses.
III. Sodium chloride Treatment
IPSCs cells were treated with completely different concentrations of
binary compound, sodium chloride (50, 100, 200, 300, 400 mM) and for various
incubation times (0, 15, 30, and 60 & 120 minutes) to check the result of the
treatment on iPSCs pluripotency and on SG formation. Since sodium arsenite
(250 mM) stimulates SGs formation it'll be used as a positive control. On the
opposite hand H2O2, that didn't stimulate SGs in iPSCs were used as a
negative control (16).
IV. Immunocytochemistry
In a 6-well plate, cover slips were coated with Matrigel for ½ hrs, 2 hrs or
overnight; then incubated with StemFlex medium for ½ hrs or 2 hours to
equilibrate the cover slips before cells are going to be seeded on them. hiPSC
cells were cultured onto 6 cover slips. Once colonies reached the suitable size,
that is typically concerning 4-5 days post passage, the immunocytochemistry
procedures can begin by washing the cells doubly with PBS. Then, cells are
fastened by using 4% paraformaldehyde (Sigma) for 15–30 mins at RT with
continuous shaking. Later, cells were washed doubly from the fixative chemical
agent for 5-10 minutes every by TBST solution (TBS with 0.2% Tween).
Fastened cells were then permeabilized by using PBST (PBS with 0.2% Triton)
for 10 mins and followed by washing cells doubly with TBST solution (all
immunocytochemistry steps were performed with continuous shaking on a
microtiter shaker). To decrease the non-specific binding of antibodies, colonies
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were incubated in an exceedingly obstruction buffer (5% horse blood serum
(Hyclone) in PBS with sodium azide) for a minimum of 1 hr at RT. Once
obstruction, cells were incubated with the required primary antibodies diluted
̊ (Either
within the obstruction buffer with continuous rocking nightlong at four C
antibodies to substantiate pluripotency like SOX2, Oct4, and Nanog or those to
notice SG formation like G3BBP and TIAR). Afterwards, cells were washed
thrice with TBST; then, cells were incubated in dark with the suitable
secondary antibodies that were diluted in PBS for 1 hr at RT. After this time,
cells were washed thrice with TBST and incubated finally with 0.5μg/ml
Hoechst 33258 dye (Molecular probes; 1:30000 dilution in PBS) for 3-5 mins to
permit nuclear staining. Then, cells were washed doubly with TBST and coated
with PBS to avoid cover slip dryness. Finally, these cover slips were mounted
by using Mowiol (Poly(vinyl alcohol)) mounting medium. Cells were then shown
and photographed by a fluorescence (Zeiss Axio Imager 2) by using 40X
objective. (see fig. II)

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V. SG formation quantification
Cells containing SGs were detected as explained previously by using
immunocytochemistry. A manual investigation of SGs cells were done close to
ten representative fields among the treated or non-treated cells. three or
additional clear and visual granules will be considered positive. Differentiated
cells additionally as dead ones were excluded. The proportion of cells with SGs
are going to be quantified by investigation ~500 cells/experiment.
Efficiency of Methodology
We planned our methods in a way to be effective in achieving the goals
of the study. We used the experimental method based on the use of practical
experience in studying how stress factors affect the fate of pluripotent stem
cells. The research method is designed with right procedures as well as
accurate positive and negative controls.
Research Results (Display and Analyze data and Information)
I. Hyperosmotic stress induces SGs assembly in IPSCs
To determine whether hyperosmolarity induces SGs formation in PSCs,
iPSCs (IMR-90-1) were treated with different concentrations of sodium chloride
(Fig. 1A and B in the appendices). Each treatment were incubated for 1hr, then
cells were fixed, stained with the robust SGs marker (G3BP), and quantified for
SGs formation using fluorescence microscopy. Sodium arsenite treatment was
used as a positive control and LIN28 was used a pluripotent marker that is
known to be recruited to SGs under sodium arsenite and heat shock
treatments (Palangi et al, 2017). As compared to non-treated cells that
showed no granules (Fig 1A), 50mM of sodium chloride treated cells showed
no granule formation (Fig. 1B). In contrast, at concentration of 100mM of
sodium chloride, ~5% of the cells induce granule formation (Fig. 1C and H in
the appendices). An abrupt and significant increase in the number of granules
were observed in 100% of the cells treated with 200mM sodium chloride (Fig.
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1D and H in the appendices). Interestingly, this increase did not last with the
elevation of sodium chloride concertation, however, the number of cells with
granules gradually decreased to reach 80% at 300mM sodium chloride
treatment (Fig. 1E and H in the appendices) till it completely disappeared in
cells treated with 400mM sodium chloride (Fig. 1F and H in the appendices).
The granules formed in sodium chloride treated cells resemble those observed
in the positive control cells treated with 250uM sodium arsenite (Fig. 1G in the
appendices). This data indicate that certain concentrations of hyperosmotic are
capable to induce SGs in PSCs.

