Optimization of culture conditions for artificial

blastocysts

Maria Inês Baptista Leite

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisors: Prof. Maria Margarida Fonseca Rodrigues Diogo

PhD Erik Jacob Vrij

Examination Committee:

Chairperson: Prof. Joaquim Manuel Sampaio Cabral

Supervisor: Prof. Maria Margarida Fonseca Rodrigues Diogo

Member of the Committee: Dr. Tiago Paulo Gonçalves Fernandes

June 2014

II

III

“If someone offers you an amazing opportunity

and you’re not sure you can do it, say yes

- then learn how to do it later”

Richard Branson

IV

V

Acknowledgments

First of all I would like to thank my supervisor, Erik Vrij, for the support and inspiration during the

last months. For his constant guidance, confidence and patience. For teaching me on being

precise and accurated in work. For the opportunity to work in a challenging, multitasking and

groundbreaking project.

I also present my thankfulness to Professor Clemens van Blitterwijk and Professor Roman

Truckenmüller for giving me the opportunity of developing my master thesis at the Department

of Tissue Regeneration of the University of Twente.

To my supervisor Prof. Margarida Diogo for all the confidence and her prompt and efficient

support.

To Dr. Hugo Fernandes, Prof. Lorenzo Moroni and Eng. Rik Akse for helping me and my family

in an especially difficult time that I had during my stay in Enschede.

To the Tissue Regeneration team, the actual people and the ones that already left, for the

collaboration, good environment (specially afterhours) and for making me part of the dwarf

team.

To my friends in Portugal for all the unforgettable moments but especially for being present,

even while being abroad.

To my friends in Enschede for making me feel home abroad and for being my family. I could not

had so much success if I did not have all the laughs, great moments and their true friendship.

To my family for celebrating my successes, their support in bad times and for teaching me that

unity is strength.

Finally, my heartfelt gratitude to my parents for their unconditional love, support, patience and

dedication. For being there for me anytime I needed and all the strength that they gave me to

carry on. For the opportunity and encouragement for studying abroad. In particular, I would like

to thank my sister, Sofia, for all the love, inspiration, complicity and wise advices. For being my

person, my role model and for our unique friendship built day by day during the last 25 years.

For making me feel that nothing is complete without them and nothing is too serious as long as

they are around. This thesis is dedicated to them.

VI

VII

Abstract

The main goal of this thesis was to optimize the method to develop artificial blastocysts (also

called blastoids). In parallel different studies were developed with the aim of ensuring that the

compounds tested are not harmful to the blastoids and reveal some details of biological

mechanisms that take place during blastocyst formation.

The stem cells were seeded on a platform and their seeding cell densities were optimized. Dose

responses of blastoids to several compounds of the blastoids medium were performed to

address the optimum concentration of each one.

With the different studies was possible to conclude that one specific pathway was important

during the preimplantation stage of the mouse blastocyst and a screening of a specific library

revealed 15 potential hits that promote cell differentiation.

Key words: mouse blastocyst, stem cells

VIII

Resumo

O objectivo desta tese foi optimizar o método de desenvolvimento de blastócistos artificiais

(também denimonados blastoides). Simultaneamente, foram desenvolvidos diferentes estudos

para assegurar que os compostos testados não seriam prejudiciais para os blastoides e revelar

alguns detalhes biológicos que ocorrem durante a formação do blastócisto.

As células estaminais foram semeadas numa plataforma e as suas concentrações iniciais

foram optimizadas. Para além disso, foi também estudada a resposta dos blastoides a

diferentes doses de vários constituintes do meio de cultura dos blastoids para determinar a

concentração óptima de cada um.

Através dos estudos desenvolvidos foi possivel concluir que uma via específica é importante

nesta fase de preimplantação do blastócisto de ratinho e o rastreio de uma biblioteca de

compostos revelou 15 potenciais hits que promovem a diferenciação celular.

Palavras-chave: células estaminais, blastócisto de ratinho

IX

List of Contents

Acknowledgments ......................................................................................................................... V

Abstract ....................................................................................................................................... VII

Resumo ...................................................................................................................................... VIII

List of Contents............................................................................................................................. IX

List of Figures ............................................................................................................................... XI

List of Abbreviations .................................................................................................................... XII

1 Introduction........................................................................................................................... 15

1.1 Events leading to formation of the blastocyst .............................................................. 16

1.1.1 The first lineage decision: segregation of the TE and ICM ..................................... 18

1.1.2 The second lineage decision: segregation of the PE and EPI ................................ 20

1.2 Developmental signaling pathways in preimplantation mouse embryo ...................... 22

1.2.1 Hippo pathway ......................................................................................................... 22

1.2.2 FGF signaling pathway ............................................................................................ 23

1.2.3 Wnt/β-catenin signaling pathway............................................................................. 25

1.3 Stem cell populations representative of the Early Embryonic Lineages ..................... 27

1.3.1 Embryonic stem cells (ES) ...................................................................................... 27

1.3.2 Trophoblast Stem Cells (TS) ................................................................................... 29

1.3.3 Extraembryonic endoderm cells (XEN) ................................................................... 29

1.4 Postimplantation period ............................................................................................... 30

2 Materials and Methods ......................................................................................................... 31

2.1 Mouse fibroblast feeder layer (mEF) culture ............................................................... 31

2.2 Cell culture ................................................................................................................... 31

2.3 Immunostaining of blastoids ........................................................................................ 31

2.4 Image acquisition and processing ............................................................................... 32

2.5 Statistical analysis ....................................................................................................... 32

3 Results and Discussion ........................................................................................................ 33

3.1 Method’s Optimization ................................................................................................. 33

3.1.1 Study the influence of stem cell number in blastoids development ........................ 33

3.1.2 Study of the influence of different medium compounds in blastoids development . 34

X

3.1.3 Optimization of staining of blastoids ........................................................................ 35

3.2 Studies about blastocyst development ........................................................................ 36

3.2.1 Z signaling in A treatment ........................................................................................ 36

3.2.2 Interaction of A with different growth factors ........................................................... 36

4 Conclusions and future perspectives ................................................................................... 38

5 References ........................................................................................................................... 39

XI

List of Figures

Figure 1 – Stages of mouse preimplantation development. ........................................................ 16

Figure 2 - Polarity in the mouse preimplantation embryo. .......................................................... 17

Figure 3 - Models of TE specification in the mouse embryo. ...................................................... 19

Figure 4 - Models of PE and EPI segregation in the second cell fate decision. ......................... 21

Figure 5 - Schematic of the Hippo pathway. ............................................................................... 23

Figure 6 - Schematic of FGF signaling ........................................................................................ 24

Figure 7 – Schematic of canonical Wnt/β-catenin pathway. ....................................................... 25

Figure 8 - Stem cell types that can be derived and propagated in culture representing the three

blastocyst lineages. ..................................................................................................................... 27

XII

List of Abbreviations

2i 2 Inhibitors

APC Adenomatous Polyposis Coli

aPKC Atypical Protein Kinase C

BIO 6-Bromoindirubin-3’-Oxime

BMP4 Bone Morphogenic Protein 4

BSA Bovine Serum Albumin

Cdx2 Caudal-Type Homeodomain Transcription Factor

CHIR Chir99021

DMEM Dulbeco’s Modified Eagle Medium

DMSO Dimethyl-Sulphoxide

DVL Dishevelled

E Emrbyonic Day

EB Embryoid Body

Eomes T-Box Transcription Factor Eomesodermin

EPI Epiblast

ERK Extracellular Regulated Kinase

ES Embryonic Stem

ExE Extraembryonic Ectoderm

FBS Fetal Bovine Serum

FGF Fibroblast Growth Factor

FGFR Fibroblast Growth Factor Receptor

Gata3, Gata4, Gata6 Transcription Factor Member Of The GATA Family

GFP Green Fluorescent Protein

GSK3 Glycogen Synthase Kinase

ICM Inner Cell Mass

JNK C-Jun N-Terminal Kinases

Klf5 Kruppel-Like Zinc-finger Transcription Factor

Lats1/2 Large Tumor Suppressor

LIF Leukemia Inhibitory Factor

MAPK Mitogen-Activated Protein KINASE

XIII

MeEM NEAA Minimum Essential Medium Non-Essential Amino Acids

mEF Mouse Embryonic Fibroblast

Mob Mps1 Binder

mRNA Messenger Ribonucleic Acid

Mst1/2 Mammalian Sterile 20 Like

Nanog Homeobox Transcription Factor

NDR Nuclear Dbf2-Related

Oct4 POU Domain Protein

PBS Phosphate Buffered Saline

Pdgfrα Platelet-Derived Growth Factor Receptor Alpha

PDMS Polydimethylsiloxane

PE Primitive Endoderm

Pen/Strep Penicillin Streptomycin

PKA Protein Kinase A

PMMA Poly(Methyl Methacrylate

RA Retinoic Acid

Ras Rat Sarcoma

RFP Red Fluorescent Protein

RPMI Roswell Park Memorial Institute Medium

RTK Receptor Tyrosine Kinases

RT-PCR Reverse Transcription Polymerase Chain Reaction

Sav1 Salvador Homolog 1

Sox2, Sox7, Sox17 Member Of SOX (SRY-Related High Mobility Group Box) Transcription Factor Family

