Modelling embryonic development and disease using pluripotent stem cells Flashcards
What are pluripotent cells?
Cells that can generate cells from all 3 germ layers, including germ cells
What are the germ layers- and give a couple of examples of cells generated from each
Ectoderm- CNS, PNS
Endoderm- gut, internal organs
Mesoderm- muscle, blood
Where are pluripotent cells found in the embryo? And what signals do they express?
The inner cell mass
Nanog, Oct4, Sox2
How do we test that a cell is pluripotent?
- Does it express pluripotent signals
- Graft cells onto the kidney of a host mouse- should prouce a teratocarcinoma- cancer comprised of cells from each germ layer
How and why do we produce iSC?
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
How do we capture pluripotency in vitro?
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
Examples of signals which keep cells in a self-renewing, undifferentiated state in mice and humans
Mice- leukaemia inhibitory factor, BMP
Humans- FGF2, TGFbeta
How do we know ESC are pluripotent?
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
What are two ways of developing differentiated cells in vitro?
3D approach and 2D/adherent approach
Describe the 3D approach and its advantages and disadvantages
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
Describe the 2D approach and its advantages and disadvantages
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
What are the two applications of in vitro cell engineering
Disease modelling
Cell replacement
What does disease modelling consist of ?
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
What is microcephaly ?
Neurodevelopmental disorder where the brain is abnormally small
Patients have neurological defects and experience seizures
The zika virus casues microcephaly
What are the challenges with disease modelling in this way?
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
What is trying to be done in terms of cell replacement and parkinsons?
Implanting reprogrammed stem cells into patients with parkinsons- to create new dopaminergic neurons etc
Not yet perfected
Drawbacks with cell replacement therapy
Immune response (avoided with iPSC)/ posible oncogenicity of grafted tissue do we use progenitor cells of differentiated cells
Which cells can we produce hESC from in the nervous stem?
Can produce hESC from neural progenitors of the anterior CNS but not any lower
