L9: Human Genetic Engineering to model Neurodevelopmental Disorders

Question 1

What are some human specific aspects of cortical developement?

Answer 1

1. Extensive Cortical Progenitor Cells amplification
2. Outer Radial Glia Cells

Question 2

What is a problen with such a complicated cortical development?

Answer 2

Such complicated structure
 could easily lead to
 Diseases

Question 3

What are neurodevelopmental disorders?

Answer 3

Neurodevelopmental disorders (NDDs) are a group of conditions characterized by impairments in the growth and development of the nervous system, particularly in the brain’s structure and function.

These disorders typically manifest early in childhood and involve difficulties in cognitive, motor, social, and emotional functioning.

They’re commonly defined as disorders of multivariate origin where the cause is unknown or complex.

Question 4

Name some disorders neurodevelopmental disorders manifest in

Answer 4

intellectual disability (ID),
attention-deficit/hyperactivity disorder (ADHD),
autistic spectrum disorder (ASD),
communication disorders (CD), specific learning disorder (SLD),
motor disorders (MDs),

Question 5

What is the prevalence rate of NDDs?

Answer 5

New studies conclude that the prevalence rate of NDDs fluctuates globally between 4.70 and 88.50%

Question 6

What are some problems associated with NDDs? (3)

Answer 6

Misdiagnosis
Too complicated mechanisms
Lack of treatments

Weird combinations of complex syndromes we don’t know how to treat

Question 7

What are type A and type B neurodevelopmental disorders?

Answer 7

Type A: Anatomical Malformations

Type B: Neuronal Connectivity unbalance

Question 8

How may genetic mutations resulting in these disorders present themselves?

Answer 8

1 Mutation in 1 gene (“Monogenic” - mendelian disorder)

Complex genomic (Rearrangement, SNPs..) Certain variants in the genome can be ok but when matched with other mutations/ variants can lead to pathology

Question 9

What is angelicas (<3) two research aims?

Answer 9

First research aim:
Investigate how Humans evolved such unique enlarged Cortex

Second research aim:
Investigate Mechanisms and Treatment of Neurodevelopmental Disorders

Question 10

In what 3 experimental steps does she intend to study the cortex?

Answer 10

1. Genetic manipulation:
   Sample with GOI (WT)
   Sample without GOI (KO)
2. Model generation
   WT Phenotype
   KO phenotype
3. Analyses
   Measure and comparison

Question 11

What are three forms of possible genetic manipulation?

Answer 11

STABLE (Knock-Out)
- Before CRISPR/Cas9 (B.C.)
- CRISPR/Cas9-mediated knock-out

TRANSIENT (Knock-Down)
- CRISPR/Cas9-mediated silencing
- RNA interfering

“Non-manipulation”
- Patient’s cells

Question 12

What are the steps involved in a knockout?

Answer 12

Stable genetic manipulation: Knock-Out
0. Know the target gene sequence
1. Design and clone a vector (e.g. plasmid)
2. Transfect the vector
3. Selection of the cells
4. Screening of the cells
5. Expansion of the cells, etc..

Question 13

What is the concept behind CRISPR/Cas9-mediated knock-out?

Answer 13

Concept:
Your target sequence:
Around 20 nucleotides + PAM sequence (a short DNA sequence (usually 2-6 base pairs in length))
(NGG)

You transfect:
a sgRNA (single guide RNA; single RNA molecule that contains both the custom-designed short crRNA sequence fused to the scaffold tracrRNA sequence), to match exactly your target sequence

Cas9, an enzyme able to cut the DNA

Optional: a donor template

Question 14

Following cleavage of the DNA, what are the possible outcomes?

Answer 14

Non-homologous end joining (NHEJ): Randomly inserts nucleotides no longer in frame, knocks out the gene

Homology guided repair: Can incorporate donor DNA to insert a complimentary region

Question 15

How is transient genetic manipulation achieved through CRISPR/Cas-9?

Answer 15

Concept:
Your target sequence:
Around 20 nt + PAM sequence (NGG)

You transfect:
a sgRNA, to match exactly your target sequence

dCas9, a defective Cas9 enzyme fused with a transcription activator/silencer (or other e.g GFP)

Question 16

How can you achieve genetic analysis of cell culture through “Non-Manipulation” means?

Answer 16

Using patient-derived cells, you take a somatic cell and subject to to the yamanaka cocktail of Oct3/4, Sox2, Klf4 and c-Myc to reprogram it to an IPS cell. Then you subject it to a cocktail of signals to differentiate it to a specific cell type.

Question 17

What are two problems with patient-derived cells?

Answer 17

Lack of isogenic control
Not always accessible

Question 18

What are two possibilities for model generation?