Taz Transcriptional Co-Activator With Pdzbinding Motif

TCF/LEF T-Cell-Specific Factor/Lymphoid Enhancer-Binding Factor

TE Trophectoderm

Tead4 TEA-Domain Transcription Factor

TEF Transcriptional Enhancer Factor

TGFβ Transforming Growth Factor Β

TS Trophoblast Stem

VE Visceral Endoderm

XIV

Wnt Wingless

XEN Extraembryonic Ectoderm

Yap Yes-Associated Protein 1

15

1 Introduction

How a single fertilized egg gives rise to a complete embryo is a complex and intriguing process

for biologists to resolve in detail, already since Aristotles’ times [1]. Besides its complexity, the

challenge is also difficult because of embryo small dimension and its inaccessibility in the

maternal uterine environment. However, the advent of modern tools to study the molecular

biology has enabled a steady increase in our understanding of morphogenetic processes and

embryonic development.

Apart from these drawbacks, the mouse embryo has several features that make it an attractive

subject for study. For instance, mouse embryos offer a biological tool to generate and correct

genetic mutations that resemble similar phenotypes to human disease mutations. This gives us

useful indications about molecular mechanisms, regulating both embryonic development and

disease progression and draws parallels between these two apparently disparate processes [2].

Furthermore, mouse embryos have been an invaluable model for reproductive strategies such

as the in vitro fertilization studies [3]. Additionally, the mouse embryo breeds rapidly, which

speed up studies. The pluripotent embryonic stem cells obtained from mouse embryos also

have had an invaluable importance on understanding the dynamics of pluripotency and cell fate

decisions [2].

In this way, the mouse has become the leading model organism for experimentation in

mammalian genetics and the most studied alternative for humans [4]. Studies using the

preimplantation mouse embryo as a model organism have improved cellular replacement

therapies in regenerative medicine. In fact, due to these pioneering studies is now possible to

obtain somatic cells and reprogram them back to the early pluripotent state. [2]. Yet many

questions remain unanswered, for example, is still unknown how the embryo controls cell

lineage choice.

For in vitro study of preimplantation development, the mouse embryos are flushed from the

uterus or oviduct of a pregnant mouse. Presently, preimplantation development can be

recreated in vitro in a chemically-defined culture medium, using stem cells under a controlled

environment without significantly impairing the developmental potential of embryos [5].

However, animals are normally sacrificed in the process of harvesting embryos [6-8] and natural

embryos can only be obtained in relatively small numbers compared to large-scale generation

of artificial blastocysts. Hence, one way to overcome previous limitations is to develop artificial

blastocysts. Those artificial embryos could be also used as biological models for drug screening

and potentially for cloning [9]. In this thesis, a method is described that allows the formation of a

cellular model that resembles the development of a natural blastocyst, including its stereotypical

morphogenetic processes.

16

During the initial stage of natural embryonic development three distinct cell types are formed.

Here are described the main events that occur between fertilization of the egg cell and the

implantation of the blastocyst, with special focus on the formation of the three distinct tissue

lineages (the epiblast (EPI), the primitive endoderm (PE), and the trophectoderm (TE) [10]) and

stem cells that represent each cell lineage.

1.1 Events leading to formation of the blastocyst

The dividing cells of the totipotent zygote are progressively specialized, generating all

subsequent cell-types of the future embryo and the supportive extraembryonic tissues. The first

three distinct tissues (TE, EPI and PE) can be detected in the late blastocyst [10, 11] and the

morphological changes that lead to its formation provide insight into the cellular origins of those

three lineages (Figure 1).

Figure 1 – Stages of mouse preimplantation development.

Zygote (fertilized egg). The polar body is the by-product of the second meiotic division of the oocyte and degenerates during preimplantation development. E4.5 (peri-implantation blastocyst) notes the altered morphology of the mural

trophectoderm cells at this stage as they transform into trophoblast giant cells. Arrows denote the migration of the PE over the TE to form parietal endoderm. The descendant of the PE covering the epiblast at later stages is the visceral

endoderm (VE). The timeline indicates the time elapsed since fertilization in embryonic days (E). Timeline not to scale. Scale bars= 20 µm. Adapted from [12]

During the first three cell divisions, that take place between E0,5 (embryonic day 0,5) and E2,5,

the fertilized egg produces an embryo consisting of 8 cells. These cells, also called

blastomeres, are morphologically indistinguishable and retain the potential to form all cell

lineages [5, 13].

17

One day before blastocyst formation, the 8-cell embryo undergoes compaction. At this time the

cell-cell contact regions become enriched in E-cadherin, which is the main component of

adherens junctions [14]. As compaction proceeds, blastomeres become polarized along their

apical-basal axis with enrichment of subcellular components. The apical domain, which faces

outside and is free of cell-cell contacts, is enriched in atypical protein kinase C (aPKC), the

portioning defective proteins (Par3 and Par6), F-catin and other molecules (Figure 2A). On the

other hand E-cadherin and other proteins are restricted to the basolateral surfaces (Figure 2A,

[13]).

It has been proposed that, in subsequent cell divisions, from 8-cell stage to morula stage,

cleavage plane orientation dictates cell fate, according with cell position within the morula

(Figure 2B). When the cleavage plane is parallel to the basolateral axis the cell division will

produce two outer polarized cells that will form the TE. Otherwise it will generate an outer

polarized cell and an inner non-polarized cell that will become TE and inner cell mass (ICM),

respectively [3, 10].

Figure 2 - Polarity in the mouse preimplantation embryo.

Blastomeres polarize along the axis of cell contact, forming apical and basolateral domains (A). From 8 to 16-cell stages two populations of cells are formed: outside, polar cells and inside, nonpolar cells (B). Adapted from [3].

After the morula stage the activity of sodium pumps promotes blastocyst cavitation. They

establish ionic gradients across the TE promoting a fluid filled cavity formation, also called

blastocoel. Promoting blastocoel expansion, tight junctions prevent water leakage and

aquaporins contribute to water movements across the TE [15]. This cavity is essential for the

proper formation of the ICM.

Blastocoel formation marks the segregation of the two lineages of ICM - pluripotent epiblast

(EPI) and primitive endoderm (PE) lineages. The PE appears as a layer of cells on the surface

of the ICM lining the cavity, with deeper cells comprising the pluripotent epiblast (EPI) [3, 14]. At

this point the mouse embryo is called blastocyst and is ready to implant into the uterine wall.

18

Besides their position within the embryo these three cell lineages can also be defined by their

expression of lineage-specific markers, which provide insight into cell lineage commitment and

segregation. It is believed that early lineage segregation occurs in two successive phases. In

the first phase (the segregation of TE and ICM), is characterized by the co-expression of the

markers in individual cells different levels. On the other hand, during the second phase

(segregation of EPI and PE, within the ICM) the markers expression becomes mutually

exclusive as cells are lineage committed. In the following section these two phases are

described in close detail.

1.1.1 The first lineage decision: segregation of the TE and ICM

The cell position within the morula dictates the cell fate: the inside-cells give rise to the ICM fate,

whereas the outside-cells become TE. This divergence can be a consequence of differences in

the nature of the cell-cell contacts (Inside-Outside Model) or differences in the

microenvironment (Cell Polarity Model) and promote two distinct cell fate decisions based on

the two different models.

The Inside-Outside Model hypothesized that blastomeres are equivalent and totipotent until

around the 32-cell stage, when some blastomeres are surrounded by other blastomeres,

resulting in microenvironmental positional differences that would then dictate cell fate (Figure

3A) [10, 14, 16]. This model was supported by several studies. Lineage-tracing in intact

embryos showed that individual cells that contribute to TE and ICM are progressively restricted

as an inner and an outer compartment are formed, at 32-cell stage morula. This model is also

supported by the observation of labelled blastomeres that when positioned outside contributed

to TE and the inside-placed labelled blastomeres contributed to ICM [17], demonstrating that

blastomeres respond to positional cues. Still, it remained unknown what, if anything, governed

cell position.

In an alternative, Johnson et al. proposed that TE and ICM formation takes place before the late

morula stage, by the establishment of cell polarity along the radius of the early morula [13]. The

Cell Polarity Model defends that the different developmental potential of polar and apolar cells

sets up TE and ICM lineages (Figure 3B) [14]. In fact, experimental perturbations of cell polarity

affect cell position and probably cell fate.

