cliapter 1shodhganga.inflibnet.ac.in/bitstream/10603/15774/6/06...embryonic germ layers (ectoderm,...
TRANSCRIPT
Cliapter 1
Introtfuction
Chapter 1: Introduction
Chapter 1: Introduction
1.1. Stem cells in development and differentiation
The development of the mammalian embryo begins with the fertilization of
the mature oocyte by the sperm-the zygote, the ultimate stem cell. It is totipotent
with the ability to produce all the cell types of the species including the trophoblast
and the embryonic membranes. Development begins when the zygote undergoes
several successive cell divisions, each resulting in doubling of the cell number and
reduction in the cell size. At the 32- to 64-cell stage called morula, each cell is
called a blastomere, which retains totipotency. The next stage is the blastocyst
(Figure 1.1) - consisting of a hollow ball of cells, which is the primary differentiation
event during mammalian development leading to the delineation of the inner cell
mass (ICM) and the trophectoderm (TE) (Adjaye et a/., 2005). The ICM is
comprised of pluripotent cells, which subsequently differentiate into the three
embryonic germ layers (ectoderm, mesoderm and endoderm) (Figure 1.2) the
founders of all adult tissues (Ohtsuka and Dalton, 2008). The formation of organs
from these germ layers is achieved by asymmetric cell division of the multipotent
germ layer cells whereby they segregate to produce appropriate numbers of stem
cells and differentiated daughters (Morrison and Kimble, 2006; Knoblich, 2008).
Molecular and cellular interactions between germ layers, combined with the
development potential of the cells, prompt further differentiation of organ-specific
cell types during organogenesis. The development of individual organ in animal
embryos involves the formation of tissue-specific stem cells, multipotent in nature,
that sustain cell renewal and differentiation of their own tissue (Slack, 2008).
Differentiation is a common process in adults as well; an organism after
development and growth undergoes cell division for renewal and repair of the tissue
adapted from Kirschstein, 2001
Figure 1.1: Blastocyst. Blastula stage of the embryo is characterized by the presence of trophectoderm surrounding a cavity called blastocoel. At the apical region of the cavity
lies a bunch of cells called inner cell mass, which are pluripotent in nature, which divide
and differentiate to develop cells of the three germ layers, fmally metamorphosing into an organism.
Zygote
~
Blnstocyst
~
muscle m uscle of the blood muscle cells kklney cells (in ut)
Lung cell (alveolar
cell)
adaptedfrom Kirschstein, 200 /
Figure 1.2: ES cell differentiation. Fertilization of germ cells results in the totipotent cell, zygote, which undergoes series of divisions to form morula and differentiates into blastula. The inner mass cells of the blastocyst, under in vivo conditions, undergo division and differentiation into cells of three germ layers, namely, ectoderm, mesoderm and endoderm. Cells from these layers undergo further division and differentiation to
develop different tissues and organs.
lA
Chapter 1: Introduction
for the lifetime of the organism and the cells with such properties are termed adult
stem cells.
Developmental biologists are in search for understanding: How can the
fertilized egg generate so many different cell types? How can the cells form ordered
structures, as the differentiated cells are not randomly distributed, but organized into
intricate tissues and organs? How do our cells recognize when to stop dividing and
how cell division is so tightly regulated? How the sperm and egg, very specialized
cells which can transmit instructions for making an organism from one generation to
the next, set apart to form the next generation and what are the instructions in the
nucleus and cytoplasm that allow them to function this way?
Earlier approaches such as anatomical, exp~rimental or genetic were
commonly used to study development. Most of the studies on embryo development
were based on the defect experiment wherein one destroys a portion of the embryo,
the isolation experiment wherein one removes a portion of the embryo, the
recombination experiment, wherein one observes the development of the embryo
after replacing an original part with a part from a different region of the embryo and
the transplantation experiment, wherein one portion of the embryo is replaced by a
portion from a different embryo. Later, cloning experiments, differential gene
expression, RNA localization techniques, generation of transgenic cells and
organisms were some of the techniques used to study development of higher
organtsms. The most successful model systems for animal development have been
established in species in which genetic manipulation is relatively easy such as
Drosophila melanogaster, Caenorhabditis e/egans, and most recently the zebra fish
Danio rerio. C. e/egans development is characterized better and the complete cell
lineage of the organism is recorded. The development pattern of each somatic cell
2
Chapter 1: Introduction
from zygote to adult organism is known, which facilitates the identification of the
fate of any particular cell at any point of development. However, genetic analysis of
mammalian development has been restricted due to relatively low rates of
reproduction, long gestation periods, and limited access to embryos, which has made
analysis of development complicated even in the most commonly used mammal, the
mouse, Mus musculus. A variety of mutations in the mouse have been described
which are lethal at some stage or the other in the embryonic development. The
biochemical nature of these mutations cannot be easily studied in vivo because
resorption or abortion inevitably results. In order to understand further the molecular . properties of cells that have self-renewal and differentiation properties, which leads
to the embryo development, embryonic stem cell lines were established from
blastocyst in early 1970s. The embryonic stem cells (ESCs) are useful to study
embryo development as they can be established from individual blastocyst of any
genotype, ease of establishment, advantage of working with cells, which are
temporally close to the embryo, and in vitro expression of a large degree of
development and differentiation potential. Studies of embryonic stem cells will yield
information about the complex events that occur during animal development and
understanding the causes of various birth defects.
3
Chapter 1: Introduction
1.2. Stem cells in biomedical applications
Stem cells offer unprecedented opportunities to study development, replace
damaged cells, study and treat various diseases and as a resource for testing new
medical treatments. Stem cell transplantation was pioneered using bone-marrow
derived stem cells by a team at the Fred Hutchinson Cancer Research Center from
the 1950s through the 1970s led by E. Donnall Thomas, whose work showed that
bone marrow cells infused intravenously could repopulate the bone marrow and
produce new blood cells. His work also reduced the likelihood of developing a life
threatening complication called graft-versus-host disease (Thomas et a/., 1957) and
now it is a well-established treatment for blood cancers and other blood disorders.