II. The morphology of induced pluripotent stem cells are not altered
under hyperosmotic stress conditions.
In the previous experiment LIN28 expression did not change under
different sodium chloride treatments conditions, suggesting that the cells
maintain its pluripotent status.
As another way to assess pluripotency under these conditions, we tested the
shape and size of non-treated and treated iPSCs colonies using phase
contrast microscope.
Stem cell colony morphology, is one of the main characteristics that
determine stem cell pluripotency visually. We chose 200mM (Fig. 2B in the
appendices) as a concentration that induced 100% SGs formation and 400mM
(Fig. 2C in the appendices) that induce no SGs formation. The expected
pluripotent morphology was detected in all treated and non-treated cells (Fig.
2A-D in the appendices). The colonies appear with distinct, circular, and
homogenous borders and are composed of small, compact, uniform, and
healthy cells that are densely packed together. Similarly the positive control
cells treated with sodium arsenite did not show any changes in cell morphology

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(Fig. 2D in the appendices). These results indicate that hyperosmotic stress
ranging from 50mM-400mM did not alter the shape and morphology of iPSCs.
III. Hyperosmotic stress induced SGs have a specific pattern of assembly
in induced pluripotent stem cells.
Since the formation of SGs is known to be time dependent (Anderson
and Kedersha, 2009), so we used immunostaining to examine the kinetics of
SGs assembly in iPSCs treated with NaCl 200mM and 400mM for various
incubation intervals of 15, 30, 60, and 120 min. Whereas the first observation
of SGs in 200mM treated cells was as early as 15 min after treatment, 400mM
did not induce SG formation at any time point of incubation (Fig. 3A and B in
the appendices). In 200mM, the number of cells showing SGs gradually
increases over time till appear in the majority of cells by 60 min and remain
constant over 2 hours of incubation time (Fig. 3A in the appendices). These
results show that 200mM NaCl is capable to form SGs that lasts for long period
of times, however with high concentrations of NaCl (400mM), iPSCs are
resistance to SGs formation at any time.
IV. Hyperosmotic stress negatively affect cell morphology and capacity
of MDA-MB-231 breast cancer cells
It has been previously shown that hyperosmolarity may affect cancer cell
proliferation (33). Since our data showed the formation of SGs in specific
concentration of iPSCs with keeping cell adherence and morphology, it was of
our interest to test if the same phenomenon occur in cancer stem cells. MDA-
MB-231 breast cancer cells were used as a model, where cells were treated for
one hour with different concentrations of NaCl 100mM, 200mM, 300mM, and
400mM and then stained with Coomassie brilliant blue to check their
morphology and cell capacity. As shown in (Fig. 4 in the appendices) the
gradual increase in sodium chloride concentration has significant effect on
cancer cells as they gradually lose their morphology and showed a high degree
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of detachment. The highest effect was observed with 400mM NaCl treatment
where the majority of cells are detached from the plate (Fig. 4E in the
appendices). Interestingly, at the same concentration, the cells lost their
morphology as compared to the non-treated ones (compare Fig. 2A to 2E in
the appendices). These results suggest that hyperosmolarity lead to MDA-MB-
231 breast cancer cell detachment and may be death, which may also be the
same effect on cancer stem cells.
Conclusions (Research Discussion)
In this study we were able to show that hyperosmotic stress induces
SGs in pluripotent stem cells. Hyperosmotic stress is stimulated in iPSCs by
NaCl was used as a natural chemical that is essentially used by the human
body in many of its metabolic process. The physiological concentration of
NaCl in the human body is 100-150mM (34). In our study, we noticed that SGs
is initiated within this normal range and reached its maximum in a
concentration little beyond the normal range (200 and 300 mM). However,
when the concentrations increase to reach 400 mM, no SGs formation was
observed. This indicate that SGs are able to maintain cell function and survival
by possibly sequestering antiapoptotic genes as a way to protect them from
apoptosis. However, when the hyperosmolarity reach toxic levels cells are
unable to induce SGs and thus these could enter into the apoptotic phase.
Indeed, SGs formation under 200mM NaCl lasts for long period of times (lasts
up to 2 hr), however with high concentrations of 400mM iPSCs are resistance
to SGs formation at any time of treatment. The ability of the cells to form SGs
for longer period of times in iPSCs indicate the possibility that these cells is
able to preserve its self-renewal ability for this period. A potential scenario that
genes responsible for differentiation are sequestered to SGs and thus keep the
stemness status of these cells.

