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