To summarize, the Inside–Outside Model indicates that cell position promotes cell fate, whereas

the Cell Polarity Model predicts that cell fate drives cell position. However, as the orientation of

polarity may be influenced by regions of cell–cell contact, the Inside–Outside and Cell Polarity

Models are not completely in contradiction with each other [14].

More recently, several studies have revealed that initial outer cells can adopt a final inner

position, suggesting that the process of inner and outer cells formation is more dynamic than

previously assumed [11]. Still, it remains unclear if cell movement is random or actively

19

controlled, but it is known that several transcription factors are required for the proper

segregation of each lineage (Figure 3C).

Figure 3 - Models of TE specification in the mouse embryo.

Inside-Outside Model: inner and outer cells receive different amounts of cell contact, and this is translated into differences in transcription factor expression (A); Cell Polarity Model: presence or absence of an apical domain is

translated into differences in transcription factor expression (B); Transcription factors that are required for the proper segregation (C). Adapted from [3]

Firstly, TE and ICM lineages can be distinguished by the expression patterns of several

lineage-specific transcription factors. Cdx2 encodes a caudal-like transcription factor that is

expressed in the TE and is required to promote TE fate. Oct4, a POU-domain transcription

factor, is ICM-specific. In the beginning they are co-expressed in all blastomeres and it is

believed that interactions between them, during blastocyst formation, reinforce TE and ICM

fates [14, 18-20].

Cdx2 is the earliest transcriptional factor identified so far to be involved in specification of TE

fate. It is first detectable in the nuclei of select cells at the 8-cell stage and becomes restricted to

outer cells of the late morula [21]. Besides being crucial in epithelial integrity maintenance [15] it

is also required for repression of Oct4. Interestingly, Niwa et al. showed that overexpression of

Cdx2 is enough to stimulate the differentiation of embryonic stem (ES) cells into trophoblast

stem (TS) cells [21].

20

Moreover, on the basis of apical aPKC localization, the blastomeres with high levels of Cdx2

expression appeared to be more highly polarized [22]. In addition to this, polarity was found to

influence localization of Cdx2 mRNA.

While the cell polarity can be correlated with Cdx2 expression, the cells response to their

position within the embryo can be associated with the Hippo pathway.

In mammals, Hippo signaling controls proliferation through cell contact (see section 1.2.1-Hippo

pathway). Yap (Yes-associated protein 1) accumulates in the nucleus only in the outer cells and

can directly interact with its transcriptional coactivator Tead4 to stimulate transcription of Cdx2

(Figure 3C). Tead4 is specifically required for the critical aspects of TE formation: blastocyst

cavity formation and trophoblast differentiation [23].

Taken together, by establishing a link between Cdx2 and polarity, Hippo pathway and

cell-position and Cdx2 and Hippo pathway it is possible to demonstrate the interplay between

position, polarization and cell fate.

Besides Cdx2 and Tead4 there are other transcription factors that promote TE differentiation

like Eomes, Klf5, Gata3 and Sox2 [5]. To understand ICM differentiation and the segregation of

EPI and PE lineages in detail, we must address the second cell fate decision in preimplantation

development.

1.1.2 The second lineage decision: segregation of the PE and EPI

The PE and EPI are two lineages derived from ICM in the second cell fate decision. They start

to differentiate by mid blastocyst (E3.5) and, in late blastocyst (E4.5), they form morphologically

distinct layers [24]. As mentioned before, the PE forms a monolayer on the surface of the ICM

directly facing the blastocoel, while the EPI lies between the PE and the TE. By this stage both

lineages appear to be lineage-restricted, because they contribute only to their respective

lineages in chimeric embryos [23]. Once again, the mechanisms that lead to the establishment

and segregation of both lineages have been unclear until recently.

Numerous possibilities came out to describe this fate decision. One of the earliest was the

Induction or Positional Hypothesis, similar to the Inside-Outside Model described above. It

proposed that ICM cells of the early blastocyst were a homogeneous population of bipotential

cells with the capability to become either EPI or PE and their position dictates their fate [14, 23].

Consistent with this, the outside cells of isolated ICM or embryoid bodies (EBs) formed by ES

cells differentiate into endoderm when cultured in vitro [25].

Later studies revealed that the PE marker, Gata6, and the EPI marker, Nanog, were expressed

in a random “salt-and-pepper” pattern in early blastocyst, previously to PE formation [26]. This

gave rise to a new model – the Sorting Model – that defends that a mixed population of EPI and

PE progenitors segregates into their composite layers. Only later they become segregated to

21

their respective layers by several mechanisms that lead to random cell movements, positional

signals and selective apoptosis [27-31]. Several studies support this model, including the first

time-lapse study, starting at early blastocyst stage, where it was demonstrated that at least

some deep ICM cells expressing platelet-derived growth factor receptor alpha (Pdgfrα), a

marker of the late PE, move to the ICM surface [28]. Unfortunately, the origins of this pattern

had never been addressed, but it was widely taken to have stochastic origins (Figure 4A) [10,

23, 31].

Figure 4 - Models of PE and EPI segregation in the second cell fate decision.

EPI and PE segregation according with Induction, Cell sorting or a combination of both models (A). The influence of developmental history on lineage segregation in the ICM. EPI is derived from wave 1 internalized cells, whereas PE is

derived from the later, wave 2 internalizations (B). Adapted from [23].

In summary, the both discussed cell fate decisions demonstrate the remarkable capacity for the

early mouse embryo to maintain the flexibility to adjust to changing circumstances during its

development. Through examining these ways of patterning, we may build towards a more

complete understanding of patterning in the early embryo.

To uncover the genesis of ICM lineages, Morris et al. followed the origin, behavior and fate of

each and every individual cell as it developed into either PE or EPI. It revealed that the

developmental timing with which cells become set aside in the inside part of the embryo dictates

their fate. Thus, inside cells generated by the first wave during the 8- to 16-cell transition give

rise to the majority of EPI cells, while the majority of PE cells are progeny of inside cells

generated by the second cell divisions [30]. To sum up, the asymmetric divisions, which play

such an important part in the first cell fate decision, also have a major impact upon the second

cell fate decision (Figure 4B).

Once again several transcription factors are required for the proper segregation of each lineage.

As mentioned before, the EPI-specific factor Nanog and PE specific Gata6 are initially

22

expressed in both lineages and at E3.5 the patterns of Gata6 and Nanog expression are

mutually exclusive [3].

Gata4 and Gata6 are expressed at the early blastocyst stage and drive PE formation when

overexpressed in ES cells [31, 32]. On the other hand, several lines of evidence indicate that

the fibroblast growth factor (FGF) family of receptor tyrosine kinases (RTK) is required for Gata6

expression and PE formation [14] (see section 1.2.2-FGF signaling pathway). Still, these are not

the sole transcription factors required to specify the PE. For example, Sox17 and Sox7 protein

are important for endoderm differentiation in different model systems [33, 34]. On the other

hand, EPI cells express pluripotency factors such as Nanog, Sox2 and Oct4 [20, 35, 36].

1.2 Developmental signaling pathways in preimplantation mouse

embryo

Several pathways such as Hippo, FGF and Wnt/β-catenin Signaling Pathways have crucial

importance in cell differentiation during mouse blastocyst development. In fact, experimental

disturbances of these pathways lead to variations in embryonic geometry and tissue

morphogenesis, proving that they can control cell lineage. Interestingly, despite initial gross

architectural differences of early embryos these pathways are conserved in different vertebrate

species [10, 37-39].

1.2.1 Hippo pathway

The Hippo pathway is a signaling pathway that is essential for the proper regulation of organ

growth, proliferation, apoptosis, and is critical for cell fate decisions. It is composed of a highly

conserved core kinase cascade that comprises four types of tumor suppressors: the Ste20-like

kinases Mst1/2, its regulatory protein Sav1, the NDR family kinases Lats1/2 and its regulatory

protein Mob1A/B. The Mst1/2–Sav1 complex phosphorylates and activates Lats1/2-Mob1A/B

complex, which in turn phosphorylates oncogenic proteins Yap/Taz (Figure 5). These last two

proteins promote cell growth, proliferation and survival when, in the nucleus, Yap/Taz activate

TEAD/TEF family transcription factors. However, after phosphorylation they are retained in the

cytoplasm. Hence, the Hippo pathway activation inhibits transcriptional activity of Yap/Taz [38,

40].

23

Figure 5 - Schematic of the Hippo pathway.

