10. Neurodegeneration: Pluripotent cells to Model Disease Flashcards
What is the definition of pluripotency?
Ability to generate all cells of an embryo including the germ cells
What is the definition of a stem cell?
Stem cells have the capacity to:
- Generate themselves for an indefinite number of generations
- Differentiate into a specialised cell type
Where do stem cells reside?
Stem cells reside in a stem cell niche:
- In niche, stem cells receive signals that promote self-renewal
- When stem cells leave niche, they receive signals that promote differentiation
What are pluripotent stem cells used for?
To recreate the events in early embryonic development in vitro
When are mouse embryonic cells pluripotent?
What happens at E6.5?
Mouse gestation lasts 19 days
- Between E3.5 and E7.5 embryonic cells are pluripotent
At E6.5 embryo undergoes gastrulation to generate 3 germ layers that will form all cells of the body
- Ectoderm - Nervous system, Skin
- Mesoderm - Heart, Muscles, Blood
- Endoderm - Gut
What is the descriptive defining feature of pluripotency?
Expression of pluripotency factors that are only expressed by pluripotent cells (Nanog, Oct4, Sox2)
- Can be identified using in situ hybridisation - expression restricted to inner cell mass
What is the functional defining feature of pluripotency?
- Pluripotent cells can be grafted onto the kidney of host mouse and will form a teratocarcinoma (tumour containing cells from all germ layers)
- Non-pluripotent cells do not form teratocarcinomas but instead form mass of cells that reflect the cell of origin
Why are embryos difficult to study?
What can offer a solution?
- They have small cell numbers and ethical issues
- Capture of pluripotent cells in vitro
How are embryonic stem (ES) cells obtained?
- Microdissect inner cell mass of an embryo and plate ES cells on layer of feeder cells (irradiated stroll cells from later stage embryo) which support ES cell growth
- After ES cells divide few times, disaggregate and re-plate (can identify ES cells from feeder cells by making ES cells express GFP transgene)
- Alternatively critical signals that maintain ES cells in self-renewing state can replace feeder cells (mouse = Leukaemia Inhibitory Factor, BMP / human = FGF2, TGFb)
Why are ES cells pluripotent?
- They express pluripotentcy factors (Nanog, Oct4, Sox2)
- They form teratocarcinomas when grafted onto kidney of host mouse
What is an additional test to show mouse ES cells are pluripotent?
Chimera formation
- Mouse ES cells can be reintroduced into normal blastocyst and contribute towards normal development
What is tetraploid complementation?
- ES cells can be labelled with GFP and then injected into an embryo that has had its inner cell mass removed
- Generate a transgenic animal, in which all cells express GFP
How are induced pluripotent stem (iPS) cells obtained?
- Take somatic cell from adult (e.g. skin fibroblast)
- Cell treated with reprogramming factors (c-Myc, Oct4, Sox2, Klf4)
- Cell is reprogrammed to generate an iPS cell
Why are iPS cells pluripotent?
- They express pluripotentcy factors (Nanog, Oct4, Sox2)
- They form teratocarcinomas when grafted onto kidney of host mouse
How can pluripotent stem cells be used to recapitulate normal embryonic development?
- Remove signals that maintain stem cells in self-renewing state
- Provide signals that promote differentiation to a specific cell lineage
How are target cell types defined?
- Expression of specific markers
- Functionality (e.g. if neurone fires action potential, if cardiomyocyte beats)
What is the 3D approach to in vitro differentiation?
- Remove signals that maintain stem cells in self-renewing state (mouse = LIF, BMP / human = FGF2, TGFb)
- Grow cells in aggregates in presence/absence of signals
- Cells self-organise into embryos bodies which contain many different cell types
What are the advantages and disadvantages of the 3D approach to in vitro differentiation?
+ Recapitulates embryonic development more accurately
+ Can study how different cell types interact with one another
- Hard to dissect the role of individual signals
How can embryoid bodies be used as reporter lines?
- In embryo, see expression of the Wnt-signalling reporter (Axin2:lacZ) in the primitive streak (posterior)
- 3 days following removal of LIF and BMP, embryoid bodies express Axin2:lacZ in a posterior region
- Cells can generate anterior-posterior structures in embryoid bodies
What is an alternative method to generating embryoid bodies?
