L4 - In Vitro Stem Cell Models Flashcards
Define pluripotent cell
Potency to be able to generate all of the cell types of an embryo including the germ cells
Define multipotent cell
Capable of differentiation into only a few lineages
What are the cells called that surround the inner cell mad
trophectoderm
Give examples of pluripotent markers expressed by pluripotent cells
Nanog
Oct4
Sox2
What are the two hallmarks of pluripotency
Descriptive and functional
Descritptive test for pluripoteny
Expression of various pluripotency markers (Nanog, Oct4, Sox2)
Functional test for pluripotency
Grafting of pluripotent cells onto a kidney of a host mouse will give rise to a teratoma
What cell types are containined in a teratoma
All of the cell types
3 types of ectoderm
Surface
Neural
Neural crest
What does the ectoderm go on to form
Skin
Nervous system
4 types of mesoderm
Axial
APraxail
Intermediate
Lateral plate
What does the mesoderm go onto form
Blood
Heart
Muscle
Kidney
Whar happens to potency as devlopment proceeds
Becomes restricited
What are used to mimich the niche in
MOUSE
HUMAN
Leukaeamia inhibitory facotr (LIF), BMP
FGF2 and TGFB
Describe one method of capturing ES cells
Obtain the cells of the inner cell mass and dissociate
Plate on a layer of feeder cells
Once eSCs have divided a few times then disaggregate and replate
What factors would be used to re-program an adult somatic cell to a pluripotent fate
Oct4
Sox2
Klf4
C-myc
Describe the gene expression in iPS cells
Express the main pluripotency markers (Nanog, Oct4 and Sox2)
No expression of any genes which are indictaive of differentiation
Describe some of the characteristics of iPS cells
Single cell able to generate many identical daughters (self-renewal)
Formation of teratoms in permissive environment
When reintroduced into an embryo they contribute to normal development
What is a marker expressed by neural stem cells
RC2
Describe a method for capturing multipotent stem cells
Dissociate cells
Plate on laminin in the presence of cytokines FGF2 and EGF
Describe the 3D method for in vitro differentiation
Remove signals keeping cells in an undifferentiated state
Grow in aggregates (embryoid bodies/organoids)
Cell have functionality and express the right markers at the right time dependent on their fate
Advantage of the 3D method of in vitro differentiation
Resembles embryonic development more closely
Disadvantage of the 3D method of in vitro differentiation
Diffiuclt to observe/dissect the roles of individual signals
Describe the 2D (Adherent) method of in vitro differentiation
Plate defined number of cells on the right substrate
Remove the signals keeping cells from differentiating
Grow in defined medium with appropriate levels of signals
Advantages (2) of 2D/Abberate in vitro differentiation
More traceable system for live imaging
Easier to diisect roles of specific signals
Disadvantage of 2D/Abberant in vitro differentiation
Don’t get same cell-cell interactions which would be seen in vivo
What is microcephaly
Neurodevelopmental disorder in which infants are born with an abnormally small brain
Why may in vitro differentiation be needed to model microcephaly
Mice models fail to replicate the condition
How may microcephaly be modelling in iPS cells
Take a skin biopsy and culture with Oct4, Sox2, Klf4 and myc to get to the iPS cells
Formation of a cerebral organoid
What can be seen when iPS cells are made to model microcephaly
fewer DCX neurones and fewer nerual progenitors
Potential applications of microcephaly models using iPS cells
Small molecule screens to identify suitable drugs
What is familiail dysautonomia
Genetic disorder affecting the development and survival of the neruones controlling involuntary actions
May also affect the NS in activites related to senses
Early symptoms of familial dysautonomia
Poor muscle tone Feeding difficulties Poor growth Lack of tears Frequent lung infections Difficulty maintaining body temperature
Late symptoms of familial dysautonomia
Delayed speech and walking Spinal curvature Reduced perception of pain/temp Poor growth UNstable BP
familial dysautonomia usually caused by a mutation in which gene
IKBKAP
Parkinsons affects how many
1:500
Symtpoms of parkinsons
Resting temor Slowness of movement Rigidity Dementia Anxiety
Parkinsons characterised by the …
Loss of mDA neurones of the substantia nigra
How can cell replacement be used in parkinsons
Generate h-ES derived mDA neurones
Transplant into mouse model
Evidence of improvement in motor function
What may be used as a marker in Parkinsons treatment
TH
Tyrosin hydroxylase - enzyme involved in the synthesis of dopamine
Cell replacement has also be used in the treatment of
AMD - replacing RPE cells