Cells (outlined in grey, nuclei in green) are shown with adherens junctions (AJ). Pointed and blunt arrowheads indicate activating and inhibitory interactions, respectively. Continuous lines indicate direct interactions, whereas dashed lines indicate unknown mechanisms. Abbreviations: Ajub, Ajuba; Ex, Expanded; Lats, Large tumor suppressor; Mer, Merlin;

Mob1A/B, Mps1 binder; Mst, Mammalian sterile 20 like; Rassf, Ras-associated factor; Sav, Salvador; Taz, transcriptional co-activator with PDZbinding motif; TEAD, TEA domain protein; Yap, Yes associated protein; Adapted

from [40]

1.2.1.1 Hippo signaling in mouse embryo

As mentioned before (see section 1.1.1-The first lineage decision: segregation of the TE and

ICM) the Yap/Taz phosphorylation results in Tead4 inactivation in inner cells, which are fated to

form ICM cells. In opposition, in outside cells, where the Yap/Taz were not phosphorylated,

activated Tead4 induces Cdx2 expression, leading to acquisition of TE fate [2].

Actually, the outside cell restriction of Cdx2 can be explained by the connection between cell

density and Yap/Taz regulation. ICM cells might not express Cdx2 due to their apparent sense

of surrounding cells, resulting in phosphorylation of Yap/Taz, which may fail to activate

downstream targets, such as the Cdx2 gene. Thus, the complex arrangement of transfactors,

cell position, cell polarity, and signaling may control TE fate within the early embryo [2].

1.2.2 FGF signaling pathway

Fibroblast growth factor (FGF) signaling pathway has been involved in several phases of early

embryogenesis such as the induction and maintenance of several cell lineages. Here is

summarized the regulation and several roles of this pathway.

FGF family of extracellular ligands is characterized by a conserved core of 140 amino acids and

their strong affinity for heparin sulphate (HS). The members of this family were grouped into

subgroups according to their sequence homology and function. Fibroblast growth factor

receptor (FGFR) is one subgroup that undergoes alternative splicing in their extracellular

domain to generate a vast variety of receptors. FGF ligands, FGFRs and HS form a dimer that

24

results in the transphosphorylation of specific intracellular tyrosine residues (Figure 6). This

activates several cytoplasmic signal transduction pathways, such as the Ras/ERK pathway,

associated with proliferation and differentiation, or the protein kinase C (PKC) pathways, related

with cell morphology and migration [41].

Figure 6 - Schematic of FGF signaling

Cell response to dimerization of the FGFR leads to the activation of several intracellular signalling pathways, including

the PLCγpathway (A), PI3K/PKB pathway (B) and the Ras/ERK pathway (C). The cell responses to these different

pathways are shown. Abbreviations: CamKII, calcium/calmodulin-dependent protein kinase II; DAG, diacylglycerol; ERK, extracellular-signal related kinase; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FRS2, fibroblast growth factor receptor substrate 2; Gab, Grb2-associated protein; Grb2, growth factor receptor-bound protein

2; HSPG, heparan sulphate proteoglycan; IP3, inositol (1,4,5)-trisphosphate; MEK, mitogenactivated protein kinase kinase; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PKB, protein kinase B; PKC,

protein kinase C; PLCγ, phospholipase C γ; cRaf, v-raf-leukemia viral oncogene homologue 1; Ras, rat sarcoma;

SHP2, SH2 domain-containing tyrosine phosphatase 2; SOS, son of sevenless; Src, sarcoma proto-oncogene tyrosine kinase. Adapted from [41].

1.2.2.1 FGF signaling in mouse embryo

FGF signaling is essential for PE formation (as it was mentioned in section 1.1.2-The second

lineage decision: segregation of the PE and EPI), where loss of the FGFR results in the

absence of PE differentiation. Still, the exact mechanism by which FGF signals participate in

ICM patterning is unknown.

In fact, the Sorting model, which describes the second lineage decision, was developed from

the finding that chemical inhibition of FGF signaling with small molecules blocks differentiation

into PE and promotes the formation of an ICM consisting purely of EPI cells.

Yamanaka et al. showed that modulating FGF signaling levels shifts the fate of ICM cells to

become either Nanog-expressing (EPI specific) or Gata6-expressing (PE specific) progenitors,

respectively. Furthermore, the fate of the either Nanog- or Gata6-expressing cells can still be

controlled by FGF signaling, during blastocyst maturation [23, 29].

Recently, Krupa et al. observed regulatory mechanism adjusting the proportion of outer to inner

cells within the embryo that might be mediated by FGF signaling. The cells that are initially

25

scattered randomly within the ICM differ in their level of Fgf4/Fgfr2 expression and hence they

differ in the competence to respond to Fgf4 signaling [42].

1.2.3 Wnt/β-catenin signaling pathway

The canonical Wnt/β-catenin signaling pathway plays essential roles in a wide range of

biological processes including cell proliferation and determination of embryonic patterning [39,

43, 44].

β-catenin is a cytoplasmic protein that functions in cell-cell adhesion by linking cadherins to the

actin cytoskeleton. It also acts as an intracellular signaling molecule of the canonical Wnt

signaling pathway.

In resume, binding of Wnt ligands to the cell surface receptor Frizzled results in signaling

through Dishevelled (DVL) avoiding β-catenin phosphorylation by inactivating the β-catenin

destruction complex, which consists of adenomatous polyposis coli gene (APC), Axin, and

glycogen synthase kinase (GSK3β). This way, β-catenin translocates to the nucleus where

interacts with the T-cell-specific factor/lymphoid enhancer‑binding factor (TCF/LEF)

transcription factors to regulate target gene transcription (Figure 7A).

Alternatively, in the absence of Wnt activation, β-catenin is phosphorylated by the complex

(Figure 7B). Phosphorylated β-catenin is rapidly ubiquitylated and degraded by proteasomes.

The nucleus becomes depleted of β-catenin and TCF‑mediated complexes silence Wnt target

genes via transcriptional repressors [37, 45].

Figure 7 – Schematic of canonical Wnt/β-catenin pathway.

Wnt signaling activation leads to β-catenin accumulation (A), Wnt signaling inactivation leads to β-catenin degradation

(B). Abbreviations: APC, adenomatous polyposis coli; CK1γ, casein kinase 1γ; CTBP, carboxy‑ terminal binding protein; DKK1, Dickkopf protein; DVL, Dishevelled; GRO, groucho; GSK3β, glycogen synthase kinase 3β; HDAC, histone

deacetylase; Krm, kremen; LRP, low‑density‑ lipoprotein receptor‑ related; PEE, proximal epiblast enhancer; sFRP,

soluble frizzled‑ related protein; TCF/LEF, T‑ cell‑ specific factor/lymphoid enhancer‑binding factor; WIF1, Wnt inhibitory factor 1; βTRCP, β-transducin repeat‑containing protein;. Adapted from [37]

26

1.2.3.1 Wnt signaling in mouse embryo

Though the function of several Wnt molecules is well known during postimplantation embryonic

development, there is no solid evidence for Wnt signaling functional activity during

preimplantation of mouse blastocyst.

Another study in mouse has reported that β-catenin was localized in the nucleus of the outer

cells in morula and in the TE cells of preimplantation blastocyst. However, it disappeared after

this stage. Hence, Wnt signaling is probably involved in the hatching or TE differentiation during

preimplantation embryonic development [43]. Recent findings reveal that Wnt signaling can

direct the movement of the visceral endoderm [46]. In fact, reciprocal signaling between visceral

endoderm (VE), extraembryonic ectoderm (ExE) and EPI cell populations by secreted growth

factors of the transforming growth factor β (TGFβ) family and the Wnt and FGF families, leads

to regionalized gene-expression patterns in these three tissues [37]. Still, the molecular details

remain imprecise.

The components of Hippo signaling pathway can also interact with the Wnt/β-catenin signaling

pathway (see [47] for detailed review).

1.2.3.2 Wnt signaling in ES cells

The activation of Wnt signaling pathway promotes the proliferation and maintenance of

pluripotency in ES cells.

In fact, some studies suggested that Wnt/ β-catenin signaling may suppress differentiation in ES

cells. For example, ES cells with a mutant form of APC show impaired ability to differentiate into

the three germ layers. Likewise, when the gene expression profile of undifferentiated was

compared with the one of differentiated human ES cells it revealed an enrichment of Frizzled 5

in undifferentiated ES cells. Equally important, it was reported that 6-bromoindirubin-3’-oxime

(BIO), a newly identified pharmacological inhibitor of GSK3β, could maintain mouse ES cell

pluripotency in the absence of leukemia inhibitory factor (LIF) [48]. Still, additional experiments

are required to clarify the precise function of Wnt/β-catenin signaling in ES cells.

Apart from the canonical pathway Wnt can signal independently of β-catenin through

non-canonical pathways such as calcium flux, c-Jun N-terminal kinases (JNK) and both small

and heterotrimeric G proteins. Yet, the roles of these non-canonical Wnt pathways in

pluripotency remain elusive [48].

