Placentation & the Trophoblast I

Question 1

What is the placenta? (9)

Answer 1

• 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
• Acts as an endocrine organ releasing hormones into the maternal circulation such as hCG and progesterone

semi-allograft = the cells have genetic material from both the mother and the father

In a human pregnancy, fetal cells are in direct contact with maternal blood

This requires mechanisms to evade the maternal immune system

However the fetal and maternal circulations do not mix

Question 2

Placental development (5)

Answer 2

1) blastocyst attaches to and adheres to teh epithelial layer of the wall

2)Trophoectoderm proliferates and fuses to form a primitive syncytium
(PS) beneath the implanted embryo

3)TC migrate or invade into the decidua

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

5)Cytotrophoblasts proliferate and migrate through the syncytium to form the anchoring villi

Question 3

The Placenta -Villous (5)

Answer 3

• Highly branched with a large surface area for exchange
• Outer layer of fused cells- the syncytium
• Underlying cytotrophoblast stem cells
• Diffusion distance to vessels small
• Growth is regulated by a number of factors including IGF I and

Question 4

Villous structure - spiral arteries (5)

Answer 4

As the placenta develops, the villi start to branch to form = secondary + tertiary villus

within them, there are blood vessels that supply placenta from foetus + exchanging respiratory gases + nutrients from mother and delivered back to foetus

Spiral arteries are veins that help drain the blood from teh interspace back into the maternal circulation

vessels are located very close to the surface of the line = enables effective + rapid exchange of material

also contain macrophages - not sure why but maybe immune protection of the placenta or reg. + form. of the branching vessels

Question 5

Formation of the syncytium-MoA (6)

Answer 5

formed by combined action of hCG + Syncytin 1/2

1) hCG binds to Lhr/CGR = cAMP
2) cAMP membrane protein (scramblase)
3) cAMP inc. PKA activity = phospory. GCM1
4) GCM1 (TF) move to nucleus + reg. Syncytin 1/2
5) syncytin 1/2 transported to plasma membrane = induce self fusion + form. syncytium
6) constant regeneration of syncytium = bind w/ trophoblasts = growth, repair + formation

• 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

Question 6

Trophoblast differentiation and function

Question 7

Extravillous cytotrophoblast invasion

Answer 7

1) cells move into column + undergo epithelial mesenchymal transition (cells lose polarity+ adherence = more motile/invasive)

2) start to express diff. cell surface markers (α6β4, αVβ6 and E cadherin)

3) as the cells start away from cell column: start to express more markers (αVβ3, α1β1, VE-cadherin, VCAM-1, and PECAM-1)

Question 8

What is the purpose of the trophoblast plug? Oxygen tension and gestational age (6)

Answer 8

REDUCES AMOUNT OF O2 THT VILLU STISSUE EXPOSED TO IN EARLY GESTATION

• Up until 12th week the uterine spiral arteries are plugged with trophoblasts
• Placental development therefore occurs under relative hypoxia 2-3% O2
• While the spiral arteries are plugged nutrition is histiotrophic nutrients being secreted by the glandular cells
• Following dissolution of the trophoblast plug the placenta switches to haemotrophophic nutrition

Low oxygen early in pregnancy is important normal pregnancy progression
Prolonged low oxygen leads to placental pathologies

Question 9

Methods used to study human placental development (9)

Answer 9

Human studies are limited for ethical reasons!!!!!!!

Animal models:
There are significant differences in the placental development b/w mammals

• Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood
• However there is no deep interstitial invasion of the decidua
• Mice do not exhibit the same obstetric complications as humans
• Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood
• Deep interstitial invasion of the decidua does occur
• Some evidence that they do exhibit the same obstetric complications as humans
• Ethically unacceptable to experiment on these animals

Question 10

In vitro methods used to study human placental development- benefits (3)

Answer 10

• Human tissue can only be obtained either in the first trimester from TOPs or at term
• Trophoblast cell lines derived from choriocarcinomas JEG3, Jar and BeWo
  – grow well
  – have lost some characteristics
• Developed following transfection with oncogenes such as t- and T-antigen of SV40 or more recently hTERT
  – grow well
  – have lost some characteristics but this can depend on how they are cultured