Stem cell research contributes to a fundamental understanding of how
organisms develop and grow, and how tissues are maintained throughout adult life.
This knowledge will be useful to study the pathobiology of various diseases and
further develop diagnostics and therapeutics for treatment. The development of a
range of human tissue-specific and embryonic stem cell lines will provide
researchers with the tools to model disease, test drugs and develop increasingly
effective therapies. Currently, researchers are investigating the use of adult, fetal and
embryonic stem cells as a resource for various, specialized cell types, such as nerve
cells, muscle cells, blood cells and skin cells that can be used to treat various
diseases. Replacing diseased cells with healthy cells, a process called cell therapy, is
another promising use of stem cells in the treatment of disease. In theory, any
condition in which there is tissue degeneration can be a potential candidate for stem
cell therapies, including Parkinson's disease, spinal cord injury. stroke, bums, heart
disease, Type 1 diabetes, osteoarthritis. rheumatoid arthritis. muscular dystrophies
and liver diseases.
4
Chapter 1: Introduction
The capacity of ES cells to differentiate into almost all of the cell types of the
human body highlights their potentially promising role in cell replacement therapies
for the treatment of human diseases. The science of stem cell therapies has entered a
phase of research and development that could lead to unprecedented cures and
palliative treatments. The development of patient-specific or disease-specific
pluripotent stem cells has great therapeutic promises. These cells provide a powerful
new tool for studying the basis of human disease and for discovering new drugs.
The resulting embryonic stem cells could be developed into a required cell type, and
if transplanted into the original donor, would be recognized as 'self, thereby avoiding
the problems of rejection and immunosuppression that occur with transplants from
unrelated donors.
Kim et a/ have shown that a highly enriched population of midbrain neural
stem cells can be derived from mES cells (Kim et a/., 2002; Kim et a/., 2003 ). The
dopamine neurons generated by these stem cells show electrophysiological and
behavioral properties expected of neurons from the midbrain. These results
encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.
ES cell derived dopaminergic neurons and insulin-secreting cells normalized the
Parkinson's disease and Diabetes respectively in mouse animal model systems
(Figure 1.3 and Figure 1.4) (Soria et a/., 2000; Kirschstein, 2001 ). The same can be
extrapolated to humans after significant understanding of molecular processes of ES
cells.
There are several mES cell lines available for research purposes. Although
all mES cell lines of different origins are regarded as equally pluripotent, reports
indicate that, their in vitro differentiation potential varies, suggesting that their
5
mass or bi<J~tocyst
Undtfle ronuotod embryonic stem cells
EMBRYOID BODIES
liTFSn m edium (•nsulin/tramferrin/
rtbronectin/sele ntum)
Adherent substrate
SELECTION OF NESTIN-POSITIVE CELLS
N2 m odoumlbFGF/Iam onon I I Ex~ansion
NESTIN-POSITIVE NEURONAL Phase PRECURSOR CELLS
~~ .... ; Differentiation Phase
N2 mcdiumlbFGF/ 827 media supplem ent
NESTIN·POSITIVE PANCREATIC PROGENITOR CELLS
DOPAMINE- AND SEROTONINSECRETING NEURONS
INSULIN-SECRETING PANCREATIC ISLET-LIKE CLUSTERS
At•t»udui4od Wtlh IM"""'YOn ltnm N~n lllf'lll""tvwWlgY
Rl:>flfodUCOd Wllh J)eml!Ubl tmmSdrnrAt
adapted from Kirsclrstein. 200/
Figure 1.3: ES cell differentiation in vitro. ES cells are pluripotent which can differentiate into cells
of all the three germ layers, namely, ectoderm, mesoderm and endoderm. These cells initially, in the absence of LIF, undergo divisions and form aggregated cell structures called 'embryoid bodies ' and after treatment with specific cytokines and growth hormones these embryoid bodies differentiate and develop into neurons, pancreatic islet cells etc.
SA
Diabet.ic mouse
Mou•e embryonic cells
;
Insulin secreted
Mouse blastocyst
adapted/rom Kirschstein, 2001
Figure 1.4: Cell replacement therapy in mouse model. Soria eta! 2000, directed differentiation of mES cells with growth hormones, cytokines and specific conditions, into pancreatic islet like cells in vitro which are then assayed for their function, activity and purity. These cells when transplanted into artificially induced diabetic mice, recovery from diabetes
was observed as these implanted cells vascularized and started producing insulin.
5B
Chapter I: Introduction
response to developmental signals is different. Examples of mES cell lines used for
in vitro differentiation:
Cardiogenic differentiation: 03, R1, 8117, AB1, AB2.1, CCE, and E14.1.
Myogenic differentiation: 03, BLC6, AB 1, AB2.1.
Neuronal differentiation: BLC6, 03, CGR8, Jl.
Endothelial and vascular smooth muscle cell differentiation: 03, AB 1, AB 2.1.
Epithelial differentiation: 03.
Stem cell treatments have the potential to change the face of human disease
and alleviate suffering and technologies derived from adult and embryonic stem cell
research can treat many of these diseases. More research is needed concerning both
stem cell behavior and the mechanisms of the diseases they could be used to treat
before most of these experimental treatments become realities (Singec et al., 2007).
Limitation of stem cell therapy New clinical applications for stem cells are currently
being tested therapeutically for the treatment of musculoskeletal abnormalities,
cardiac disease, liver disease, autoimmune and metabolic disorders (amyloidosis),
chronic inflammatory diseases (lupus) and other advanced cancers. There are
currently several limitations to using adult stem cells. Although many different
kinds of multi potent stem cells have been identified, adult stem cells that could give
rise to all cell and tissue types have not yet been found. Adult stem cells are often
present in only minute quantities and can therefore be difficult to isolate and purify
and adult stem cells may contain more DNA abnormalities--caused by sunlight,
toxins, and errors in making more DNA copies during the course of a lifetime.
