Modelling embryonic development and disease using pluripotent stem cells

Question 1

What are pluripotent cells?

Answer 1

Cells that can generate cells from all 3 germ layers, including germ cells

Question 2

What are the germ layers- and give a couple of examples of cells generated from each

Answer 2

Ectoderm- CNS, PNS
Endoderm- gut, internal organs
Mesoderm- muscle, blood

Question 3

Where are pluripotent cells found in the embryo? And what signals do they express?

Answer 3

The inner cell mass

Nanog, Oct4, Sox2

Question 4

How do we test that a cell is pluripotent?

Answer 4

1. Does it express pluripotent signals
2. Graft cells onto the kidney of a host mouse- should prouce a teratocarcinoma- cancer comprised of cells from each germ layer

Question 5

How and why do we produce iSC?

Answer 5

Why- so we can create clinically relavant populations of cells
How- take a biopsy (skin fibroblasts) -> introduce repogramming factors (Oct4, Soz2, cMyc) -> induction of pluripotency

Question 6

How do we capture pluripotency in vitro?

Answer 6

Take blastocyst- microdissect the ICM- plate it on a layer of feeder cells- provide with trophic factors found in its niche- once the cells have divided a few times- disaggregate and replate

Question 7

Examples of signals which keep cells in a self-renewing, undifferentiated state in mice and humans

Answer 7

Mice- leukaemia inhibitory factor, BMP

Humans- FGF2, TGFbeta

Question 8

How do we know ESC are pluripotent?

Answer 8

Can form genetic chimeras
mouse ESC can be introduced into a host blastocyst can can contribute to normal development- produce cells of all 3 germ layers

Question 9

What are two ways of developing differentiated cells in vitro?

Answer 9

3D approach and 2D/adherent approach

Question 10

Describe the 3D approach and its advantages and disadvantages

Answer 10

remove signals that keep the cells in an undifferentiated state
form embryoid body
ADV= more accurately capitulates embryonic development
DISADV= difficult to observe the role of single cells

Question 11

Describe the 2D approach and its advantages and disadvantages

Answer 11

plate a defined number of cells on the right subsrate/ECM
remove signals that keep the cells undifferentiated
ADVS= more tractable- can use live imaging, can make a more homogenous population of cells, can test the role of specific ligands
DISADV=loss of cell interactions that may occur in vivo

Question 12

What are the two applications of in vitro cell engineering

Answer 12

Disease modelling

Cell replacement

Question 13

What does disease modelling consist of ?

Answer 13

can make an organoid from diseased iPS cells
look at expression and cells in the organoid
can also make a healthy organoid and infect it with the disease- see what happens

Question 14

What is microcephaly ?

Answer 14

Neurodevelopmental disorder where the brain is abnormally small
Patients have neurological defects and experience seizures
The zika virus casues microcephaly

Question 15

What are the challenges with disease modelling in this way?

Answer 15

Usually multiple genetic caused behind a disease- autism etc
Complex phenotype- cleft palata (hard to see in an organoid)
Late onset- parkinsons etc
Lack of efficient differentiation protocols

Question 16

What is trying to be done in terms of cell replacement and parkinsons?

Answer 16

Implanting reprogrammed stem cells into patients with parkinsons- to create new dopaminergic neurons etc
Not yet perfected

Question 17

Drawbacks with cell replacement therapy

Answer 17

Immune response (avoided with iPSC)/ posible oncogenicity of grafted tissue 
do we use progenitor cells of differentiated cells

Question 18

Which cells can we produce hESC from in the nervous stem?

Answer 18

Can produce hESC from neural progenitors of the anterior CNS but not any lower