Question 11

Initial promise -In vitro methods (2)

Answer 11

Human embryonic stem cell-derived trophoblast cells (hESCs)
– characterisation has proved problematic - illegal

however:
* 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
– difficult to prepare and grow

Question 12

What is a common problem w/ cell cultures? (2)

Answer 12

The phenotype of all cells grown in vitro will be dependent the culture conditions

“all models are wrong, but some are useful“ – George Box

Question 13

Culture formats (6)

Answer 13

Simple mono layer cultures

Simple co-cultures - (mixing 2 cell types + growing in monolayer or eparet using multi cell insert)

Addition of extracellular matrix (in presence/absence of matrixes)

Effect of flow (grow cells + look at effect of flow)

3D environment (steroid, perfusion )
- organoid culture - isolated but tissue is reconstructed

Question 14

What regulates trophoblast invasion? (look at ex-vivo and in-vivo) (5)

Answer 14

Ex vivo: look at 1st trimester placental tissue
1)anchoring villus identified + dissected - placed on extracellular matrix
2)migration of cells from tissue - monitored by timelapse 3)microscopy/photography

In-vivo: uses inserts - extracellular matrix + trophoblast cells +stim. in lower chamber
1) if stim. trophoblasts, they may stim. through matrix

Question 15

What factors influence trophoblast invasion? (7)

Answer 15

-Trophoblast and cancer cells have mechanisms of invasion in common
However trophoblast invasion is tightly regulated, unlike metastasing cancer (both +ve + -ve reg.).

-Growth factors and cytokine: HGF, IGF-1, Prolactin (+)

-Matrix proteases: MMP-2, 9, 10, 12 (+)

-Tissue inhibitors matrix: metalloproteinases (-)

-Inhibitory factors: TNF, TGFβ, IGFBP-1(-)

Upsetting the balance can lead to pregnancy complications.

Question 16

A major source of factors that reg. trophoblast invasion = maternal immune cells (5)

Answer 16

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

-recruited following implantation
- localise to maternal spiral arteries
- precede the invasion trophoblasts
- secrete factors that regulate trophoblast invasion (to repair spiral arteries from invading trophoblasts + release chemokines to attract invading trophoblasts)

Question 17

Placenta - what happens if goes if doesn’t dev. normally? (2)

Answer 17

• 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 (3)

Answer 18

• early in pregnancy cells of the placenta (trophoblasts) invade the
  decidua (from villus) = institial trophoblast = migrate towards spiral arteries + interact with and replace vascular smooth muscle cells
• they plug the maternal spiral arteries = endovascular trophoblast = plug spiral arteries, migrate down the lumen of vessels = interact w/ endothelium cells = their loss
• ultimately this will result in increased blood flow to the developing baby

Question 19

What are spiral arteries + how do they change? (2)

Answer 19

non-pregnant state - 200um: endothelial lumen. surrounded by connective tissue , surrounded by smooth muscle cells

pregnant state- (following remodelling) 2mm: goes from Low-flow to high-resistance vessel to High-flow, low-resistance remodelled vessel

Question 20

2 phases of Spiral Artery Remodelling (2)

Answer 20

• Trophoblast independent
  – Immune cell
  – Pregnancy hormones
• Trophoblast dependent - We know this by studying ectopic pregnancies

Question 21

How are vascular cells lost from spiral arteries? suggestions (4)

Answer 21

Endovascular trophoblast replaces endothelium + smooth muscle ells
cells can induce:
 Migration (away from vessel)
 De-differentiation (of both smooth muscle cells = loss)
 Loss of adhesion (=Anoikis - programmed cell death)
 Vascular cell apoptosis

Question 22

How are vascular cells lost from spiral arteries? Hypothesis (4)

Answer 22

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 23

Apoptosis (6)