These potential weaknesses might limit the usefulness of adult stem cells. ES cells
6
Chapter 1: Introduction
also have some limitations at present such as, they are tumorigenic, as they form
tumors when undifferentiated ES cells are injected into the organism, and when
inspecting these tumors we see cell types of all the three germ layers. Purity of
differentiated and undifferentiated cells is important. We need to fine-tune the
differentiation mechanisms for which we need to know the specific molecular
pathways active in these cells. ES cells differentiate into several cell types, even
when directed differentiation protocols are used. There are reports on genetic
selection of the differentiated cells derived from genetically modified ES cells,
however use of these genetically modified ES cell derived cells for CRT raises few
issues.
Ethical issues of ES cells The adult stem cells e.g. from bone marrow transplants;
can help cure diseases such as leukemia, but they are not near as effective to diseases
such as diabetes. The other problem with using bone marrow transplants is that they
do not help cure major organs; the stem cells just tum into scar tissue. The major
controversy is that the best stem cells come from growing embryos. Stem cell
controversy is the ethical debate centered on research involving the creation, usage
and destruction of human ES cells (de Wert and Mummery, 2003; Solomon and
Brockman-Lee, 2008). Some opponents of the research argue that this practice is a
slippery slope to reproductive cloning and fundamentally devalues the worth of a
human being. Contrarily, medical researchers in the field argue that it is necessary to
pursue ES cell research because the resultant technologies could have significant
medical potential, and that excess embryos created for in vitro fertilization could be
donated with consent and used for the research. This in tum, conflicts with
opponents in the pro-life movement, who advocate for the protection of human
embryos. The ensuing debate has prompted authorities around the world to seek
7
Chapter 1: Introduction
regulatory frameworks and highlighted the fact that ES cell research represents a
social and ethical challenge. Some organizations have issued recommended
guidelines for how stem cell research is to be conducted.
1.3. Understanding pluripotency and self-renewal- Mouse Embryonic Stem (mES)
cells as model system
Stem cells are distinguished from other cell types by two important
characteristics i.e. self-renewal and differentiation. Stem cells are classified
according to their plasticity or developmental versatility. Totipotent stern cells can
give rise to a fully functional organism as well as to every cell type of the body e.g.
zygote. Pluripotent stem cells are capable of giving rise to virtually any tissue type,
but not to a functioning organism e.g. embryonic stem cell (ESC), embtyonic germ
cell (EGC), and embryonal carcinoma cell (ECC). Multipotent stern cells (Figure
1.5) are more differentiated cells i.e. their possible lineages are less plastic/more
determined and thus can give rise only to a limited number of tissues. For example,
a specific type of multipotent stem cell called a mesenchymal stem cell has been
shown to produce bone, muscle, cartilage, fat, and other connective tissues.
In the early 1970s the stem cell lines were isolated from mouse blastocyst,
which made possible a number of new biochemical, immunological, and genetic
approaches to study early mammalian development. Few distinguished aspects of
blastocyst-derived embryonic stem cell lines such as: they can be established from
individual blastocyst from any genotype, the consistency from one cell line to the
next with respect to in vitro developmental process (important for comparative
analysis), ease of establishment and advantage of being temporally close to embryo
8
Bone
Hematopoietic stem cell
Natural killer (NK) cell
progenitor cell
Neutrophil
adapted from Kirschstein, 2001
Figure 1.5: Multipotent stem cells. A category of stem cells differentiate into a few cell types of a specific germ layer and such stem cells are termed multipotent e.g. hematopoietic stem cells. This complex process involves intermediate stages of cells called progenitors (e.g. myeloid and lymphoid progenitor cells) that further undergo division and differentiation to form specific cell types e.g. T and 8 lymphocytes, nuetrophils, basophils, eosinophils, platelets, macrophages, red blood cells, etc.
8A
Chapter 1: Introduction
and their in vitro expression of a large degree of developmental potential, which
together make them potentially useful as a model system for embryonic development
(Doetschman et al., 1985). In three-dimensional suspension culture ES cells form
highly organized cystic embryoid body structures, which are in many respects
analogous to post-implantation embryos. With these structures one should be able to
answer more easily questions concerning 'development' of the embryo rather than
simply 'differentiation' of cell types. They are also be suitable for studying the
developmental regulation of the expression of genes, normal or altered, inserted into
ES cells, thereby offering all of the analytical advantages of in vitro systems. They
can also be used to investigate the effects of drugs and environmental factors on
differentiation and cell function in embryotoxicity and pharmacology.
ES cells are amenable for in vitro genetic modifications, such as gene knock
out, gene knock in, gene duplication, inversions, cis and trans rearrangement, gene
trap and other mutations. Since they can transmit the mutations to next generation,
i.e. they are capable of germ line transmission; scientists can study the gene
expression, development, and genetics of the organisms.
Mouse ES cell isolation Pluripotent cells are present in a mouse embryo until at least
an early post-implantation stage, as shown by their ability to take part in the
formation of chimaeric animals and to form teratocarcinomas. Evans and Kaufman
reported the establishment in tissue culture of pluripotent cell lines, isolated from in
vitro cultures of mouse blastocysts (Figure 1.6) (Evans and Kaufman, 1981 ). These
cells are able to differentiate either in vitro or after inoculation into a mouse as a
tumor in vivo. They have a normal karyotype. Later reports showed that these ES
9
Cleavage stage embryo
Cells dissociated and replated
/
Irradiated mouse fibroblast feeder cells
Established embryonic stem cell cell cultures
adapted from Kirschstein, 2001
Figure 1.6: Embryonic stem cell isolation. Embryonic stem cells are derived from 'blastocyst stage' of embryo. Inner cell mass is extracted either surgically or enzymatically and dissected into single cells. These cells are then grown on feeder cells such as embryonic fibroblasts in vitro in the presence of rich growth medium, which then develop into colonies and are then propagated as mES cell line.
