what is a pluripotent cell defined as?
a cell which can generate any cell of the body (including germ cells)
what is a stem cell?
A cell which can self renew (in response to a particular signal), and can also differentiate to give rise to a variety of cell types (according to differentiation signals)
are embryonic stem cells pluripotent?
yes - early embryonic stem cells are pluripotent but begin to lose pluripotentcy/differentiate during gastrulation
what are the defining features of pluripotent stem cells?
- expression of pluripotency factors (nanog, sox2, oct4)
- teratocarcinoma formation
When does teratocarcinoma formation occur?
when pluripotent stem cells are grafted onto a mouse kidney- tumour forms containing all cell types
what are the pluripotency factors?
Nanog, sox2, oct4
-these 3 transcription facotrs in combination define pluripotent SCs
at what stage is the inner cell mass pluripotent?
E4.0 - inner cell mass contains PCs surrounded by trophectoderm
why are (human) embryos difficult to study?
- small number of cells
- develop in utero
- ethical issues
what critical signals maintain cells in a self renewing, undifferentiated state?
FGF2 and TGFb (human)
BMPs (mouse)
how are pluripotent cells captured?
- plate inner cell mass cells on layer of feeder cells (support ES cell growth - iradiated stromal cells)
- once divided a couple of times-disaggregate ESCs and replate
- get ES cells to permantly express reporter transgene e.g. GFP(so ES cells can be seen)
- critical signals (TGFb and FGF2) added to maintain in proliferative state
what is a chimeric mouse?
A mouse composed of cells with two different genotypes
- an individual derived from 2 different genotypes
e. g. mouse ESCs can be reintroduced into normal blastocyst and contribute to normal development - can be followed as are GFP labelled
how can adult somatic cells be reprogrammed to a pluripotent fate?
- introduce pluripotency factors (sox2,nanog,oct4 and cmyc)
- c-myc-proliferation
- if cultured in right environment with right factors - can resemble pluripotent cells
what are the two approaches to get in vitro differentiation?
1) 3D approach
2) 2D/adherent approach
whats happens in the 3D approach to get in vitro differentiation (and give advantages and disadvantages)?
- signals removed which keep cell in undifferentiated state
- cells grown in aggregates/embryoid bodies
- embryoid bodies contain many cell types
- advantage - more accurate representation of invivo differentiation (cell-cell interactions)
- disadvantage - difficult to observe and dissect
how is Wnt signalling labelled in embryoid bodies?
Axin:Lacz (when Wnt signalling, axin is active-lacz expressed)
- show wnt initiates formaiton of organoids/mini organs
- heart cells from EBs can beat - showing functionality
how can it be confirmed that a cell has differentiated into the correct target cell type?
- is expression of the correct markers
- has functionality (e.g. heart cell - has cardiac AP/ can beat)
what happens in the 2D/adherent approach to get in vitro differentiation (and give advantages and disadvantages)?
- plate defined number of ESCs on right substrate/ECM
- remove signals which keep ESCs in undifferentiated state
- grow in defined medium with appropriate amounts of a signal
advantage- can test roles of specific signals
disadvantage - loss of cell/cell contact/communication which may occur in vivo
what is an early mesodermal marker?
T(bra)
what happens in microcephaly?
- small brain
- get seizures - neurological defects
- autosomal recessive (CDKsrap2) gene)
what was shown when a skin fibroblast was taken from a microcephaly patient?
- skin fibroblast taken and intorduced to pluripotency factors - becomes pluripotent (iPS cell)
- was shown that patient iPS cells produced smaller cerebral organoids on a dish with less neural progenitors
what virus is associated with mucrocephaly?
- Zika virus
- when cells exposed to zika virus cerebral organoids were smaller
- zika virus increased cell death (marked by cas 3 and labelled zika virus)
- death of cells blocked by emricasan - prevented small organoids / microcephaly phenotype
what is an embryonic stem cell?
a pluripotent stem cell which is derived from the inner cell mass of a blastocyst at an early stage - pre-implantation
give some neural disease modelling problems
- some diseases have multiple genetic causes
- complex phenotypes
- late onsets
- lack of efficient differentiation protocals
what has cell replacement been used to try to treat?
parkinsons (tremors, shaking, stiffness)
- lack of dopinergic neurons in substantia nigra
- TH- tyrosine hydroxylase - enzyme derived in dopinergic synthesis
- hES cells derived from MDA neurons
- transplant hES stem cells into a mouse - model of parkinsons- evidence for improved motor function
