Placentation & Trophoblast I

Question 1

What is the placenta?

Answer 1

Is an organ unique to pregnancy
Forms the interface between the mother and the fetus.
The placenta is fetal in origin at term it weighs 500-1000g
Acts as the lungs, gut and kidneys of the fetus
It also acts as an endocrine organ releasing hormones into the maternal circulation such as hCG and progesterone

Question 2

The placenta is a semi-allograft, what does this mean?

Answer 2

the cells have genetic material from both the mother and the father

Question 3

Foetal and Matenal blood

Answer 3

In a human pregnancy, fetal cells are in direct contact with maternal blood
As it is in contact with maternal blood this requires mechanisms to evade the maternal immune system
However although the fetal cells and maternal cells are in direct contact, the fetal and maternal circulations do not mix

Question 4

How does the placenta develop?

Answer 4

Implantation is the multistep process by which the free-floating blastocyst attaches to the endometrium, invades through the epithelium and into the stroma beneath and begins to establish the placenta.

The blastocyst attaches to and adheres to the epithelial layer of the uterine wall.
The Trophoectoderm cells will proliferate and migrate through the epithelial cells through the basement membrane and into the underlying uterine wall.

Later the Trophoectoderm cells fuses to form a primitive syncytium (PS) (synctiotrophoblasts) beneath the implanted embryo.

The Trophoectoderm cells will continue to migrate or invade into the decidua.

They will form an increasing primitive syncytium (PS), and within the syncytium will form holes (Lacunae).

Lacunae (L) form by the action of proteases which later develop into the intervillous space

Behind the primitive syncytium, Cytotrophoblasts will proliferate and migrate through the syncytium and into the uterine wall to form the anchoring villi (dark blue).

Question 5

Placental formation at later stage in gestation

Answer 5

Can see the decidua basalis underneath the placenta (light blue).

Can see the anchoring villi
As the placenta develops the villi start to branch and form both secondary and tertiary villi.
Within these villi are the blood vessels the supply the placenta, from the foetus. They are responsible for exchanging the nutrients and respiratory gases that come from the mother and are delivered back from the foetus.

Can see the presence of spiral arteries that deliver maternal blood to the intervillous space.
The are also veins that will drain the blood from the intervillous space back into the maternal circulation.

The villi are highly branched with a large surface area for exchange of nuitrents and respiratory gasses.
The Outer layer of the villi is formed of fused cells- the syncytium
Underlying this are the cytotrophoblast stem cells
Diffusion distance to vessels small
Growth is regulated by a number of factors including IGF I and II

Question 6

Define the villous structure and cross section appearance:

Answer 6

Within each of the villi are a highly branched vascular network of arteries and veins which takes blood to and from the developing foetus.

Cross section:
On the outside there is a fused layer of cells called the syncytium
Underneath are the cytotrophoblast stem cells
The vessels are very close the surface of the villi, this is important for the delivery and exchange of nutrients.
Within the villi there are macrophages known as hofbauer cells. Their function may be involved in immune protection of the placenta and regulating the formation and branching of the vessels.

Question 7

Formation of the syncytium

Answer 7

It is formed by the combined action of hCG and an endogenous retroviral protein called Syncytin-1/2.
hCG binds to the LH/CRG receptor
This will stimulate the production of cAMP
cAMP will then activate a membrane enzyme called Scramblase.
Scramblase is responsible for the redistribution of phosphatidylserine from the inner surface of the plasma membrane, to the outer surface of the plasma membrane.
cAMP will also increase the activity of PKA
PKA will phosphorylate the protein GCM1 (Glial Cells Missing Homolog 1), which is a transcription factor
GCM1 will then move to the nucleus and regulate the expression of Syncytin-1/2
Syncytin 1 and 2 will then be transported to the plasma membrane where it will induce cell fusion and the formation of the syncytium.

The syncytium is being regenerated constantly throughout gestation, although it does slow down.
In order to do this the underlying cytotrophoblasts have to fuse with the syncytium, thereby increasing/replacing lost material.
Villous cytotrophoblast proliferation decreases with gestation by term the syncytium close to placental vessels
Syncytium is continually been shed in to the maternal circulation and is replaced by the underlying cytotrophoblasts
Using the stain desmoplacin you can see that the membranes between cells are lost and multinucleated cells are formed.