9A
Chapter 1: Introduction
cells could be cultured for longer periods without undergoing differentiation by
supplementing the culture medium with leukemia inhibitory factor (LIF) (Smith et
a/., 1988). This provides an alternative method for culturing ES cells, which does
not involve the feeder cells. They could be cultured in vitro, and can also be injected
back in the mouse blastocyst to get young ones, completely derived from the injected
ES cells (Nagy et a/., 1993). ES cells contribute to the characteristics of the
organism, therefore capable of germ line transmission. Some of the commonly used
mES cell lines are: CCE, D3, El4, El4.1, Rl, G-Olig2, 8117, ABl, AB2.1, HMl,
MBL-5, etc (Boheler eta/., 2005; Siva eta/., 2007).
Induced pluripotent stem (iPS) cells Use of ES cells have some ethical issues related
to their derivation from the embryos. In 2006, Takahashi and Yamanaka at Kyoto
University were able to generate pluripotent cells directly from mouse embryonic or
adult fibroblast cultures, which are termed as 'induced pluripotent stem cells' (iPS)
(Takahashi and Yamanaka, 2006). They performed genetic reprogramming with
protein transcription factors Oct3/4, Sox2, c-Myc, and Klf4 to generate pluripotent
stem cells equivalent to ES cells derived from human adult skin tissue. Yu eta/ at
the University of Wisconsin-Madison used a different set of factors, Oct4, Sox2,
Nanog and Lin28, and carried out their experiments using cells from human foreskin
(Yu et a/., 2007). Researchers are also hoping to make ES cells from human adult
cells by somatic cell nuclear transfer referred as therapeutic cloning, generating ES
cells that are genetically identical to the donor of adult cells. These genetically
matched ES cells can then be used to differentiate into specialized cell types to be
used for CRT. So far such technology has been applied to obtain ES cells from
sheep, mice, cows, monkeys and other mammals.
10
Chapter I: Introduction
1.4. Molecular pathways regulating self-renewal and pluripotency in mES cells.
The decisive, instructive and permissive signals that govern sternness are
provided by growth factors in the microenvironment or "stem cell niche" (Ying et
a/., 2003). Identification of these growth factors and defining their respective inputs
are critical to understanding the developmental and physiological regulation of stem-
cell pluripotency, self-renewal, stem cell-mediated tissue generation, turnover, and
repair (Tanaka et a/., 2002; Niwa, 2007). Furthermore, extending such knowledge to
control the expansion and differentiation of stem cells in vitro holds promise for
applications in regenerative medicine and biopharmaceutical discovery.
LIFIJAK-STAT3 signaling pathway The best-characterized effector of mES cell
self-renewal is LIF (Figure 1.7), which is a member of the IL6 family of cytokines
that plays a key role in maintaining mES cell self-renewal and functions by engaging
the LIF/gp130 heterodimeric receptor, thereby recruiting and activating STAT3, a
transcription factor that translocates to the nucleus and regulates genes required for
'sternness' (Niwa et a/., 1998; Matsuda et a/., 1999; Raz et a/., 1999; Ohtsuka and
Dalton, 2007). While LIF can activate JAK-ST AT3 and Ras-MAPK pathways in
mES cells, studies in mice indicate that genetic inactivation of LIF signaling has no
major effect on development (Nichols et a/., 2001 ). This may be due to
compensation by other IL6 family members, such as ciliary neurotrophic factor,
which can also signal through LIF/gp 130 receptors (Humphrey et a/., 2004 ).
LIF /ST A T3 is important for maintenance of the blastocyst during delayed
implantation (Nichols et a/., 2001 ). One of the most promising targets identified is
the proto-oncogene c-myc, a helix-loop-helix transcription factor that is a direct
11
Plosm a m mbra n
Embryonic stem cells
Cell receptors
Blastocyst
ERI( : acltvatlon 1 Signal I Transduction f Pathway
J Blocks Self-Renewal
adapredfrom Kirsclrstein, 2001
Figure 1.7: Self-renewal mechanism of stem cells. It is reported that stem cells undergo selfrenewal by activating leukemia inhibitory factor (LIF) receptor and glycoprotein gpl30 cell receptors, which results in activation of downstream JAK-ST AT signaling pathway and enables transcription of cell division regulatory genes and disabling molecular mechanisms which block self-renewal of stem cells. LIFR: Leukemia inhibitor factor receptor, STAT: signal transducers and activators of transcription, JAK: Janus kinase, ERK: extracellular signal-related kinase, Gab: Growth factor receptor bound protein, SHP: small heterodimer partner.
11 A
Chapter 1: Introduction
transcriptional target of ST AT3 (Cartwright et a/., 2005). Following LIF
withdrawal, c-myc transcript levels decrease due to inactivation of STAT3.
Maintenance of myc levels using inducible transgenes can maintain self-renewal in
the absence of LIF indicating that myc is a major target of the LIF-ST A T3 self-
renewal pathway in mES cells (Cartwright eta!., 2005).
Glycogen synthase kinase-3 (GSK3) pathway Another pathway that controls myc
levels involves glycogen synthase kinase-3 (GSK3)-dependent phosphorylation.
When LIF signaling ceases, GSK3 is rapidly activated and phosphorylates c-myc on
threonine 58, triggering its ubiquitination and proteasome-dependent degradation.
Suppression of GSK3 activity in mES cells is unclear, however, it is reported to
involve phosphatidylinositol 3 kinase (PBK) activity either directly or indirectly as a
consequence of LIF signaling. The efficiency of mES cell derivation was shown to
be markedly enhanced in the presence of 6-bromoindirubin-3'-oxime (BIO), a
chemical inhibitor of GSK3, suggesting its role in self-renewal (Umehara et a/.,
2007). Hence, low GSK3 activity could be an absolute requirement for pluripotency
and ES cell self-renewal.
Bone morphogenic protein (BMP) signaling pathway BMP promotes sternness of
mES cells and blocks neural differentiation by promoting inhibitor of differentiation
(ld) gene expression. Ying et a/ reported that BMP signaling inhibits differentiation
mES cells to ectoderm and in collaboration with other factors, it further inhibits
differentiation of mesoderm and endoderm (Ying et a!., 2003), however, Qi et a!
observed that BMP blocks differentiation by suppressing p38 MAP kinase (Qi eta/.,
2004; Kunath et a/., 2007).