Answer 18

2D cell culture:
Primary neurons
Differentiated Stem Cells (ESCs or iPSCs)

3D cell culture:
Spheroids
Organoids

Human vs Non-Human (?)

Question 19

How could you obtain a 2D cell culture of mouse neural cells?

Answer 19

Take an E15.5 mouse embryo, disect the cortex and dissassociate and isolate neural cells

Question 20

What are some downsides to this mouse neural primary cell culture method?

Answer 20

Mainly applicable to Animal models

Short life in culture

Not suitable to study complex interactions

Question 21

What is an alternative method for 2D cell culture? Describe the steps

Answer 21

Differentiated Stem Cells:
human iPSC dual SMAD inhibition protocol or NGN2 direct differentiation:

1. hiPSC maintenance
2. Neural induction
   
   • LDN+SD or NGN2 lentivirus
3. Neural expansion
   
   • FGF2 or BDNF
4. Neuronal culture maintenance

Question 22

What are some pros and cons to the differentiation stem cell 2D culture?

Answer 22

• Both human and mouse
• Short life in culture
• Not suitable to study complex interactions

Question 23

Elaborate on three cons of 2D cell culture

Answer 23

• They aren’t representative of real cell environments — Growing on a flat surface isn’t a good way to understand how cells grow and function in a human body, where they surrounded by other cells in three dimensions.

Lack of predictivity — 2D cell testing isn’t always predictive, which increases the cost and failure rate of new drug discovery and clinical trials.

Issues caused by the growth media and expansion of cells — As cells grow in a standard 2D culture, they consume growth media and exude waste. This can result in toxic waste products, dead cells, nutrition depletion and damage of the environment the cells are in

Question 24

Contrast the two methods of 3D cell culture

Answer 24

1. Organoid
2. Spheroid
3. Derived from stem cells
4. Derived from cell line monoculture
5. Multiple cell lineages
6. Represent single/ partial tissue compartments
7. Recaptulate organ psyiological parameters
8. Transiently resemble cell organisation
9. Long term culture
10. Difficult to maintaim long term

Question 25

How do these differences between organoids and spheroids arise aside from their derivation?

Answer 25

In organoids you follow specific steps in culturing to improve organisation. Use a complex cocktail to create signalling gradients for more long term neurogenesis and more complex organisation.

Question 26

Describe two methods regarding neural organoids

Answer 26

Cerebral organoid protocol with minimalistic culture media generates self-patterned organoids containing diverse regions of the brain.

Region-specific organoids are generated by addition of patterning factors to the culture media to direct the regional identities

Question 27

What are cortical organoids best suited for?

Answer 27

Given the resemblance of organoid models to early stages of cortical neurogenesis, they are best suited for modelling developmental disorders that manifest at embryonic or foetal stages.

Question 28

What does cortical organoids allow for when compared to other cell culture models?

Answer 28

Allow to discover human-specific phenotypes

Question 29

Describe the cortical organoids structure

Answer 29

They mature Neural Rosettes, resembling the main layers of the human Cortex with upper layer neurons, deeper layer neurons, OSVZ and VZ/SVZ. These Neural Rosettes are measurable

Question 30

Give four advantages of 3D cell culture

Answer 30

More relevant cell models — Much better biomimetic tissue models make 3D cell cultures more physiologically relevant and predictive than 2D cultures. 3D plate cultures also show a higher degree of structural complexity and retain a “steady state” (homeostasis) for longer.

Interaction between different types of cells

Better simulation of conditions in a living organism — Cells and organs grow in a more realistic way.

Reduces use of animal models — Animal models aren’t a reliable way to predict how drug treatments will affect humans. Screening drugs against human organs grown on chips is a much more reliable method.

Question 31

Give three ways of analysing organoids

Answer 31

Immunostaining

RNA-sequencing

Electrophysiology

Question 32

Describe the cencept of immunostaining organoids

Answer 32

• Fixation with PFA
• Cryostat slicing (16-20 um)
• IMMUNOFLUORESCENCE (IF) with
  specific antibodies marking different cell types

Question 33

Describe the concept of RNA sequencing

Answer 33

• Extract RNA from your COs (WT vs KO)
• Sequencing of RNA transcripts
• DATA analysis: map the “reads”
• DATA analysis: compare the differences between WT and KO COs
• Results: Differently Expressed Genes (DEGs)

Question 34

Describe the concept behind electrophysiology

Answer 34

• Microelectrode arrays (MEAs) measure changes in the extracellular electric potential and enable the recording of neuronal network activity.
• COs possess a more relevant microenvironment, tissue architecture and potential for modeling the network activity
• COs recapitulate many aspects of network function in the human brain and provide valid disease models