Question 8

Trophoblast differentiation and function

Answer 8

Have mentioned the villous pathway of the cytotrophoblast stem cells, will now focus on the Extravilous pathway:

These cells are derived from the cytotrophoblast cell columns and shell. They form two different subtypes:
The endovascular Extravilous trophoblast
The interstitial Extravilous trophoblast
These two cells types work together to remodel the maternal spiral arteries.

BUT in the initial stages of pregnancy, the endovascular extravilous trophoblasts form the trophoblast plug. This prevents maternal blood from entering into the intervillous space.

The interstitial extravilous trophoblasts invade into the decidua and migrate towards the maternal spiral arteries.

Question 9

Extravillous cytotrophoblast invasion

Answer 9

As the cells from the column move into the decidua, they undergo epithelial mesenchymal transition.
During this process cells lose the polarity they have. They loose adherence. They become more motile and invasive in nature.
As this progresses they will also start to express different cell surface markers.
Initially they express: α6β4, αVβ6 and E cadherin
As the cells migrate away from the column they express: αVβ3, α1β1, VE-cadherin, VCAM-1, and PECAM-1

Question 10

Oxygen tension and gestational age

Answer 10

The main purpose of the trophoblast plug is that it reduces the amount of oxygen that the villous tissue is exposed to in early gestation.
Up until the 12th week of gestation, the uterine spiral arteries are plugged with trophoblasts.
Placental development therefore occurs under relative hypoxia 2-3% O2
While the spiral arteries are plugged, the nutrition that is supplied to the growing foetus is through the process of histiotrophic nutrients being secreted by the glandular cells
Following the dissolution of the trophoblast plug, the placenta switches to a normal haemotrophophic nutrition. This occurs around the 12 week.

Low oxygen early in pregnancy is important normal pregnancy progression

Prolonged low oxygen leads to placental pathologies

Question 11

Methods used to study human placental development

Answer 11

Human studies are limited for ethical reasons

Animal models:
BUT There are significant differences in the placental development between mammals:

The mouse is not always a suitable model:
Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood. SMILARITY
However there is no deep interstitial invasion of the decidua. The extent of interstitial invasion is significantly less than in humans.
Mice do not exhibit the same obstetric complications as humans. DIFFERENCE

The best model of human pregnancy is the great apes:
Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood (maternal spiral artery) SMILARITY
Deep interstitial invasion of the decidua does occur. SMILARITY
Some evidence that they do exhibit the same obstetric complications as humans. SMILARITY
BUT it is ethically unacceptable to experiment on these animals.

Question 12

In vitro methods used to study human placental development

Answer 12

Human tissue can only be obtained either in the first trimester from Termination of pregnancy or at term upon delivery.
Tissues can either be used whole or cells can be isolated from them.

Also available from cell lines:
Trophoblast cell lines derived from choriocarcinomas JEG3, Jar and BeWo
grow well in culture
but have lost some characteristics

Developed cell lines following transfection with oncogenes such as t- and T-antigen of SV40 or more recently hTERT
grow well
but have lost some characteristics but this can depend on how they are cultured
An approach that showed initial promise: use of Human embryonic stem cell-derived trophoblast cells (hESCs)
BUT characterisation has proved problematic
It cannot be used in a number of regions due to legal considerations.

The most recent development showing initial promise: Use of Human trophoblast stem cells (hTSCs) derived from the trophectoderm and first trimester placentae
express characteristics of first trimester trophoblasts
can be induced to differentiate along either syncytial or extravillous lineages
BUT can be difficult to prepare and grow
Not allowed in some regions.

For ALL models: The phenotype of all cells grown in vitro will be dependent the
culture conditions used. (eg differences between monolayer and 3D cultures)

Question 13

Culture formats

Answer 13

Simple mono layer culture
Simple co-culture models
Mixing two cell types together and growing in a monolayer
Or separate them using multi cell inserts – so factors produced by each cell type can freely diffuse between the two.
Addition of extracellular matrix
Communication can take place between cells in a more direct fashion.
Effect of flow
Grow cells and look at the effects of flow
3D environment
Spheroid model or a perfusion model of dissected arteries.
Organoid cultures
More recent approach where cells are isolated but then the tissue is reconstructed.

Question 14

What regulates trophoblast invasion- how can these models be used?