12
Chapter 1: Introduction
Phosphatidylinositol 3 kinase (P/3K)IAKTJ pathway Phosphatidylinositol 3 kinase
is involved in many aspects of cell behavior such as proliferation, apoptosis and
differentiation (Takahashi et a/., 2005). A major effector of PI3K signaling is
protein kinase B (PKB)/AKTI. Inhibition of PI3K signaling by small molecule
inhibitors such as L Y294002 promotes differentiation even in the presence of LIF
(Welham et a/., 2007), which demonstrates that PI3K signaling is crucial for mES
cell self-renewal (Paling eta/., 2004; Storm eta/., 2007). As mES cells differentiate,
PI3K and AKT activities decline, which is consistent with this signaling pathways
being important for self-renewal. Sustained AKT activity, achieved by ectopic
expression of a constitutively active mutant, significantly delays differentiation of
murine and monkey ES cells (Watanabe et a/., 2006). Although PI3K/AKT is
reported to be crucial for mES cell self-renewal, the factors promoting their activity
have not been clearly defined.
Wnt signaling pathway Activation of Wnt signaling by the GSK-3 inhibitor, BIO,
maintains the undifferentiated status in both hES cell and mES cell (Sato et a/.,
2004). The receptor tyrosine kinase (RTK) pathway promotes differentiation and
the Hedgehog (Hh) signaling pathway functions on neuronal differentiation in mES
cell (Burdon et a/., 1999; Maye eta/., 2000).
Tra11scription regulators ill mES cell In the mouse, Oct3/4 expression directs
pluripotent cell lineages during embryo development (Nichols eta/., 1998), Nanog, a
homeodomain transcription factor, is essential for self-renewal in mES cell
(Chambers et a/., 2003; Mitsui et a!., 2003; Rho et a/., 2006), and Sox2, a co-
activator for Oct3/4 (Ambrosetti et a/., 1997), is expressed in multipotent embryonic
and extraembryonic lineages (Avilion eta/., 2003). Other transcription factors, such
as FoxD3, Rexl and Etsl, are required for the self-renewal of mES cell and
13
Chapter 1: Introduction
embryonic development in mice (Kola eta/., 1993; Ben-Shushan eta/., 1998; Hanna
eta/., 2002). In mouse haematopoietic stem cells, the Notch signaling pathway is
involved in the maintenance of self-renewal (Stier et a/., 2002). Transcriptional
profiles of the BMP4, transforming growth factor-f3 (TGF-f3), RTK, Wnt, Hh,
JAKJST AT and Notch signaling pathways, which are conserved in animal
development (Pires-daSilva and Sommer, 2003) have to be investigated to increase
understanding of the self-renewal of mES cell.
In summary, LIF-STAT3 is critical for mES cells self-renewal. In
conjunction with additional signals in serum, self-renewal is promoted. Besides LIF,
PI3KJAKT appears to be most critical, and may be activated as part of the LIF
signaling pathway or from other factors in media (for example insulin, IGF). The
absence of defined media formulations has compounded the definition of self
renewing signaling pathways in mES cells. Although various signaling pathways are
involved in the self-renewal of stem cells, little information is available regarding the
expression of the developmentally important signaling pathways in mES cells. The
large-scale proteome analysis may provide fundamental information to elucidate the
molecular mechanisms of self-renewal and differentiation in mES cell.
Several reports have shown that ES cells were employed to derive variety of
specialized cell types. Guan et a/ cultivated ES cells to form embryo-like
aggregates, termed 'embryoid bodies' from which they derived cells of all the three
germ layers such as cardiomyocytes, skeletal muscle, neuronal, epithelial and
vascular smooth muscle cells (Guan et a/., 1999; Gerecht-Nir et a/., 2004 ). O'Shea
used lineage selection and forced differentiation approaches to develop neuronal
progenitor cell lines which marked the start for neuronal and glial lineage
segregation (O'Shea, 2001 ). Blyszczuk and Wobus, developed strategy to derive
14
Chapter 1: Introduction
insulin-producing cells usmg specific growth and extracellular matrix factors,
involving multilineage progenitor cells (Blyszczuk and Wobus, 2006). Balconi eta/
developed protocols to derive endothelial cells from ES cells, these endothelial cells
were assessed for the expression of endothelial cell specific markers, including
growth factor receptors and adhesion molecules (Balconi et al., 2000; Siva et al.,
2007). Doetschman et al developed strategies to develop blood islands, visceral yolk
sac, and myocardium from ES cells (Doetschman et al., 1985). Hubner et al and
Kerkis et a! have demonstrated the development of reproductive cells such as
oocytes and sperms (Hubner eta!., 2003; Kerkis eta!., 2007).
Mouse ES cell lines isolated from different strains respond in an
uncharacterized manner to development and differentiation signals. Different ES
cell lines exhibit different colony morphologies, growth curves, and variable
expression levels of pluripotency markers. A number of recent studies have
indicated that individual mouse and human ES cell lines exhibit varying degrees of
efficiency when called upon to differentiate into specific cell types (Tesar, 2005;
Arufe et a!., 2006; Tavakoli et al., 2009). For example, Kramer et al reported that
the ES cell lines CCE, BLC6, 03, E 14, and R I, all of which express the stem cell
marker Oct4, exhibit varying degrees of spontaneous chondrogenic differentiation
(Kramer eta/., 2005). 03, Rl, El4, and CCE were poorly able to differentiate into
mature chondrocytes, but the BLC6 ES cell line forms chondrogenic nodules very
efficiently (Kramer eta/., 2005). Likewise, Wobus eta/ reported differences in the
ability of various ES cell lines to differentiate into cardiomyocytes and skeletal
muscle cells (Wobus et al., 2001 ). Arufe et a! suggest that mES cell lines of
different origins TSHR +/-and CCE, vary in their ability to differentiate into
thyrocytes (Arufe et al., 2006). They also suggest that within a specific ES cell line,
15
Chapter 1: Introduction
it is possible to select for the ability to differentiate into the thyrocyte lineage. The
differentiation capacity of an ES cell line might be dependent upon the mouse strain
from which it was established, or it could be due to their origins from different
blastocystic precursors (Gardner and Brook, 1997) or due to epigenetic modifications
(Santos eta/., 2002).