- 19 -
 Since our study showed a possibility that SGs protect iPSCs, we thought
to test the effect of hyperosmotic stress in cancer cells. It has been previously
shown that SGs stimulate survival in cancer cell, and hyperosmolarity stress
affect cancer cell proliferation (33). Our preliminary experiment by treating
MDA-MB-231 breast cancer cell with different concentrations of NaCl showed
that the high concentration of 400mM significantly changed the cell morphology
and cause remarkable cell detachment, as compared to 200mM that showed
mild effect. iPSCs showed 100% SG formation at the concentration of 200 mM.
Whether SG formation plays a role in cancer cell protection at low
concentration treatment (200 mM) through cancer stem cells protection. This is
an important question that need an answer.
Research Recommendations (future applications)
▪ Test the effect of hyperosmotic stress on cancer stem cells.
▪ Test the formation of SGs in cancer stem cells.
▪ Test if SGs stimulate apoptosis in stem cells (pluripotent stem cells
and/or cancer stem cells).
▪ Test the effect of hyperosmotic stress on solid tumors in laboratory
animals.
Acknowledgments
We thank Dr. Mohamed Emara and college of medicine, QU, for providing us
lab space, equipment, and reagents to perform the experiments. Special
thanks for Mr. Othman and Dr. Emara for their supervision, and guidance
throughout the work. This work was supported by HSREP-01 funded by Qatar
National Research Fund 2019/2020.

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Appendices

IPSCs background

Fig. Induced Pluripotent Stem Cells (IPSCs): meaning, function and

significance.

I
Appendices
Research Results figures

Fig. 1: Hyperosmotic stress induces SGs assembly in iPSCs.

Fluorescence microscopy images showing (A) Non treated iPSCs, (B-F) iPSC
treated with different concentrations of sodium chloride or (G) iPSC treated
with 250uM of sodium arsenite (positive control). Cells were stained with the
SGs markers (G3BP (green) and LIN28). Nucleus is stained in blue (Hoechst).
White arrows indicate SGs. (H) Quantification of the percentage of cells
showing SGs, Cells were counted in 10 separate fields. Insets show magnified
SGs.

II
Appendices
Research Results figures

Fig. 2: Hyperosmotic stress did not alter the morphology of iPSCs. The
morphology of (A) non-treated hiPSC colonies or colonies treated with (B)
200mM (C) 400mM NaCl or (D) 250μM Sodium arsenite using phase contrast
microscopy. Large insets show magnified views of cells.

Fig. 2: Hyperosmotic stress did not alter the morphology of iPSCs. The
morphology of (A) non-treated hiPSC colonies or colonies treated with (B)
200mM (C) 400mM NaCl or (D) 250μM Sodium arsenite using phase contrast
microscopy. Large insets show magnified views of cells.

III
Appendices
Research Results figures

Fig. 3: Hyperosmolarity induces SGs assembly in a specific manner in

iPSCs. Fluorescence microscopy images showing iPSCs treated with (A)
200mM and (B) 400mM of sodium chloride for different time points as
indicated. Cells were stained with the SG marker (G3BP (green)). Nucleus is
stained in blue (Hoechst). White arrows indicate SGs.

IV
Appendices
Research Results figures

Fig. 4: Hyperosmolarity induces detachment and morphology changes in

MDA-MB-231 breast cancer cells . Phase contrast microscopy images
showing (A) non treated MDA-MB-231 breast cancer cells or cells treated with
(B) 100mM, (C) 200mM, (D) 300mM, and (E) 400mM of sodium chloride. Cells
were stained with the Coomassie brilliant blue.

V
Appendices
Students work pictures

VI
Appendices
Students work pictures

VII