10. Neurodegeneration: Pluripotent cells to Model Disease Flashcards Preview

BMS336 - Modelling Human Disease and Dysfunction > 10. Neurodegeneration: Pluripotent cells to Model Disease > Flashcards

Flashcards in 10. Neurodegeneration: Pluripotent cells to Model Disease Deck (31)
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1
Q

What is the definition of pluripotency?

A

Ability to generate all cells of an embryo including the germ cells

2
Q

What is the definition of a stem cell?

A

Stem cells have the capacity to:

  • Generate themselves for an indefinite number of generations
  • Differentiate into a specialised cell type
3
Q

Where do stem cells reside?

A

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
4
Q

What are pluripotent stem cells used for?

A

To recreate the events in early embryonic development in vitro

5
Q

When are mouse embryonic cells pluripotent?

What happens at E6.5?

A

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
6
Q

What is the descriptive defining feature of pluripotency?

A

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

7
Q

What is the functional defining feature of pluripotency?

A
  • 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
8
Q

Why are embryos difficult to study?

What can offer a solution?

A
  • They have small cell numbers and ethical issues

- Capture of pluripotent cells in vitro

9
Q

How are embryonic stem (ES) cells obtained?

A
  • 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)
10
Q

Why are ES cells pluripotent?

A
  • They express pluripotentcy factors (Nanog, Oct4, Sox2)

- They form teratocarcinomas when grafted onto kidney of host mouse

11
Q

What is an additional test to show mouse ES cells are pluripotent?

A

Chimera formation

- Mouse ES cells can be reintroduced into normal blastocyst and contribute towards normal development

12
Q

What is tetraploid complementation?

A
  • 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
13
Q

How are induced pluripotent stem (iPS) cells obtained?

A
  • 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
14
Q

Why are iPS cells pluripotent?

A
  • They express pluripotentcy factors (Nanog, Oct4, Sox2)

- They form teratocarcinomas when grafted onto kidney of host mouse

15
Q

How can pluripotent stem cells be used to recapitulate normal embryonic development?

A
  • Remove signals that maintain stem cells in self-renewing state
  • Provide signals that promote differentiation to a specific cell lineage
16
Q

How are target cell types defined?

A
  • Expression of specific markers

- Functionality (e.g. if neurone fires action potential, if cardiomyocyte beats)

17
Q

What is the 3D approach to in vitro differentiation?

A
  • 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
18
Q

What are the advantages and disadvantages of the 3D approach to in vitro differentiation?

A

+ Recapitulates embryonic development more accurately
+ Can study how different cell types interact with one another

  • Hard to dissect the role of individual signals
19
Q

How can embryoid bodies be used as reporter lines?

A
  • 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
20
Q

What is an alternative method to generating embryoid bodies?

A
  • 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
21
Q

What is the 2D approach to in vitro differentiation?

A
  • 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)
22
Q

What are the advantages and disadvantages of the 2D approach to in vitro differentiation?

A

+ More tractable system (e.g. for live imaging)
+ Easier to study role of individual signals

  • Loss of cell interactions that occur in vivo
23
Q

What is a use of 2D approach to in vitro differentiation?

A

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
24
Q

What is the method for using iPS cells for disease modelling?

A
  • 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
25
Q

What is microcephaly?

A
  • A neurodevelopmental disorder, in which infants are born with abnormally small brains
  • This results in severe neurological deficit and seizures
26
Q

What was done in Lancaster et al (2013) study of microcephaly?
What were the results?

A
  • 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

27
Q

What was done in the study of microcephaly following Zika Virus outbreak?
What were the results?

A

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

28
Q

What were the results of the drug screen on ZKV organoids?

A

Found that emricasan (a drug that blocks apoptosis) prevented the increased cell death caused by ZKV infection

29
Q

What are the challenges associated with modelling disease with pluripotent stem cells?

A
  • 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)
30
Q

How can cell replacement be used to treat Parkinson’s?

A
  • 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
31
Q

What are the challenges associated with cell replacement using pluripotent stem cells?

A
  • 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?