Modelling embryonic development and disease using pluripotent stem cells - 2

Question 1

What was shown by the Wnt reporter Axon2-lac2 during embryonic development? How about after removal of self-renewal signals?

Answer 1

Wnt reporter was found at the primitive streak of the de developing embryo.
After removal of self-renewal signals (eg LIF/BIF) regionalised expression of Wnt was seen in embryoid bodies

Question 2

What is a cerebral organoid?

Answer 2

Organoid which ‘mirrors’ the structure of the cerebrum and is derived from an embryoid body.
Cerebral organoid example of when an embryo is directed slightly in generating cells of one specific type
It expresses markers that are in the same place as the cerebrum (as shown by staining).

Question 3

Outline the 2D approach of in vitro differentiation

Answer 3

• Plate a number of cells on an ECM/matrix
• Remove ‘undifferentiated’ signals (BMP/LIF for mouse ES cells or FGF2, TGFb for human ES cells)
• Grow in refined medium with appropriate signals (e.g. FGF, WNT etc)

Question 4

Advantages and Disadvantages of 2D approach

Answer 4

ADVANTAGE: more tractable system (e.g. for live imaging), easier to test the role of specific signals

DISADVANTAGE: loss of cell interactions that may occur in vivo

Question 5

What is an organoid?

Answer 5

An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy.

Question 6

What is microcephaly?

Whats the problem w microcephaly regarding mouse models?

Answer 6

Neurodevelopmental disorder that results -from recessive mutations- in an abnormally small brain
Results in neurological impairments and seizures
Cannot be recapitulated in mice – need human model!

Question 7

What was shown in the study by Lancaster et Al 2013 – attempted to recapitulate the mini brain organoid

Answer 7

• Skin fibroblasts of patients with mutations in the CDK5RAP2 gene (resulting in microcephaly)
• Sample blasted with pluripotency factors
• Induced iPS cells! (characterised by normal ways teratocarcinomas etc)
• When using immunofluorescence for DCX (marker for committed neurons) and SOX2 (marker for neural progenitors) FEWER NEURAL PROGENITORS WERE SEEN IN MICROCEPHALY PATIENT ORGANOIDS

Question 8

Relevance of the zika virus? What was shown regarding the drug identified in the drugs screen?

Answer 8

• Zika virus in mothers correlated with an increased number of infants with microcephaly
• Drugs screen carried out whereby cerebral organoids were produced (microcephalic brains) and EMIRCASAN identified in drugs screen as blocking the zika virus, demonstrated by an inhibition of cell death (apoptosis) caused by the zika virus

Question 9

Features of Parkinson’s disease?

Answer 9

RIgidity, anxiety, dementia and tremors.
Progressive loss of dopaminergic neurons
effects 1 in 500 people

Question 10

CELL REPLACEMENT IN PARKINSON’S DISEASE using the TH enzyme —- outline process?

Answer 10

-The TH enzyme synthesises dopamine. TH and FOXA2+ expressed in neurons
-Neurons are transplanted back into mouse model and this results in the REGENERATION OF DOPAMINERGIC NEURONS !!!
This has been taken forward for clinical trials

Question 11

What is TBra linked to in the 2D approach? What does it act as?

Answer 11

GFP is linked to TBra in the 2D approach.

TBra is an early MESODERMAL marker

Question 12

Challenges of modelling diseases?

Answer 12

• Often multiple genetic causes to a disease
• Some diseases are late onset so are not feasible to study during development
• Complex phenotypes are often seen in diseases

Question 13

Challenge of cell replacement technique?

Answer 13

• Immune reaction of body
• Possibility of the method resulting in tumours?
• Positional identity of cells is important - human E.S cells can only be produced with a ANTERIOR identity