1.6. mES cell transcriptomics and proteomics.
Each specialized cell type in an organism expresses a subset of all the genes
that constitute the genome of that species and is defined by its particular pattern of
regulated gene expression. Gene regulatory pathways are important for maintenance
of undifferentiated state and their differentiation to various cell types. A regulatory
gene and its cis-regulatory modules are nodes in a gene regulatory network; they
receive input and create output elsewhere in the network.
The basic mechanisms involved in the stem cell biology remain elusive and
to understand ES cell mechanisms, such as cell-cell communication, signal
transduction, transcription regulation there is a strong necessity for the gene
expression analysis. Expression of some of the genes/proteins reported for sternness
in mES cells include Dpp5a (Esg 1 ), Nanog, NrOb 1, Nr5a2, Pou5fl (Oct3/4 ), Sall4,
Utfl, Stat3, Gata4, Myc, Sox2, Wnt3, Piwil2, Piwil4, Gm397, Zscan4d,
Dppa3/Stella, Whsc2, etc. A better understanding of the molecular pathways,
regulatory networks and their dynamics, which determine their diverse
differentiation fates, is needed for therapeutic approaches to be successful. And
hence there is a need for global gene/protein expression analysis of ES cells. Global
expression analyses gives us the profile of genes/proteins expressed in ES cells and
16
Chapter 1: Introduction
by annotating these proteins to biological functional, cellular localization and
network mapping to various regulatory pathways and processes we can have
enhanced understanding of ES cell biology. It can also discover putative Open
Reading Frames (ORFs), and annotating these proteins could lead to discovery of
new ES cell specific genes/proteins.
1.6.1. Transcriptomics
Several attempts have been made to obtain the expression profiles of mES
cells at the RNA level. Expressed sequence tag (EST) based DNA microarray
experiment led to the construction of large scale EST database for mES cells with
9099 ESTs (Boguski eta!., 1993). To quantify the functionally active genome of R1
mES cells Anisimov et al performed SAGE analysis and sequenced a total of
140,313 SAGE tags which mapped to 44,569 unique transcripts (Anisimov et a/.,
2002). The data from this study provided the starting point for detailed
transcriptome analysis.
Ramalho-Santos et a/ have carried out transcriptional profiling of mouse
embryonic, neural, hematopoietic stem cells to define a genetic program for stem
cells (Ramalho-Santos et a/., 2002). In this study, they identified 1787 genes from
mES cells using Affymetrix DNA microarray. Transcriptome datasets of mES,
neural and hematopoietic stem cells were compared to define a genetic program for
stem cells. A total of 216 genes were enriched in all three types of stem cells (Table
1.1 ), and several of these genes were clustered in the genome. As represented by
ESTs, it was reported that stem cells express a significantly higher number of genes
compared to differentiated cell types, whose functions are unknown. They also
17
Table 1.1: The genes defined as "sternness genes" expressed in mES, neural, hematopoietic stem cells (Ramalho-Santos eta/., 2002).
Category Genes Signaling (35) F2r (Thrombin R), Growth Hormone R, Integrin a6/b1, Adam9, Bystin, Ryk, Pkd2, shaker K channel b3, Gnb1,
Gab1, Kras2, Cttn, Cop9 4/7a, Smadl/2, Tbrg1, Starn, Statip1, Cish2, Rock2, Yes, Yap, Ptpn2, Ppp1r2, Ywhab (14-3-3b), Ywhhb (14-3-3e), Axotrophin, Trip6, Gfer (ALR), Upp, ESTs highly similar to Gap, ESTs highly similar to PPP2R1B, ESTs moderately similar to Jak3
Transcriptional regulation ( 14) MyoD family inhibitor, Tead2, Yap, Four and a half LIM, Zfx, Zfp54, Rnf4, Chromodomain Helicase 1, Etll, Rmp, 4 ESTs highly similar to Zfp
DNA repair (4) Ercc5, Xrcc5 (Ku80), Msh2 (MutS2), Rad23b Cell cycle regulation (13) Cyclin 01, P21, Cdkap1, Cell cycle progression 2, Gas2, CenpC, Wild-type p53 induced 1, Trnk, Umps, Sfrs3,
ESTs highly similar to exportin 1, ESTs highly similar to CAD, ESTs similar to Mapkkkk3 Cell death (3) Gas2, Pdcd2, Wild-type p53 induced 1 RNA processing (9) Sfrs3, Snrp1c, Phax, NOL5, RNA cyclase, ESTs highly similar to Sfrs6, ESTs highly similar to Prp6, ESTs
highly similar to Nop56, ESTs highly similar to Ddx 1
Translation (6) Eif4ebp 1, Eif4g2, Mrps31, Mrpl17, Mrpl34, ESTs highly similar to Eif3s 1 Protein folding, chaperones (8) Hspall (Hsc70t), Hspa4 (Hsp110), Dnajb6 (Mr Dnaj), Hrsp12, Tcp1-rs1, peptidylprolyl isomerase C, FKBP 9,
ESTs moderately similar to Fkbp 13
Ubiquitin pathway ( 12) Ube2d2, Ariadne 1, F-box only 8, Ubiquitin Protease 9X, Uchrp, Axotrophin, Tpp2, Cop9 4/7a, Nyren18 (Nub 1 ), ESTs moderately similar to Ubc 13 (bendless), ESTs highly similar to proteasome 26S subunit, non-ATPase, 12 (p55)
Vesicle traffic (5) Rab18, Rabggtb, Stxbp3, Sec23a, ESTs moderately similar to Coatomer delta Toxic stress response (6) Abcb 1 (Mdrl ), Gsta4, Gslm, Thioredoxin reductase, Thioredoxin-like 32kD, Laptm4a Other (8) Reticulocalbin, Supl15h, Pla2g6, Acadm, Suclg2, Pex7, Tjp 1, Gcat
Unknown (100) EST clusters with little or no homologies ..