Answer 14

Ex vivo model using human placental tissue.
The anchoring villous can be identified and dissected, then placed on an extracellular matrix and the migration of cells from the villous tissue can be monitored by time-lapse microscopy. These processes can be manipulated by using both pharmacological can molecular techniques.

An alternative approach:

In vitro model using a multicell insert.
Have an extracellular matrix, on top of that are seeded cells: Extravillous trophoblast.
A stimulus can then be put into the lower chamber and if this stimulates the trophoblast, it may stimulate the trophoblast to migrate through the matrix.

Using a combination of approaches, we have identified a number of factors that regulate trophoblast invasion:
hCG
EGF
HGF
IGF-I/II

Question 15

What factors influence trophoblast invasion?

Answer 15

Trophoblast and cancer cells have mechanisms of invasion in common
However trophoblast invasion is tightly regulated unlike metastasis. There are both positive and negative regulators of trophoblast invasion.

Positive regulators of trophoblast invasion:
Growth factors and cytokines
HGF
IGF-1
Prolactin
Matrix proteases
MMP-2, 9, 10, 12 – that will digest the extracellular matrix

Negative regulators of trophoblast invasion:
Tissue inhibitors of matrix metalloproteinases
Inhibitory cytokines and growth factors:
TNF
TGFβ
IGFBP-1

Upsetting the balance between stimulatory and inhibitory factors can lead to pregnancy complications

Question 16

Major source of factors that regulate trophoblast invasion are the maternal immune cells

Answer 16

Immune cells in decidua basalis
70% uterine natural killer cells (uNK cells)
20% are macrophages

They are recruited following implantation
They localise to maternal spiral arteries
They precede the invasion trophoblasts
They secrete factors that regulate trophoblast invasion that have two roles:
1) to prepare the spiral artery for the invading trophoblast
2) to release chemokines that will attract the invading trophoblast.

Question 17

Failure of placental development

Answer 17

Failure of the placental to develop normally can lead to common pregnancy disorders such as early pregnancy loss, pre-eclampsia and fetal growth restriction.

Failure to thrive in utero has lifelong consequences including increased risk of developing hypotension and diabetes

Question 18

Overview of extravillous trophoblast function

Answer 18

Early in pregnancy cells of the placenta (extravillous trophoblasts) invade the decidua
They migrate from the anchoring villi into the decidua
They will form plugs that block the maternal spiral arteries
These two functions lead to two different phenotypes:
Interstitial extravillous trophoblasts
Endovascular extravillous trophoblasts

The invading interstitial extravillous trophoblasts migrate towards the spiral artery and will interact with the vascular smooth muscle cells, line the vessels, and replace the cells of the vessel wall

The endovascular extravillous trophoblasts will initially plug the spiral arteries and will then migrate down the lumen of the vessels interacting with the endothelial cells, ultimately will lead to their loss

The physiological reason for this interaction is that the spiral arteries under normal circumstances will respond to vasoconstrictors and dilators, these arteries will be under control of the maternal needs. By removing the ability of these vessels to contract, the foetus removes the ability of the mother to regulate the flow of blood into the intervillous space.
Ultimately this will result in increased blood flow to the developing baby

Question 19

Spiral artery remodelling

Answer 19

In a non pregnant state they have a diameter of 200um. They consist of an endothelial cell lumen, surrounded by both connective tissue (elastin and/or collagen) and then surrounded by smooth muscle cells.

Following the remodelling process, interaction with trophoblasts and the stimulation by pregnancy related hormones:

The diameter of the vessel increases significantly (2mm diameter).
This results in a change of system from a low flow high-resistance vessel -> High flow, low resistance vessel.

There are two phases of spiral artery remodelling:
Trophoblast independent phase
Maternal Immune cells (specifically the uterine natural killer cells (UNK))
Pregnancy hormones (Such as progesterone)

Trophoblast dependent phase

We know about the trophoblast independent phase by studying ectopic pregnancies, as when the blastocyst implants in a place other than the uterine wall, it still has spiral artery remodelling.

Question 20

How are the vascular cells lost from spiral arteries?

Answer 20

Mechanisms from the contractile phenotype of smooth muscle cells to a vessel that is highly modified with loss of smooth muscle cells and endothelial cells. These are replaced by invading foetal cells (invading Extravilous trophoblasts).

Some of the influences are:
dNK cells
dMacro (decidual macrophages)
Stromal cells (within the decidua)
EVT (Extravilous trophoblasts)

The extravillous trophoblasts will replace both the endothelial and smooth muscle cells within the wall of the spiral artery.

