Differentiation Flashcards
Define differentiation
The process where an specialised cells acquires specialised features and functions, and becomes committed, this makes it a differentiated cell
How can differentiation be triggered?
Removal of extrinsic factors which stimulate self-renewal
what are embryoid bodies?
heterogeneous aggregates of pluripotent cells that were initially obtained from embryonic carcinoma cells.
what can mediate axis formation and self-organisation in embryoid bodies?
- Wnt signalling
- shown by Axin-Lacz reporter
- can see loaclised areas of Wnt signalling in EB
- if add wnt inhibitor- dkk1 get opposite effect
Give pros and cons of embryoid bodies
Pros- cheap to produce and from 3 germ layers
- cons - difficult to control aggregation in a reproducible way
- number of days before have to be collected
what are different EB morphologies best at producing? e.g. cavities..
Hollow - best at producing endoderm
Bright cavity - 3 germ layers, best organised, closer to real embryo, faster differentiation
Dark cavity - good production of 3 germ layers
- EBs show variable differentiation potentials depending on size
What techniques were tried to try and control the reproducibility of EBs?
need to make sure EBs come from a single cell
- hanging drop method
- controlled aggregation
What happens in the hanging drop method?
1 cell in each drop, no contact with anything else - each drop produces an embryoid body
- used in mESCs
What happens in the controlled aggragation method?
Have a definitive number of cells in each well, is a similar size and shape per well (homogenous)- wells have special geometry
- tried with hESCs
What can we use to get directed differentiation?
- growth factors
- how much, conc, when, how long for, and combination added
- also ECM - some lineages differentiate with different components, e.g. laminin or collagen
How can desired cell types be isolated?
FACs
- sort for specific cell markers
- can also use density gradients - cells have different boyancies with different properties
(if direct differentation will still not get completely pure population - will still have a mixture of cells)
Give a method that was used to create a purified population of neural precursors from ES cells
- attach AB resistant gene, e.g. neo to makrer of neural precurosers (Sox2)
- when cells are cultured in a AB medium (G418), cells thsat express Sox2 will also express AB resistant gene (neo), so will survuve
- allows identification and isloation of Sox2+ cells - neural precursors
give the steps in development that can result in Bcells (pancreas dev) and the markers
- foregut patterning - Fox2a, Sox17
- pancreas specification - Pdx1
- budding - Ptfa
- branching - Ngn3
- B and A cell development - Ins and Gl expression
what happened when scientists tried to produce pancreatic hormone expressing cells (B cells) from hESCs the first time?
- Cells produced could secrete C-peptide (insulin precursor), but were only a small % of insulin expressing cells
- were not glucose responsive
- didnt maintain expression of B cell markers for long - more like fetal B cells
What happened the second time scientists tried to produce hormone expressing B cells from hESCs?
improved protocol - mini pancreases were transplantes into mice - got granules specifc to B cells - cells were glucose responsive in vivo (shows cells are functional in vivo)