(the numbers m the parenthesis md1cate number of genes under the respective category)
17 A
Chapter 1: Introduction
could infer that embryonic and neural stem cells exhibit similarities at the
transcriptional level. These results provide a foundation for a more detailed
understanding of stem cell biology. Rosenkranz et a! constructed a large
transcriptome database of 14,434 mouse RefSeq genes from F1 mES cells using next
generation RNA-Seq technology (Rosenkranz et a!., 2008). These genes were
analyzed for GO annotations, which revealed large number of genes involved in cell
cycle, signal transduction, transcription regulation and translation regulation.
In an attempt to understand the crucial molecular switches that regulate early
mES cell differentiation, Sampath et a! have carried out large-scale transcriptome
analysis combined with global assessment of ribosome-loading during mES cell
differentiation into embryoid bodies (Sam path et a!., 2008). This study revealed that
mES cells during self-renewal synthesize proteins parsimoniously, however
differentiation induced anabolic switch, with global increase in transcript abundance,
polysome content, protein synthesis and protein content. Furthermore 78%
transcripts showed increased ribosome loading, thereby enhancing translational
efficiency. Transcripts under exclusive translational control included the
transcription factor A TF5, the tumor suppressor DCC, and the b-catenin agonist
Wntl. A hierarchy of translational regulators, including mTOR, 4EBP1, and the
RNA-binding proteins DAZL and GRSFI, are shown to control global and selective
protein synthesis during ES cell differentiation. Parsimonious translation in
pluripotent state and hierarchical translational regulation during differentiation may
be important quality controls for self-renewal and choice of fate in mES cells.
The information about the extent to which RNAs are truly expressed and the
steady state levels of protein species in the cellular repertoire, extent and level of
post-translational modifications, such as phosphorylation, glycosylation, alternative
18
Chapter 1: l11troduction
splicing, isoforms, etc. is critical to understand how these molecules interact with
each other and the functional properties of ES cells. Proteomics is a powerful
approach and has the potential to gain more insight into the molecular mechanisms
and regulatory pathways in stem cells.
1.6.2. Proteomics
Proteome is the entire complement of proteins expressed by a genome, cell,
tissue or organism at a given time under defined conditions. The term is a
portmanteau of proteins and genome, which was coined by Marc Wilkins in 1994 in
the symposium: "20 Electrophoresis: from protein maps to genomes" in Siena, Italy
(Wasinger et al., 1995; Wilkins, 2009). It has been applied to several different types
of biological systems and also been used to refer to the collection of proteins in
certain sub-cellular biological systems.
Gel-based approach The classical proteomic approach includes the separation of
proteins by two-dimensional (2-D) gel electrophoresis. In the first dimension, the
proteins are separated by isoelectric focusing, which resolves proteins on the basis of
charge and second dimension, separation is by molecular weight using SDS-PAGE.
The gel is stained to visualize the proteins. Protein spots of interest are excised and
digested with a proteolytic enzyme, generally trypsin. The tryptic digest is then
subjected to mass spectrometric analysis for protein identification. Peptide mass
fingerprinting (PMF) analysis then deduces the protein identity by matching the
observed peptide masses against protein sequence database available in NCBI,
Swissprot etc using search engines such as Mascot. Tandem mass spectrometry, on
19
Chapter 1: Introductio11
the other hand, can get sequence information by fragmentation of the selected
peptides (Pandey and Mann, 2000; Takahashi and Isobe, 2007).
The 2-DE-MS approach yields expressiOn information with significant
clarity, including clues on post-translational modifications and protein isoforms.
Although 2-D gel electrophoresis provides unprecedented separation power for
proteins, this approach suffers several limitations, including the difficulties of
resolving proteins with extreme size, pi or hydrophobicity, identifying relatively less
abundant proteins, and the difficulties associated with automation and
reproducibility.
Liquid chromatography (LC)-based approach Proteome analysis faces challenges
because of the great complexity of protein species and the large dynamic range of
protein levels. In the past few years, some new separation techniques were
developed for peptide separation and significantly improved the overall sensitivity,
dynamic range, throughput and general effectiveness of proteomic analysis. Liquid
chromatography-mass spectrometry (LC-MS) is frequently used in drug
development at many different stages including Peptide Mapping, Glycoprotein
Mapping, Natural Products Dereplication, Bioaffinity Screening, In vivo Drug
Screening, Metabolic Stability Screening, Metabolite Identification, Impurity
Identification, Degradant Identification, Quantitative Bioanalysis, and Quality
Control. Reverse phase (RP)-LC coupled on-line with electrospray ionization (ESI)
MSIMS is typically used for proteomic analysis because of the good compatibility of
the mobile phase with MS detection. Although relatively complex mixtures can be
separated well in RPLC, however, the analysis of mixtures containing thousands of
peptides, which is extremely complex, requires multi-dimensional separation.
