Lecture 3 - Pluripotent stem cells and their applications Flashcards
How do we capture pluripotent cells in vitro embryonic stem cells ?
We use a blastocyst. These scientists took ut the embryo and replanted the ICM cells onto a dish. Put them on feeder (fibroblast) cells. The pluripotent cells glowed green and there is a difference in morphology. Feeder cells provided the tropic signals that keep the pluripotent cells.
ES cells expressing permanently display a transgene encoding green fluorescent protein (GFP)
This is growing pluripotent embryonic stem cells on a dish
Are Embryonic stem cells (ESCs) pluripotent ?
Yes
ES cells express the main pluripotency factors Oct4, Nanog, Sox2 (markers)
No expression of genes indicative of differentiation
A single cell can generate identical daughter cells (=stem cell)
ESC form teratocarcinoma when transplanted in permissive environments
Is it possible to reintroduce ESC into embryos ?
Yes. Take pluripotent stem cells and inject them back into the embryo.
How are adult somatic cells reprogrammed to a pluripotent cell fate ?
Adult cells (already differentiated) are extracted from the doner. Then reprogramming factors are used to allow an induction of pluripotency. This results in self-renewal creating IPS cells
Diffrentiated to Pluripotent
What is the 3D appraoch in in vitro differentiation ?
Remove self renewing signals that keep cells in an “undifferentiated” state
(e.g. BMP/LIF for mouse ES cells or FGF2, TGFb for human ES cells)
Removes signals that make the pluripotent cell specialize.
grow in aggregates (=Embryoid bodies OR organoids) in the presence or absence of signals
ADVANTAGE: recapitulates more accurately the embryonic environment
DISADVANTAGE: difficult to “observe”/dissect the role of individual signals
What is the 2D approach in in vitro differentiation?
Plate a defined number of cells on the right substrate/extracellular matrix
Remove signals that keep cells in an “undifferentiated” state
(e.g. BMP/LIF for mouse ES cells or FGF2, TGFb for human ES cells)
grow in defined medium with appropriate amounts of signals (e.g. FGF, WNT etc)
ADVANTAGE: more tractable system (e.g. for live imaging), easier to test the role of specific signals
DISADVANTAGE: loss of cell interactions that may occur in vivo
What is Microcephaly ?
Neurodevelopmental disorder in which infants are born with an abnormally small brain.
Due to various autosomal recessive mutations
Neurological defects, seizures
Mouse mutants fail to recapitulate the condition
Caused by a variety of autosomal recessive mutations, it is difficult to remodel this disease using animals, the brains are too different.
How can we capture multipotent stem cells ?
NSCs
Dissociate cells
Plate on laminin in the prescence of the cytokines FGF2 and EGF
NSC express undifferentiated markers (e.g.RC2)
No expression of genes indicative of differentiation (Flia= GFAP, Neuron= TUJ1)
A single cell can generate identical daughter cells (=stem cell)
What signals that keep the ESC cells in their niche?
FGF2 and TGFbeta
What gene causes microcephaly?
A Mutated Cdk5RAP2
What do you see in the skin cells of microcephaly patients?
Skin cells taken from patient
You will see fewer neural progenitors (less SOX2)
What is Parkinson’s disease?
Loss of dopaminergic neurones in substantial nigra
Symptoms - tremors slow movement etc
What is the 3D approach in vitro differentiation?
Remove self renewing signals that keep cells in an “undifferentiated” state
(e.g. BMP/LIF for mouse ES cells or FGF2, TGFb for human ES cells)
Removes signals that make the pluripotent cell specialize.
grow in aggregates (=Embryoid bodies OR organoids) in the presence or absence of signals
ADVANTAGE: recapitulates more accurately the embryonic environment
DISADVANTAGE: difficult to “observe”/dissect the role of individual signals