Mechanisms that could be involved:
Migration – cells could induce vascular cells to migrate away from the vessel
De-differentiation – the trophoblasts could induce de-differentiation of both the smooth muscle and endothelial cells.
Loss of adhesion (Anoikis) – Anoikis is a form of programmed cell death.
Vascular cell apoptosis

The hypothesis is that:
Extravillous trophoblasts in uterine spiral arteries bring about changes leading to the loss of vascular cells - crucial for the vascular remodelling

Mechanisms of vascular cell loss
Vascular cell apoptosis
Migration
Dedifferentiation
Loss of adhesion

Question 21

What is apoptosis?

Answer 21

Programmed cell death
Cell death without the inflammatory response

Question 22

Apoptosis is characterised by distinct morphological changes, what are they?

Answer 22

Characterised by distinct morphological changes:
Cell shrinkage
Chromatin condensation
DNA fragmentation

Question 23

Apoptosis is characterised by distinct biochemical changes

Answer 23

Cleavage of lamins and actin filaments in the cytoskeleton
The breakdown of chromatin in the nucleus leading to nuclear condensation
Translocation of phosphatidylserine to outer membrane
Cleavage of key enzymes such as poly ADP ribose phosphate (PARP) involved in DNA repair

Question 24

What is apoptosis induced and mediated by?

Answer 24

Induced by cellular stress such as nutrient deprivation, hypoxia and viral infection

Mediated by a family of enzymes called caspases. Although caspase independent apoptosis does occur

Question 25

Can trophoblasts induce apoptosis in vascular cells?

Answer 25

YES – Trophoblasts can induce apoptotic changes in both endothelial and vascular smooth muscle cells

Experiment co cultured endothelial cells and vascular smooth muscle cells with trophoblasts.
Camera takes pictures every 15 mins to create kinetic curves
With time there is an increase in the amount of apoptotic cells present in both (inc control) but there is significantly more apoptosis in the presence of trophoblasts. This indicates that trophoblasts are secreting factors which can induce apoptosis in vascular endothelial cells and in vascular smooth muscle cells.

Question 26

De-differentiation and migration

Answer 26

Experiment looking at the effects of the interaction trophoblasts with vascular smooth muscle cells.
Smooth muscle cells secrete a factor (chemokine) that attracts trophoblasts.
This indicates that trophoblasts and endothelial cells interact.

Question 27

What is the nature of the factors produced by trophoblasts?

Answer 27

There are a family of cytokines that induce apoptosis. This consists of members of the TNFa family.
Experiment looked to identify whether a particular member of the TNFa family is involved in trophoblast induced vascular cell apoptosis.
Molecule called FasL binds to Fas on either the endothelial or vascular smooth muscle cells
In the presence of the inhibitory antibody NOK2, there is a reduction in the ability of trophoblasts to induce both endothelial cell and vascular smooth muscle apoptosis.
Shows that Trophoblasts use Fas/FasL to induce apoptosis in EC and VSMC

Question 28

Can the factors produced by trophoblasts be replicated in vivo?

Answer 28

An experiment carried out on an in vivo model of human placentation.
They took placental villi and transplanted them into the fat pads of a strain of mouse called Scid mice. These mice have a defective immune system so they will not reject the human placental villi.
Initially you can the see the interaction between the EVT and the endothelial cells.
They used a tunnel assay to detect apoptosis (one of the features of apoptosis is the cleavage of DNA) the ends of the fragments of the DNA can be detected.
Showed that the data found in vitro can be to some extent be replicated in an in vivo model of vascular remodelling.

The data shown above suggest that vascular cell apoptosis, induced by invading EVT may play a role in the remodelling process. Will now show how vascular cell de-differentiation is involved.

Question 29

Vascular smooth muscle cell de-differentiation: Modulation of VSMC phenotype

Answer 29

Unlike many cells, vascular smooth muscle cells have a plastic phenotype. They can move from a contractile phenotype (seen in a normal functioning vessel) to a de-differentiated non-contractile phenotype (seen primarily in pathological conditions)
This switch from a differentiated to a de-differentiated phenotype is stimulated by a number of factors:
TGFb Transforming growth factor-β will a push a cell towards the differentiated phenotype (differentiated SMC) which are:
Non-migratory
Low growth rate
Primary function is to carry out contraction

PDGF-BB Platelet-derived growth factor –BB will push a cell towards the de-differentiated phenotype (dedifferentiated SMC) which are:
Migratory
Proliferative
Produce and secretes extracellular matrix components
This change in phenotype is characterised by loss of contractile proteins: α-smooth muscle Actin and Smooth muscle Calponin.