Multidimensional separation can be achieved by 2-D LC separation of proteins or
20
Chapter 1: llltroductioll
peptides. Another approach includes gel-based separation of proteins combined with
separation of peptides, after digestion, with RP-LC. To avoid labor-intensive
operations and to obtain highly reproducible results, automated proteome-analysis
systems have been developed. LC-MS is an analytical chemistry technique that
combines the physical separation capabilities of liquid chromatography with the
mass analysis capabilities of mass spectrometry. Mass spectrometer consists of
ionization source, analyzer and detector. For LC-MS approaches ESI technique is
best suited. The sensitive detection and identification of components within complex
proteomics samples is crucial for the characterization and understanding of proteome
dynamics, which requires increased speed of acquisition and sensitivity. The
analyzers used in these instruments are quadrupole, ion trap, time of flight, and
hybrid mass analyzers such as triple quadrupoles, QTOFs, quadrupole-ion trap,
lYf a- Orbitrap and FT-ICR, which have facilitated high throughput peptide analysis and
G (::() protein identifications. --l
F Proteome analysis of mES cells Proteome analysis usmg diverse proteomic
approaches have been reported for mES cell lines. In stem cells, nuclear proteins
like transcription factors have been accessed by 2-D gel based approach, presumably
due to their higher abundance in these cells (Nasrabadi et al., 2009). Elliott et al
performed two-dimensional polyacrylamide gel electrophoresis of Rl mES cell
proteins and identified 231 proteins, and classified them based on their functions,
which consisted of genes involved directly or indirectly with expression or
maintenance along with many housekeeping proteins (Elliott et a/., 2004). Nagano
et a/ isolated E 14 mES cell line proteins and separated the total protein digest using
micro-scale online two-dimensional liquid chromatography (lon-exchange-LC
followed by Reverse-phase-LC) followed by Q-TOF based data-dependent CID
21
{v ''', ,\.1( ·' ' ...
Chapter 1: Introduction
tandem MS analysis, which resulted in the identification of 1364 proteins (Nagano et
a/., 2005). Further functional classification of these was in concordance with earlier
findings, which consisted of proteins belonging to gene expression regulation, and
housekeeping. Van Hoof et a/ used a hybrid protein fractionation approach, where
D3 mES cell proteins were first separated by general SDS-PAGE. Gel bands were
cut down to smaller pieces and in-gel digested using trypsin and the digest was used
for analysis by nanoflow LC and FT-ICR-MS/MS and identified 1871 proteins (Van
Hoof et al., 2006). Graumann eta/ carried out similar hybrid fractionation approach,
with Rl and G-Olig2 (Rl derivative) mES cell proteins, however they performed
their analysis with SILAC labeled proteins to take step further towards protein
quantitation by MS (Graumann et a/., 2008). They also performed subcellular
fractionation (nuclear), where each of the three gel lanes was cut into 15 slices and
were in-gel digested. The extracted peptides separated by nanoflow LC and
analyzed by L TQ-Orbitrap-MS/MS led to the identification of 5111 proteins, and
were then compared with the DNA microarray data set of mES cells by Hailesellasse
Sene et a! (Hailesellasse Sene et a!., 2007). This quantified mES cell proteome
consists of prominent mES cell markers such as OCT4, NANOG, SOX2, and UTFl
along with the embryonic form of RAS (ERAS). They have also quantified the
proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and
membrane/chromatin fractions. Bioinformatics analysis of the mES cell proteome
revealed a broad distribution of cellular functions with overrepresentation of proteins
involved in proliferation. On comparison with a recently published map of
chromatin states of promoters in ES cells and excellent correlation between protein
expression and the presence of active and repressive chromatin marks was observed.
22
Chapter I: Introduction
I. 7. Rationale of tlze study
Thus ES cells are of paramount importance as a renewable source capable of
differentiating into virtually all cell types under appropriate conditions, and have the
potential for regenerative therapies (Doss et a/., 2004; Murry and Keller, 2008).
Therefore, developmental biologists have increasingly focused on both
understanding how ES cells maintain pluripotency, and how defined signals lead to
their differentiation into various lineages. Understanding the active pathways in
stem cell biology that lead to differentiation can be facilitated by knowledge of the
expressed gene repertoire in ES cells (Tanaka et a/., 2002; Niwa, 2007), hence this
effort.
Some of the distinguished aspects of blastocyst-derived mouse ES (mES) cell
lines such as the isolation from any genotype, the consistency from one cell line to
the next with respect to in vitro developmental process, and advantage of being
temporally close to embryo and their in vitro expression of a large degree of
developmental potential, together make them potentially useful as a model system
for embryonic development and stem cell biology research (Doetschman et a/.,
1985).
In view of the heterogeneity observed in mES cell lines, it is important to
have an in-depth understanding of the proteins expressed, their molecular
mechanisms and regulatory pathways active in various mES cell lines. In view of
the dynamic range of proteins, to achieve the above objective, there is a need to
identify large cohort of proteins using high throughput and sensitive techniques. In
addition, the protein expression profiles from various mES cell lines may be
23
Chapter 1: Introduction
compiled to have an integrated dataset, which is a representative protein expression
dataset for mES cell lines.
Transcription profiling by microarray analysis is a mature technology that has
been applied in numerous studies of mouse embryonic stem cell lines, and has been
useful to infer ES cell-specific genes, including many transcription factors
(Ramalho-Santos et a/., 2002; Tanaka et a/., 2002; Sharov et a/., 2003; Ko, 2006;
Sharova et a/., 2007; Efroni et a/., 2008) and the global regulatory processes
(Sampath eta/., 2008).
For comprehensive protein profiling to assess the steady state levels of
protein species and the translation yield of mRNAs, mass spectrometric (MS)
methods have demonstrated the power to become the approach of choice (Baharvand
eta/., 2007; Yocum eta/., 2008). LC-MS approach can in principle assess protein
species over a broad dynamic range and provide an independent method to detect
protein expressions at the levels comparable to the sensitivity of mRNA assessment.
Several MS platforms have been previously used to study mouse ES cell proteome
(Elliott et a/., 2004; Nagano et a/., 2005; Van Hoof et a/., 2006; Graumann et a/.,
2008), and in one study 5,111 proteins (Graumann eta/., 2008) were identified, thus
far the largest collection. An effort is made, through the analysis presented here, to
expand the understanding of expressed proteins further.
24
Chapter 1: llltroductioll
1.8. Objectives of the thesis
In view of the above, the thesis aims at a comprehensive understanding of protein
expression and molecular pathways of mES cells, R l-9 and AB 1, through large-scale
proteomic analysis with the following specific objectives.
1. Compilation of experimental and published mES cell transcriptome dataset
2. Identification of proteins expressed in two mES cell lines- R 1-9 and AB 1
using LC-ESI-MS approach
3. Integration of objective 1 and 2 above and compilation of comprehensive
mES cell protein expression database
25