Answer 23

• Programmed cell death
• Cell death without the inflammatory response
• Characterised by distinct morphological
  – Cell shrinkage
  – Chromatin condensation
  – DNA fragmentation
  – Membrane blebs and blisters
• Characterised by distinct biochemical changes
  – 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
• 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 occu

Question 24

Can trophoblasts induce apoptosis in vascular cells? - experiment (5)

Answer 24

YES! in both endothelial + Vascular smooth muscle cells

1) trophoblasts in insert + endothelium/vascular muscle cells grown on base of plate

2)camera take pics every 15mins

3)using these seq.’s = generate kinetics

w/time: there is an increase of apoptotic cells present in both compared to control - w/ endothelial being higher
= trophoblasts sec. factors inducing apoptosis

Question 25

Caspase use in apoptosis proof of trophoblasts (3)

Answer 25

1)checked after 36hrs + 60hrs
2) to confirm morphological changes = apoptosis, inhibitor of Caspase used (zVAD-non-specific)
3) if they are used - then should be detected and they were

Question 26

Cleavage as proof of Apoptosis

Answer 26

Cleavage of Harp at the end of apoptosis - seen on western blot to prove the apoptosis

Question 27

Interaction between trophoblasts w/ vascular smooth muscle cells

Answer 27

smooth muscle cells sec. a factor that attracts trophoblasts : can analyse persistence of movement, directionality of movement + speed of movement

Question 28

nature of factors produced by trophoblast - experiment (Fas/FasL) (4)

Answer 28

family of cytokines that induce apoptosis (including TNFalpha)
-testing to see if member of family is involved in this apoptosis

1) trophoblasts in insert + endothelium/vascular muscle cells grown on base of plate

2) NOK2 (inhib.) binds to FasL = prevents Fas activation

3)= sig. reduction in ability to induce both cells + to lesser extent

Question 29

Trophoblast-induced apoptosis - in vivo co-culture model (4)

Answer 29

Red-Horse et al. 2006. Cytotrophoblast induction of arterial apoptosis and
lymphangiogenesis in an in vivo model of human placentation.

1) Transplanted placental villi into the mammary fat pads of Scid mice for 3
weeks.

2)CK+ve TC invaded and interacted with vessels.

3)Induction of vascular cell apoptosis in vivo and in vitro co-cultures

Question 30

Modulation of VSMC phenotype (De-differentiation): (8)

Answer 30

smooth muscle cell shave a plastic phenotype = move from contractile to noncontractile (path. conditions)

stim. by no. of factors: TGFbeta + PDGF-BB

TGFbeta: Differentiated SMC
* Non-migratory
* Low growth rate
* Primary function is to carry out contraction

PDGF-BB: Dedifferentiated SMC (loss in Actin + Calponin)
* Migratory
* Proliferative
* Produce extracellular matrix components

Question 31

Trophoblast dependent remodelling (5) -3D

Answer 31

1) Trophoblast conditioned media generated (3D)

2) Gene expression altered by trophoblast examined by Illumina Bead-
chip array

3) Control or conditioned media added to vascular spheroids for 24 hours

4) RNA extracted from vascular spheroids.

5)= no. of genes identified:
- PDGF, KLF4, CXCL10, IL-6 (Involved in VSMC dedifferentiation)
- MMP10 (Matrix degradation and possibly elastin derived peptides
which stimulate trophoblast invasion)
- IL-8 (Stimulates trophoblast invasion)
- IL-11 (Regulates VSMC phenotype)
- CCL20 (Chemokine)

Question 32

vascular remodelling - Gene ontologies (6)

Answer 32

Blood vessel morphogenesis
Inflammatory response
Angiogenesis
Blood vessel development
Vasculature development
Response to stress

Question 33

Working Model - remodelling (5)

Answer 33

1) communication w/ vascular wall + trophoblasts
2) wall releases factors to recruit more tropho.
3) dNK associated apoptosis (Fas/FasL)
4) interact with : either vascular cells or smooth muscle cells
5) release tropho. derived factors = dedifferentiation

Question 34

Summary (4)

Answer 34

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.