Question 30

Is there any evidence for de-differentiation in VSMC in a remodelling spiral artery?

Answer 30

In a non-remodelled spiral artery there is a tight ring of vascular smooth muscle cells in the vessels.
However in a spiral artery undergoing remodelling, even though the muscle cells are still present, they are much more loosely packed with gaps in the circumference of the vessels:

This suggests that in vivo there is some evidence for vascular smooth muscle cell de-differentiation

In vitro: trophoblast conditioned media containing factors secreted by trophoblasts is used to stimulate smooth muscle cells causing a dose dependant drop in the expression of smooth muscle actin and also calponin, suggesting that de-differentiation is taking place.

One of the markers of de differentiation is that they are more motile, in this experiment, there is also a Dose dependant rise in cell motility

Question 31

Trophoblast dependent remodelling

Answer 31

In order to examine the interaction between trophoblasts, vascular smooth muscle and endothelial cells in more detail:
There is production of trophoblast conditioned media using a 3D culture of trophoblasts.
This trophoblast media was then used to stimulate a 3D culture model where both endothelial cells and smooth muscle cells are co-cultured together in a hanging drop system.
This allows the cells to form without adhering to a plate.
When they are grown like this, the endothelial cells migrate to the outside of the small ball of cells that form, this forms an inside out lumen.
If these spheroids are then stimulated with the trophoblast conditioned media, and a gene array is performed, you can then identify which genes are up/down regulated in response to stimulation by trophoblasts.

Question 32

Trophoblast dependent remodelling- genes increased and involved in smooth muscle cell dedifferentiation:

Answer 32

PDGF
KLF4 – A TF which is expressed only in VSMCs which are undergoing de-differentiation
CXCL10
IL6
ALL ABOVE are involved in VSMC dedifferentiation
MMP10 – A matric metalloprotease = Matrix degradation and possibly elastin derived peptides which stimulates trophoblast invasion. SO… trophoblasts will stimulate VSMC to produce MMP10 to breakdown the matrix that forms the vessel wall, and in addition to reducing the structure of the vessel, they may also have an effect in recruiting more trophoblasts to the remodelling vessel as elastin is a chemoattractant.
IL-8 – Is known to act as a chemokine to trophoblasts to stimulate trophoblast invasion.
IL-11 – Regulates VSMC phenotype
CCL20 – Chemokine

Question 33

If you look at all the genes altered in response to trophoblast conditioned media, you can see a number fall into categories that may be involved in vascular remodelling

Answer 33

Gene ontologies:
Blood vessel morphogenesis
Inflammatory response
Angiogenesis
Blood vessel development
Vasculature development
Response to stress

Question 34

Working model

Answer 34

In conclusion we have a working model of how we think spiral arteries are remodelled by trophoblasts:
Initially there will be communication between the vascular wall and the trophoblasts.
It is likely that the trophoblasts will stimulate vascular smooth muscle cells to release factors that will begin to recruit more trophoblasts.
We know that trophoblasts can induce vascular cell apoptosis, both smooth muscle cells and endothelial cells. (fas/fasL interactions are important for this)
We know that endovascular trophoblasts will interact with the vascular endothelial cells primarily and the interstitial trophoblasts will produce factors that interact with the vascular smooth muscle cells.
We know that trophoblast conditioned media stimulates the release of a number of factors including PDGF which is important for VSMC differentiated state (pushing into a more de-differentiated motile state). It also stimulates the realise of MMPs which will have two functions to break down the extracellular matrix in the vessel wall and to synthesise elastin derived peptides which are chemoattractant for trophoblasts.

Question 35

brief summary

Answer 35

Trophoblast invasion and spiral artery remodelling (SpA) requires the coordinated interaction between the fetal cells of the placenta and maternal cells of the decidua.
SpA are primed for remodelling by maternal immune cells
Remodelling requires vascular cell loss and/or changes in phenotype
Trophoblasts replace the vascular cells leading to increased blood flow to the fetus.