To sum up, there is still some questions to be answered and the need to accurate some details.

To do so, instead of analyzing each single pathway it will be better to integrate them and do it at

the same time, as they happen simultaneously.

27

1.3 Stem cell populations representative of the Early Embryonic

Lineages

Stem cells can be derived from each one of the first three lineages (Figure 8) and they have

served as in vitro cellular models [11]. Embryonic stem (ES) cells and epiblast stem cells are

likely derived from and represent the EPI, the trophoblast stem (TS) cells from the TE and

extraembryonic endoderm (XEN) cells from the PE [13, 14, 19, 49]. These last cells are

considered as stem because they can remain undifferentiated or differentiate into

lineage-specific cell types, when cultured under specific conditions.

These stem cells can overcome some of the already mentioned limitations of mouse blastocyst

studies such as the limited number of embryos that can be recovered. In this context, the role of

a given gene in these early lineages can be determined by the ability to establish stem cell lines

from mutant embryos [13]. With this technique it was possible to confirm that lineage choice is

governed by the expression lineage-specific transcription factors. In fact, ES cells can be

converted into TS-like or XEN-like cells by misexpression of appropriate transcription factors

[21, 37, 46, 50].

Figure 8 - Stem cell types that can be derived and propagated in culture representing the three blastocyst

lineages.

Embryonic stem (ES) cells represent the epiblast (EPI), trophoblast stem (TS) cells represent the trophectoderm (TE) and extraembryonic endoderm (XEN) cells represent the primitive endoderm (PE) cell lineage. Scale bars 100 µm.

Adapted from [51]

1.3.1 Embryonic stem cells (ES)

The ES cells are the most pluripotent stem cells known. As mentioned above, ES cells can be

derived from the preimplantation EPI. On a practical level, they can be a versatile source for

repairing damaged tissues in the adult body. Additionally, ES cells are the most accurate

28

controlled forms of genetic modification, allowing the creation of practically any desired

alteration in animals genome [4, 52].

Nichols et al. concluded that, in mouse, naive EPI and ES only differ by their environment [52].

In fact, when injected into a normal blastocyst, ES cells become incorporated in the ICM of the

blastocyst and can contribute to the formation of an apparently normal chimeric mouse [4].

Recent studies have suggested that Oct-4, Nanog and Sox-2, transcriptions factors important

for PE/EPI lineage restriction, are also important for ES cell fate [7].

1.3.1.1 ES culture conditions

ES cells have been derived from single EPI cells, but at low efficiency largely because of culture

conditions that were poorly defined. Initially the cells were cultured in a feeder layer of

mitotically inactivated fibroblasts. An understanding of the requirements for propagating ES cells

has evolved and the feeder layer can now be replaced by supplementing the serum-containing

medium with cytokine leukaemia inhibitory factor (LIF) [13, 52]. However, in some cases, the ES

cell culture may be impaired with this replacement.

1.3.1.1.1 2i conditions

It has been demonstrated that self-renewal require the inhibition of proteins involved in

differentiation, such as FGF/MAPK/ERK pathway and of GSK3 [13, 52-54]. This inhibitor

strategy permits the blocking of ES cells in a naive state.

Therefore ES cells can be maintained in an undifferentiated state in the presence of FGF

receptor inhibitor SU5402, and MAPK inhibitors, PD184352, PD0325901, or PD173074.

Additionally, survival and expansion are enhanced by the addition of CHIR99021, an inhibitor of

GSK3. In 2 inhibitors (2i) treatment the ES cells are cultured with CHIR99021 and PD032901.

The last one has a higher affinity than PD184352 for the FGF receptor and is more selective

than SU5402 [52]. However, recent reports demonstrated that culture of mouse ES cells with 2i

medium causes global hypomethylation but spares key regulatory elements [54].

1.3.1.2 Embryoid bodies

When cultured in 3D dimensions, in non-adherent conditions, the ES cells form small

aggregates called embryoid bodies (EBs). EBs differentiation has a multistep process that

mimics the differentiation of the ICM. As a consequence, EBs are often used as a first step in

ES cell differentiation protocols. When they are cultured in the presence of LIF, the outer layer

acquires a ExEn layer, a PE-like identity, but it do not differentiate into either parietal or visceral

endoderm-like derivatives, suggesting that LIF pathway prevents PE differentiation. Additionally,

the formation of the ExEn layer during EB formation is a sequence of events that might reflect

aspects of the in vivo segregation of PE and EPI lineages within the ICM [13].

29

1.3.2 Trophoblast Stem Cells (TS)

Trophoblast stem cells (TS) can be derived from the polar trophectoderm of preimplantation

embryos. They retain the capacity to differentiate into all trophoblast derivatives of the later

placenta in vitro. Moreover, when injected into blastocysts, they chimerize the embryo placental

portion [15, 49, 54]. As already mentioned, Cdx2 is a transcription factor that is necessary for

the maintenance of these cells [7, 15].

1.3.2.1 TS culture conditions

TS cells are propagated under complex but poorly defined cell-culture conditions, due to

presence of 20% fetal bovine serum (FBS). The medium is supplemented with FGF4 and

heparin. Further, they require either growth-inactivated feeder cells or feeder-cell-conditioned

medium by mouse embryonic fibroblast (mEFs), with no need of using feeders but always with

serum. However, serum and feeder cells are highly variable and, due to their animal origin, they

are a usual source of contamination. As these instabilities hamper the differentiation of TS, a

chemically and standardized media must be defined [13, 15, 49, 54].

Erlebacher et al. identified two active components secreted by feeder cells, TGFβ and activin,

that maintain TS cell proliferation [8]. Still, the remaining unknown factors secreted by mEF cells

lead to ill-defined culture conditions.

Kubaczka et al. developed the TX medium - a simple medium consisting of ten chemically

defined constituents for culture of TS cells on growth-factor-deprived Matrigel. TX medium

supports long-term self-renewal of TS cells and it is believed that this medium is capable of

propagating the genuine TS state [54]. The greatest drawback of this method is Matrigel price,

which makes this method too expensive for regular use, and possible influence of

Matrigel-derived factors. Furthermore, as these medium was unsuccessful with some tested TS

lines, it might be true that exist other factors, present in serum, which, along with FGF4, inhibits

the proapoptotic factor BIM [54].

1.3.3 Extraembryonic endoderm cells (XEN)

XEN cells derived from blastocysts continuously propagate in vitro. Just as in previous cases,

they can contribute to ExEn lineage cells after injection into blastocysts [55]. In this way, XEN

cells may increase our understanding of the molecular mechanism behind endoderm behavior

and function during development [14].

Although derived from primitive endoderm, initially, in chimeras, XEN cells contribute to parietal

endoderm but not to visceral endoderm. This lack of contribution could be caused by many

factors, including the alteration of a signaling pathway in the establishment of the cell line based

on the derivation process. More recently it was found that treatments with Nodal and bone

30

morphogenic protein 4 (BMP4), both members of the TGFβ superfamily direct XEN cells toward

a visceral endoderm phenotype in vivo and in vitro [55].

Once again, the important transcription factors for the PE/EPI lineage restriction are also

important for XEN cell fates. Extensive microarray analysis on XEN cells revealed that these

cells express PE markers Sox7, Gata4, Gata6. In fact, forced expression of the Gata4 and

Gata6 in mouse ES cells mimic XEN cell characteristics, suggesting that GATA factors play a

crucial role in XEN cell specification [55]. Although little is known about the signaling pathaways

required for XEN cells maintenance, it is known that PDGF signaling is required for XEN

isolation and/or cell expansion because inactivation of PDGFRα in established XEN cells

impairs their propagation [13, 50].

1.4 Postimplantation period

After implantation, the TE gives rise to several subtypes of trophoblast cells that facilitate

interaction with the uterus. The differentiation of TE cells that are not in contact with ICM results

in trophoblast giant cells. These cells are postmitotic, migrate into the uterine tissue, and line

the developing implantation site. They proliferate to give rise to the extraembryonic ectoderm

(ExE) providing progenitors for the ectoplacental cone. Later, all of the different trophoblast

subtypes are required to form the mature placenta [56].

The PE gives rise to the parietal endoderm and the visceral endoderm. The first one migrates

from the surface of the ICM and directly contacts the maternal tissue, while the visceral

endoderm remains in contact with the embryo and expands along the surface of the ExE and

EPI. Later, it gives rise to the endoderm of the visceral yolk sac. The visceral endoderm is a

tissue of significant interest because of its crucial function in mediating the activity of TGFβ and

Wnt signaling pathways [46].

Finally, the EPI retains pluripotency and gives rise to both the somatic tissues and the germ cell

lineage of the embryo proper [11, 37].