- Cells can generate organoids - similar to embryoid bodies but directed to generate 1 cell type
- Cells grown in aggregates in presence of signals that promote generation of a single germ layer
- Cells self-organise into organoids that resemble the in vivo organ
- E.g. cerebral organoids have differentiated neurones in outer layer and neural progenitors in inner layer
What is the 2D approach to in vitro differentiation?
- Plate defined number of cells on correct ECM
- Remove signals that maintain stem cells in self-renewing state (mouse = LIF, BMP / human = FGF2, TGFb)
- Grow cells in defined medium with appropriate amounts of signals (Wnt, FGF)
What are the advantages and disadvantages of the 2D approach to in vitro differentiation?
+ More tractable system (e.g. for live imaging)
+ Easier to study role of individual signals
- Loss of cell interactions that occur in vivo
What is a use of 2D approach to in vitro differentiation?
Follow differentiation
- Can follow differentiation with live imaging
- Pluripotent stem cells contain transgene in which GFP is expressed under control of Brachury promoter (mesodermal marker)
- When ES cells differentiate into mesodermal cells and Brachury expression is activated, cells begin to express GFP
What is the method for using iPS cells for disease modelling?
- Obtain biopsy from patient and isolate skin fibroblast
- Cell treated with reprogramming factors (c-Myc, Oct4, Sox2, Klf4)
- Cell is reprogrammed to generate an iPS cell
- iPS cell then cultured in conditions that promote differentiation into cell types affected in disease of interest
What is microcephaly?
- A neurodevelopmental disorder, in which infants are born with abnormally small brains
- This results in severe neurological deficit and seizures
What was done in Lancaster et al (2013) study of microcephaly?
What were the results?
- Took skin fibroblast from a carrier of a mutation in CDK5RAP2 gene (causes microcephaly) and treated with reprogramming factors (c-Myc, Oct4, Sox2, Klf4) to generate iPS cells
- Generated cerebral organoids to recreate brain development in microcephaly
- Looked for expression of DCX (neuronal marker) and Sox2 (neural progenitor marker) in cerebral organoids generated from control iPS cells and patient-derived iPS cells
- Patient-derived organoids showed decreased expression of Sox2 suggesting fewer neural progenitors
Suggests few neural progenitors can cause microcephaly
What was done in the study of microcephaly following Zika Virus outbreak?
What were the results?
Generated cerebral organoids from human ES cells in the presence and absence of Zika Virus
- In presence of ZKV cerebral organoids were smaller (as seen in microcephaly)
- Also evidence of cell death in presence of ZKV as cell pinches at edge of organoid
Then analysed organoid expression of CAS3 (apoptosis marker)
- In presence of ZKV there is increased cell death
What were the results of the drug screen on ZKV organoids?
Found that emricasan (a drug that blocks apoptosis) prevented the increased cell death caused by ZKV infection
What are the challenges associated with modelling disease with pluripotent stem cells?
- Some diseases have multiple genetic causes ie cannot be pinned down to a single gene (e.g. Autism)
- Some diseases involve complex phenotypes involving interactions of many cell types (e.g. Cleft palate)
- Some diseases develop in later life (e.g. Parkinson’s) but pluripotent cells recreate events of embryonic development
- Lack of differentiation protocols for some cell lineages (e.g. Blood)
How can cell replacement be used to treat Parkinson’s?
- Parkinson’s is a neurodegenerative disorder characterised by progressive loss of dopaminergic neurones in the substantia nigra
- Protocols have been developed to differentiate human ES cells into dopaminergic neurones (involves culturing with Shh)
- These neurones express the correct markers (e.g. Tyrosine Hydroxylase - required for dopamine synthesis)
- When transplanted into mouse model of Parkinson’s they promote regeneration of dopaminergic neurones and an improvement in motor function
What are the challenges associated with cell replacement using pluripotent stem cells?
- Use progenitors or differentiated cells fro transplant?
- Chance of immune response/tumour formation
- What is required: replacement or regeneration?
- Positional identity - e.g. current protocols can only generate neural progenitors of anterior CNS - can these promote functional recovery of lower spinal cord injuries?
