Lecture 3- Pluripotent stem cells Flashcards
what is a stem cell
a cell type that sits in a niche which keeps the cells in a self renewing state
when removed from the niche, they differentiate due to responding to a different set of signals
how do you capture pluripotent cells and grow them in a petri dish
1) add ES cells to a layer of feeder cells
2) once they have divided a few times, disaggregate and replace them so they maintain pluripotency
you need LIF, BMP(mice) or FGF2, TGFb(humans) to keep the cells in this state by blocking its ability to differentiate
what are feeder cells
irradiated stromal cells derived from later embryos which support ES cell growth
how can you test if cells are pluripotent
- must have Oct4, Nanog, Sox2
- single cell generates identical daughter cells
- can form teratocarcinomas
no expression of differentiation genes
describe the developing of iPS cells
force expression of reprogramming factors into adult skin cells which makes them pluripotent
describe 3D differentiation of pluripotent stem cells
remove signals that keep cell in undifferentiated state
grow in aggregates(organoids) in the presence or absence
what are the pros and cons of 3D differentiation
pro- accurate environment
con- difficult to observe individual signals
what are the different signals that keep cells in an undifferentiated state
mouse ES cells - BMP / LIF
human es cells - FGF2/ TGFb
describe 2D differentiation of pluripotent stem cells
-plate a defined number of cells on the right extracellular matrix
- remove signals that keep cells in undifferentiated
- grow in a defined medium with appropriate amounts of signals ( eg. WNT/FGF)
what are the pros and cons of 2D differentiation
pro- more traceable so can test for specific signal roles
con- loss of cell interactions that might occur in vivo
describe the hybrid approach for differentiation of pluripotent stem cells
-take ES cells and push back into undifferentiated state
-use signals to grow or express TFs
- make
Trophectoderm/epiblast/primative endoderm cells to mimic epiblast
- mix these in specific ratios and grow in 3D
what is the pro about the hybrid approach
can mimic processes before and after implantation
this allows us to study what goes wrong in failed pregnancies
what are the 2 main uses of differentiation of pluripotent stem cells
disease modelling
cell replacement
what is microcephaly
small brain
autosomal recessive
cant use animals to study as its very complex
how can you model microcephaly using pluripotent stem cells
- take mutant cells
- make iPScells
-make 3D organoids
what was found from the disease modelling of microcephaly
was found that fewer neural progenitors are found in patients organoids
this means they can do small molecule screenings of these cells to find suitable drugs
what is parkinsons
progressive loss of dopaminergic neurons
shown by tremor
how can cell replacement be used for parkinsons using pluripotent cells
-express TH ( the enzyme for synthesising dopamine) and then express this in human embryonic stem cells
how can we capture later stem cells in vitro
eg. in neural stem cells
- dissociate the cells
-plate on laminin in presence of FGF2 and EGF
- when you remove FGF2 ad EGF they then differentiate into glia and neurons