31

2 Materials and Methods

2.1 Mouse fibroblast feeder layer (mEF) culture

Both embryonic and trophoblast stem cells were seeded on a mouse embryonic fibroblast

(mEF) feeder layer. Before seeding the mEF, the well plates (Nunclon Delta Surface by Thermo

Scientific Nunc) were coated with a gelatin solution (0,1%) by incubating for at least 15 minutes

at 37 ºCelsius. After removing the remaining gelatin, the fibroblasts were seeded in mEF

medium (Dulbeco’s Modified Eagle Medium (DMEM, Gibco by Life Technologies) supplemented

with 1 % L-glutamine (200mM in stock); 0,5 % Penicillin Streptomycin (Pen/Strep, Gibco by Life

Technologies); 10 % fetal bovine serum (FBS, Hubrecht) and 1 % Hepes Buffer Solution (Gilbco

by Life Technologies)) and incubated at 37 ºC and 5% CO2. All the used fibroblasts were till

passage 4 (maximum 5) and seeded at an approximate confluent density of 1x104 cells/cm2.

2.2 Cell culture

The cells were previously isolated from preimplantation blastocysts from pregnant mice,

separated from the others and seeded on well plates on the top of a mEF feeder layer. These

culture plates were incubated at 37 ºC and 5% CO2.The cells were usually seeded at a density

of 10 000 cells/cm2.

2.3 Immunostaining of blastoids

Blastoids were fixed in 4% paraformaldehyde in PBS for 30 minutes or overnight (depending on

the cells sensibility) at 4ºC and rinsed in PBS containing 0,1% TritonX-100 (Sigma Aldrich)

three times. The membrane’s permeabilization was also achieved by adding 0,25% TritonX-100

for 30 minutes. Then they were rinsed with 0,05% Tween-20 (KWR BDH Prolabo) in PBS for 15

minutes each time, and blocked in PBS with 10% serum from secondary antibodies’ host and

0,1 % TritonX-100 for 1 hour at room temperature.

After this, the blastocysts were incubated in the appropriate antibody solution, at 1:250 at 4°C

overnight. Primary antibodies were rabbit Cdx2 (EPR-2764y, rabbit anti-human/mouse) and

rabbit Nanog (abcam ab84447 Lot GR53934). Secondary antibodies were goat anti rabbit,

labelled with Alexa fluorophores (Invitrogen, Paisley, UK) diluted 1:500 in PBS+0,1% TritonX-

100. Sometimes, for control, it was also used a non-specific primary antibody, Mouse IgG

isotype.

32

2.4 Image acquisition and processing

The 96-well plates with the blastoids were imaged using a BD pathway 435 from BD

Biosciences. The images obtained were processed and analyzed using Cell Profiler Software.

Several parameters were measured, like circularity, area and relative positions of the different

fluorescently labeled cellular regions. Using this information it was possible to distinguish

cavitated blastoids from the others due to their distinct periphery radial intensity profiles (Radial

Distribution MeanFrac). The used threshold was 0,9. The ICM and the blastoid size correspond

to the sum of the pixel intensities within the ICM cells and the area of the blastoid, respectively.

When using 10x Objective of the BD Pathway and a 4x size reduction, the conversion in µm2 is

based on a resolution of 0,408 pixel/µm (equivalent to 6,0 µm2/pixel).

2.5 Statistical analysis

Statistical analysis was performed using the GraphPad Prism software. Statistical significance

was established with P-value<0,05 and determined by ANOVA with Bonferroni post hoc tests.

P-value is shown wherever the difference between compared groups was significant. All data

are presented as the mean ± standard deviation.

33

3 Results and Discussion

The aim of this work was the optimization of methods to produce blastoids (also called artificial

blastocysts) and to use this culture system to perform studies on the molecular mechanisms

that drive morphogenesis during blastoid development. The method’s optimization is discussed

in the next section (3.1) and the following section (3.2) contains a detailed description of some

studies that were developed to better understand some biological mechanisms during

blastocyst development.

3.1 Method’s Optimization

3.1.1 Study the influence of stem cell number in blastoids development

To study the effect of cell number on blastoids developmental potential, the cell number was

modulated by seeding different concentrations of cells per well. One hour after seeding pictures

of the platform were acquired and cells were counted (using the ImageJ software) showing a

good correlation with the seeded amount (data not shown).

In addition, the effect of a specific compound (A) in the blastoids development was also

examined in this experiment. A previous work had shown that this compound promotes

cavitation in blastoids and its concentration was previously optimized. In order to use it in future

screenings it was important to test its effect on blastoids development, considering the ICM and

the cavitated blastoids sizes.

Consistent with the earlier observations, in this experiment the yield of cavitation was higher

when the blastoids were cultured in the presence of the tested compound.

As expected, the yield of cavitation (formation of the blastocyst’s cavity), when cultured with A,

increased continuously with cell number, reached a plateau and decreased from this point on.

Probably this is due in part to the fact that at a high cell density the blastoid forms a big ICM

which impaired the blastocoel formation.

The results show that ICM size increased with cell number. While in the cavitated blastoids the

ICM increased, with and without A. Finally, the size of cavitated blastoids increased with cell

number.

From these observations is possible to conclude that there is an optimum cell density for

culturing blastoids, when cultured with A.

34

3.1.2 Study of the influence of different medium compounds in blastoids

development

The addition of compounds that will stimulate a good blastoid development is mandatory. In this

context, it was decided to study the influence of adding two different compounds to the blastoid

culture medium, B and C, that are crucial during the preimplantation stage of the mouse

blastocyst. Individual dose responses were performed to determine the optimal concentration of

each mentioned compound.

The effect of combining A with B was assessed at a range of concentrations. Gradual and

progressive increase of yield of cavitation with the increase of B concentration was observed

with and without A. Once again, higher yields were obtained when the blastoids were cultured

with A. The difference between the conditions with or without A is easily noted in higher

concentrations of B. Furthermore, the difference between concentrations of B is more significant

in the samples with A. In simple terms, together they promoted cavitation more efficiently than

either did separately. Consequently it might be true that A and B act together to promote

blastocoel formation.

The B both ICM’s and cavitated blastoids’ sizes increased gradually. Interestingly, with higher

concentrations of B they both decreased. The sizes reduction might indicate cell toxicity at a

combination of A with high concentrations of B.

To examine the influence of C in the blastoids development the blastoids were cultured with

increasing concentrations of C.

Surprisingly, the yield of cavitation did not increase in a dose dependent manner. On the other

hand a gradual increase in ICM size of both cavitated and non cavitated blastoids was observed

with increasing doses of C, which is essential for a good blastoid formation.

At this point a more complex experiment was performed with a combination of different

concentrations of A, B and C. The data was not as accurate and consistent with the previous

studies as it was hoped.

Among all the tested compounds, the yield of cavitation and the size of cavitated blastoids are

more influenced by B. In general, higher concentrations of A or B corresponded to the highest

yields of concentration and the biggest cavitated blastoids, with few exceptions. Besides that,

statistical analysis revealed that the results were not significantly different. Consequently, to

firmly establish the dose response of blastoids to these compounds it is recommended to repeat

this experiment with a control condition and more samples per condition.

35

3.1.3 Optimization of staining of blastoids

As it was mentioned before, the three germ layers can be easily identified by its transcriptional

factors. Therefore, to be able to study their expression along the blastoids development, the

Cdx2 (expressed in TE [15]) and Nanog (expressed in epiblast [15]) staining was optimized.

The available protocols were optimized for the staining of 2D monolayers being necessary its

adaptation to 3D cultures.

3.1.3.1 Nanog Staining

Nanog is a EPI-specific factor that maintains the pluripotency by inhibiting PE formation [13]. It

starts being expressed in all cells at the 8-cell stage, and at around 32-cell stage, it is co-

expressed in all cells along with the Gata6. Nanog persists within the ICM, and becomes

exclusive in EPI cells until the late blastocyst stage when it starts to decline and later becomes

re-established in the germline [3, 10].

In the case of the Nanog, stainings in the 2D and 3D cultures were performed separately,

starting by investigating the presence of this protein in the 2D cultures. Two different controls

were used to verify the accuracy of the antibody staining: a non-specific primary antibody

instead of the specific one and a secondary antibody without adding a primary. As expected, the

Nanog expression was undetectable in both cases (data not shown).

Likewise, the difference between the positive and negative control is clear. In the positive there

was an intense Nanog expression while in the negative control the levels of expression were

significantly low (data not shown).

It is known that the non-ionic detergents can reduce non-specific hydrophobic interactions.

These give large enough pores for antibodies to enter the cells without dissolving plasma

membrane [62]. Therefore, 0,05% Tween 20 in PBS or ice cold methanol were used to improve

permeabilization and, consequently, optimize the staining. However, the results were not

improved.

