Lecture 4 Flashcards
What are the best model organisms for studying embryonic stem cells and how can they be observed?
Mice- can be genetically modified and have a new born pup after 20 days. Oct4, nanog and SOX2 are used as pluripotent markers (descriptive test)
Describe a functional test for pluripotency
Transplant the cells into kidney in mouse= gives rise to teratocarcinoma if pluripotent, if not it only generates a small mass of cells
What happens to pluripotent stem cells in the embryo?
At E6 the embryo undergoes gastrulation and gives rise to:
1) ectoderm= neural and epidermis
2) Mesoderm= paraxial, axial, lateral, heart, muscle, bone, kidney
3) Endoderm= gut and internal organs
Pluripotency becomes extinct by E7
Why are stem cells studied in vitro?
Only a small number found in embryos- need to be captured and expanded in a dish. Also due to ethical reasons. Can be used for drug screenings and cell replacement therapies.
How are embryonic stem cells captured in vitro?
They are placed on feeders- cells that secrete factors to prevent the stem cells from differentiating (LIF and BMP= mice, TGFB and FGF2= humans). GFP is used to distinguish between the 2 cell types. Once they have divided a few times, disaggregate them and place in dish with BMP and LIF- the feeder cells are no longer needed.
How are adult somatic cells reprogrammed?
1) perform biopsy on skin fibroblasts
2) Ectopically overexpress Oct4, SOX2, c-Myc and KIF4 (pluripotent factors)= results in IPS cells that are pluripotent and resemble ES cells
What determines a stem cell (as in its features)?
If a cell can give rise to an identical daughter cell and if it expresses Oct4, SOX2 and nanog
How can multipotent stem cells be captured in vitro?
1) repress differential signals and promote self renewal signals
2) Dissociate brain cells and capture cells and grow on laminin with FGF2 and EGF cytokines
3) Can capture single cells that self renew and express RC2 marker
What markers indicate differentiated neural cells?
Neural= TUJ1 and glia= GFAP
What is 3D stem cell recapitulation and what are the pros and cons?
Is removing the self renewal signals and allowing the cells to differentiate on their own with/without signals to form organoids- forms 3D structure without signals
Pros= recapitulates the embryonic environment more accurately
Cons= difficult to direct observational microscopy and to dissect or observe signals
Give examples of 3D recapitulation
1) heart organoids are made which have a heart beat- derived from cardiomyocytes
2) cerebral organoids= resemble small brain with outer and inner neuronal precursors (expresses the right markers in the right place)
How is 2D in vitro differentiation carried out?
specific number of cells are grown in a defined medium on substrate/ ECM that mimics the environment. The self renewal factors are removed and the cells are grown in FGF and Wnt to promote differentiation into the 3 germ layers
What are the pros and cons of 2D?
Pros= more tractable system- can easily track signals cons= loss of cell interaction- doesn't really resemble what happens in vivo
What experiments can be done on 2D cells?
Have GFP marker for t(bra) which is an early mesoderm marker. Not all cells turn green- some differentiate and some don’t. Gives rise to many cell types (chaotic manner)
How can stem cells be used for disease modelling?
Microcephaly= very small brain- mouse KOs fail to replicate the system so it is difficult to study. Take skin cells from affected individual and unaffected sibling and reprogram into brain organoids to compare. Can look at markers- much less DCX (neural markers) and SOX2 (neural progenitors). Can perform a drug screen
What are the symptoms of familial dysautonomia?
Is an inherited disease mostly caused by mutations in the IKBKAP gene- effects the development of autonomous neurons
early symptoms= lack of tears, hypotonia, unregulated temperature, poor growth, frequent lung infections
late symptoms= problems breathing, delayed speech, spinal curvature, unstable BP, delayed walking, reduced pain perception
50% die by 40
How can familial dysautonomia be modelled?
Use remodelling by culturing neurons via reprogramming. Not many express the TUJ1 marker. Can be used for drug screen- TUJ1 cells can increase if kinetin added
What is Parkinson’s Disease?
Most common neurodegenerative disease (1/500). Is the degeneration of dopaminergic neurons in the substantia nigra.
symptoms= anxiety, dementia, shaking, rigidity, slow movement
What can be one of the treatments of Parkinson’s?
Can culture DA neurons in a petri dish that express the normal markers= Tyrosine hydroxylase (to make DA), FOXA2, LMX1A.
Inject into patient- found to be effective in mice
Can also be used to culture retinal cells to treat age related blindness