In what concerns to the blastoids staining (3D cultures), the results were not as good. The DAPI

was very diffused probably because of the short time of incubation (data not shown). This step

should thus be repeated with a longer time of incubation.

36

3.2 Studies about blastocyst development

As previously referred, several studies were performed to better understand some biological

mechanisms during blastocyst development. In order to avoid a high complex and through

thesis, it will only be explained and discussed the three of them.

3.2.1 Z signaling in A treatment

The aim of the next experiment was to address the role of Z signaling in the blastocyst

development.

In this experiment the Z signaling was promoted through the addition of D. In the same way, Z

signaling was inhibited with E. At last, the Z secretion was inhibited with F. Once again the

blastoids were cultured with and without A and kept for 4 days.

Among the tested conditions two were associated with high levels of yield of cavitation –

addition of D and addition of D+A. Actually, the A effect was only detected when this was

combined with A. There were no statistical differences between A and control through all

conditions.

The results tend to show a synergy between D and A in the promotion of an efficient cavitation.

Besides, as D promotes Z signaling, this supports the importance of this pathway in blastocoel

formation, being a crucial step.

On the other hand, a clearly yield reduction was detected when the blastoids were cultured with

E. Consistent with the previous hypothesis, these results suggest that Z signaling is essential

during cavitation. It is worth to notice that the fact that yield in control being similar to the yield

with D may indicate that the last one can only promote cavitation when the Z signaling is

activated. It was also noticed that the size of cavitated blastoids was significantly small, which

may indicate a deficient blastoid development. Finally, the yield of cavitation with F was still

lower in comparison with the absence of both E and F but not as low as with E.

To sum up, these results showed that Z pathway is also important in the early stages of the

blastocyst and it Is crucial during cavitation.

3.2.2 Interaction of A with different growth factors

As the concentration of A increases the yield of cavitation, it was intended to use this compound

in the future to improve screening read-out. Therefore it was tested the impact of the

37

combination of A with growth factors on blastoids development. The blastoids were cultured

with a low, medium or high concentration of each growth factor.

In general, the growth factors promoted a yield of cavitation similar to the control condition.

These may indicate that these tested growth factors do not influence the blastoids cavitation.

Moreover, when comparing the results obtained between low, medium and high concentrations

no statistically significant differences were observed. This can have two interpretations. On one

hand it could mean that the concentration does not influence the cavitation of the blastoids. On

the other hand it could also mean that the tested concentrations were not sufficiently different

from each other to obtain significantly different results. As it is believed that in this case the

concentrations are already significantly different from each other, the second hypothesis

probably does not apply. But, to be firmly sure it is recommended to repeat the experiment with

even more disparate concentrations.

Surprisingly, the ICM size of cavitated blastoids was identical upon addition of each one of the

growth factors. Once again, the results obtained with different concentrations did not diverge

from each other. At last, the size of cavitated blastoids was maintained among all other

conditions.

To conclude, A can be used with these growth factors because in general it did not impair the

blastoids development.

38

4 Conclusions and future perspectives

How a single fertilized egg gives rise to a complete embryo is still an intriguing process but,

more than thirty years since the first models of lineage restriction were proposed, we have

begun to understand the molecular foundations of lineage restriction and fate commitment in the

early mouse embryo. This advance was possible due to technological improvements and

pioneering studies with stem cell lines from the first three lineages of the mouse, among others.

Actually, lessons learned through these studies extend beyond blastocyst formation, and have

contributed to advancement in our knowledge in different ranges, such as body patterning and

stem cell therapeutics.

The protocol to develop artificial blastocysts here described is included in these studies. The

stem cells are cultured in a chemically defined culture medium under a controlled environment

that promotes the blastocyst formation with a potential to reduce the amount of blastocysts

being harvested for future studies.

The main goal of this thesis was to optimize the culture conditions to produce artificial

blastocysts and good results were obtained. In parallel, different assays were developed with

the aim of ensure that the compounds tested are not harmful to the artificial blastocysts and

reveal some details of biological mechanisms that take place during blastocyst formation.

The Z signaling study suggested that this pathway has influence in the early blastoids formation

because when it was inhibited the cavitation yield was significantly low. It was also observed

that A in combination with the tested growth factors do not impair the blastoids cavitation.

Despite the good results achieved by this method there are still several improvements that can

be implemented, beside the ones already mentioned. One drawback of this protocol is the

variability introduced by serum and feeder cells. For better reproducibility of results and less risk

of contamination is crucial to develop a standardized media.

The seeding process of the cells and the pipetting steps can also be optimized. The use of

robotic systems in the protocol and in other high-throughput screens would lead to more

accurate and reliable results by reducing the variability inside the wells, between the wells and

between different plates.

Finally, to validate the method, the blastoids must be compared with naive blastocysts using

different approaches. Cytogenetic analysis, immunoprecipitation of methylated DNA, cell cycle

distribution by propidium iodide staining and flow cytometry, quantitative RT-PCR (qRT-PCR) to

analyze the expression of specific transcription factors, immunofluorescent staining and

immunoblotting against the same transcription factors are some of them.

39

5 References

1. Sarton G, von Baer KE: The Discovery of the Mammalian Egg and the Foundation of Modern Embryology: With a Complete Facsimile [no. XIII] of K.E. Von Baer's De Ovi Mammalium Et Hominis Genesi [Leipzig, 1827]: Saint Catherine Press; 1931.

2. Bergsmedh A, Donohoe ME, Hughes RA, Hadjantonakis AK: Understanding the molecular circuitry of cell lineage specification in the early mouse embryo. Genes 2011, 2(3):420-448.

3. Cockburn K, Rossant J: Making the blastocyst: lessons from the mouse. The Journal of clinical investigation 2010, 120(4):995-1003.

4. Alberts B JA, Lewis J, et al.: Development of Multicellular Organisms. In: Molecular biology of the cell. 4th edn. New York: Garland Science; 2002: 1305-1416.

5. Alarcon YMaVB: Creation of Trophectoderm, the First Epithelium, in Mouse Preimplantation Development. In: Mouse Development - Results and Problems in Cell Differentiation. vol. 55; 2012: 165-184.

6. Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying Q-L, Smith A: Capture of Authentic Embryonic Stem Cells from Rat Blastocysts. Cell 2008, 135(7):1287-1298.

7. Chuykin I, Lapidus I, Popova E, Vilianovich L, Mosienko V, Alenina N, Binas B, Chai G, Bader M, Krivokharchenko A: Characterization of trophoblast and extraembryonic endoderm cell lineages derived from rat preimplantation embryos. PloS one 2010, 5(3):e9794.

8. Erlebacher A, Price KA, Glimcher LH: Maintenance of mouse trophoblast stem cell proliferation by TGF-beta/activin. Developmental biology 2004, 275(1):158-169.

9. Moon SY, Park YB, Kim DS, Oh SK, Kim DW: Generation, culture, and differentiation of human embryonic stem cells for therapeutic applications. Molecular therapy : the journal of the American Society of Gene Therapy 2006, 13(1):5-14.

10. Xenopoulos P, Kang M, Hadjantonakis A-K: Cell lineage allocation within the inner cell mass of mouse blastocyst. In: Mouse Development - Results and Problems in Cell Differentiation. vol. 55; 2012: 185-202.

11. Lanner F: Lineage specification in the early mouse embryo. Elsevier 2013, 321:32-39.

12. Saiz N, Plusa B: Early cell fate decisions in the mouse embryo. Reproduction 2013, 145(3):R65-80.

13. Hadjantonakis JAaA-K: Troika of the mouse blastocyst lineage segregation and stem cells. Curr Stem Cell Res Ther 2012, 7(1):78-91.

14. Yamanaka Y, Ralston A, Stephenson RO, Rossant J: Cell and molecular regulation of the mouse blastocyst. Developmental dynamics : an official publication of the American Association of Anatomists 2006, 235(9):2301-2314.

15. Strumpf D, Mao CA, Yamanaka Y, Ralston A, Chawengsaksophak K, Beck F, Rossant J: Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 2005, 132(9):2093-2102.

16. Tarkowski AK, Wróblewska J: Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. J Embryol exp Morph 1967, 18(1):155-180.

17. Hillman N: The effect of spatial arrangement on cell determination during mouse development. J Embryol exp Morph 1972, 28(2):263-268.

18. F. Beck TE, A. Russell, R. James: Expression of C&-2 in the Mouse Embryo and Placenta: Possible Role in Patterning of the Extra-Embryonic Membranes. Developmental Dynamics 1995, 204:219-227.

19. Chen L, Yabuuchi A, Eminli S, Takeuchi A, Lu CW, Hochedlinger K, Daley GQ: Cross-regulation of the Nanog and Cdx2 promoters. Cell research 2009, 19(9):1052-1061.

20. Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R: Multipotent cell lineages in early mouse development depend on SOX2 function. Genes & development 2003, 17(1):126-140.

21. Niwa H, Toyooka Y, Shimosato D, Strumpf D, Takahashi K, Yagi R, Rossant J: Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 2005, 123(5):917-929.

40

22. Jedrusik A, Parfitt DE, Guo G, Skamagki M, Grabarek JB, Johnson MH, Robson P, Zernicka-Goetz M: Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo. Genes & development 2008, 22(19):2692-2706.

23. Zernicka-Goetz SAMaM: Formation of distinct cell types in mouse blastocyst. In: Mouse Development - Results and Problems in Cell Differentiation. vol. 55; 2012: 203-217.

24. Krawchuk D, Honma-Yamanaka N, Anani S, Yamanaka Y: FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst. Developmental biology 2013, 384(1):65-71.

25. Murray P, Edgar D: The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF). Differentiation 2001, 68:227-234.

26. Chazaud C, Yamanaka Y, Pawson T, Rossant J: Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Developmental cell 2006, 10(5):615-624.

27. Grabarek JB, Zyzynska K, Saiz N, Piliszek A, Frankenberg S, Nichols J, Hadjantonakis AK, Plusa B: Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo. Development 2012, 139(1):129-139.

28. Plusa B, Piliszek A, Frankenberg S, Artus J, Hadjantonakis AK: Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst. Development 2008, 135(18):3081-3091.

29. Yamanaka Y, Lanner F, Rossant J: FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development 2010, 137(5):715-724.

30. Morris SA, Teo RT, Li H, Robson P, Glover DM, Zernicka-Goetz M: Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo. Proceedings of the National Academy of Sciences of the United States of America 2010, 107(14):6364-6369.

31. Meilhac SM, Adams RJ, Morris SA, Danckaert A, Le Garrec JF, Zernicka-Goetz M: Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst. Developmental biology 2009, 331(2):210-221.

32. Q. Tian Wang, Karolina Piotrowska, Maria Anna Ciemerych, Matthew P. Scott, Ljiljana Milenkovic, Ronald W. Davis, Zernicka-Goetz M: A Genome-Wide Study of Gene Activity Reveals Developmental Signaling Pathways in the Preimplantation Mouse Embryo. Developmental cell 2004, 6:133-144.

33. Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Sherwood RI, Yamaki M, Dimos JT, Chen AE, Melton DA et al: Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes & development 2010, 24(3):312-326.

34. Artus J, Piliszek A, Hadjantonakis AK: The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17. Developmental biology 2011, 350(2):393-404.

35. Xenopoulos P, Kang M, Hadjantonakis A-K: Cell Lineage Allocation Within the Inner Cell Mass of the Mouse Blastocyst. 2012, 55(185-202).

36. Aksoy I, Jauch R, Chen J, Dyla M, Divakar U, Bogu GK, Teo R, Leng Ng CK, Herath W, Lili S et al: Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm. The EMBO journal 2013, 32(7):938-953.

37. Arnold SJ, Robertson EJ: Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nature reviews Molecular cell biology 2009, 10(2):91-103.

38. Imajo M, Miyatake K, Iimura A, Miyamoto A, Nishida E: A molecular mechanism that links Hippo signalling to the inhibition of Wnt/beta-catenin signalling. The EMBO journal 2012, 31(5):1109-1122.

39. Morris SA, Guo Y, Zernicka-Goetz M: Developmental plasticity is bound by pluripotency and the Fgf and Wnt signaling pathways. Cell reports 2012, 2(4):756-765.

40. Halder G, Johnson RL: Hippo signaling: growth control and beyond. Development 2011, 138(1):9-22.

41. Dorey K, Amaya E: FGF signalling: diverse roles during early vertebrate embryogenesis. Development 2010, 137(22):3731-3742.

41

42. Krupa M, Mazur E, Szczepanska K, Filimonow K, Maleszewski M, Suwinska A: Allocation of inner cells to epiblast vs primitive endoderm in the mouse embryo is biased but not determined by the round of asymmetric divisions (8-->16- and 16-->32-cells). Developmental biology 2014, 385(1):136-148.

43. Lim KT, Gupta MK, Lee SH, Jung YH, Han DW, Lee HT: Possible involvement of Wnt/beta-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts. Theriogenology 2013, 79(2):284-290 e281-282.

44. Nusse R: Wnt signaling and stem cell control. Cell research 2008, 18(5):523-527. 45. Wray JP: Characterisation of a novel culture condition for the establishment and

maintenance of mouse embryonic stem cells and implications for the mechanisms of self-renewal. University of Edinburgh; 2008.

46. Rossant J, Tam PP: Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 2009, 136(5):701-713.

47. Varelas X, Miller BW, Sopko R, Song S, Gregorieff A, Fellouse FA, Sakuma R, Pawson T, Hunziker W, McNeill H et al: The Hippo pathway regulates Wnt/beta-catenin signaling. Developmental cell 2010, 18(4):579-591.

48. Okita K, Yamanaka S: Intracellular Signaling Pathways Regulating Pluripotency of Embryonic StemvCells. Current Stem Cell Research & Therapy 2006, 1:103-111.

49. Tanaka S: Promotion of Trophoblast Stem Cell Proliferation by FGF4. Science 1998, 282(5396):2072-2075.

50. Artus J, Panthier JJ, Hadjantonakis AK: A role for PDGF signaling in expansion of the extra-embryonic endoderm lineage of the mouse blastocyst. Development 2010, 137(20):3361-3372.

51. Niakan KK, Schrode N, Cho LT, Hadjantonakis AK: Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells. Nature protocols 2013, 8(6):1028-1041.

52. Nichols J, Silva J, Roode M, Smith A: Suppression of Erk signalling promotes ground state pluripotency in mouse embryo. Development 2009, 136:3215-3222.

53. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A: The ground state of embryonic stem cell self-renewal. Nature 2008, 453(7194):519-523.

54. Kubaczka C, Senner C, Arauzo-Bravo MJ, Sharma N, Kuckenberg P, Becker A, Zimmer A, Brustle O, Peitz M, Hemberger M et al: Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions. Stem cell reports 2014, 2(2):232-242.

55. Moerkamp AT, Paca A, Goumans MJ, Kunath T, Kruithof BP, Kruithof-de Julio M: Extraembryonic endoderm cells as a model of endoderm development. Development, growth & differentiation 2013, 55(3):301-308.

56. Gasperowicz M, Natale DR: Establishing three blastocyst lineages--then what? Biology of reproduction 2011, 84(4):621-630.

57. Ware CB NA, et al.: Derivation of naïve human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America 2014, 111(12):4484-4489.

58. Schultz PMaRM: Histone deacetylase 1 (HDAC1) regulates histone acetylation, development, and gene expression in preimplantation mouse embryos. Dev Biol 2008, 319(1):110-120.

59. Donnard E, Barbosa-Silva A, Guedes RL, Fernandes GR, Velloso H, Kohn MJ, Andrade-Navarro MA, Ortega JM: Preimplantation development regulatory pathway construction through a text-mining approach. BMC genomics 2011, 12 Suppl 4:S3.

60. Zhao X, Ma W-g, Das SK, Dey SK, Paria BC: Blastocyst H2 receptor is the target for uterine histamine in implantation in the mouse. Development 2000, 127:2643-2651.

61. Tanaka SS, Kojima Y, Yamaguchi YL, Nishinakamura R, Tam PP: Impact of WNT signaling on tissue lineage differentiation in the early mouse embryo. Development, growth & differentiation 2011, 53(7):843-856.

62. R&D systems web page; Technical Information - Preventing Non-specific Staining, http://www.rndsystems.com/ihc_detail_objectname_preventing_nonspecific_staining.aspx

63. Wray J, Kalkan T, Smith AG: The ground state of pluripotency. Biochemical Society transactions 2010, 38(4):1027-1032.

42

64. Bilir B, Kucuk O, Moreno CS: Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells. Journal of Translational Medicine 2013, 11.

65. Blauwkamp TA, Nigam S, Ardehali R, Weissman IL, Nusse R: Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nature communications 2012, 3:1070.

66. Guzeloglu-Kayisli O, Kayisli UA, Taylor HS: The role of growth factors and cytokines during implantation: endocrine and paracrine interactions. Seminars in reproductive medicine 2009, 27(1):62-79.

67. Lanner F, Rossant J: The role of FGF/Erk signaling in pluripotent cells. Development 2010, 137(20):3351-3360.

68. Ian Chambers DC, Morag Robertson JN, Sonia Lee,, Susan Tweedie aAS: Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells Cell 2003, 113:643-655.

69. Capo-Chichi CD, Rula ME, Smedberg JL, Vanderveer L, Parmacek MS, Morrisey EE, Godwin AK, Xu XX: Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells. Developmental biology 2005, 286(2):574-586.