L9: Human Genetic Engineering to model Neurodevelopmental Disorders Flashcards
What are some human specific aspects of cortical developement?
- Extensive Cortical Progenitor Cells amplification
- Outer Radial Glia Cells
What is a problen with such a complicated cortical development?
Such complicated structure could easily lead to Diseases
What are neurodevelopmental disorders?
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.
Name some disorders neurodevelopmental disorders manifest in
intellectual disability (ID),
attention-deficit/hyperactivity disorder (ADHD),
autistic spectrum disorder (ASD),
communication disorders (CD), specific learning disorder (SLD),
motor disorders (MDs),
What is the prevalence rate of NDDs?
New studies conclude that the prevalence rate of NDDs fluctuates globally between 4.70 and 88.50%
What are some problems associated with NDDs? (3)
Misdiagnosis
Too complicated mechanisms
Lack of treatments
Weird combinations of complex syndromes we don’t know how to treat
What are type A and type B neurodevelopmental disorders?
Type A: Anatomical Malformations
Type B: Neuronal Connectivity unbalance
How may genetic mutations resulting in these disorders present themselves?
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
What is angelicas (<3) two research aims?
First research aim:
Investigate how Humans evolved such unique enlarged Cortex
Second research aim:
Investigate Mechanisms and Treatment of Neurodevelopmental Disorders
In what 3 experimental steps does she intend to study the cortex?
- Genetic manipulation:
Sample with GOI (WT)
Sample without GOI (KO) - Model generation
WT Phenotype
KO phenotype - Analyses
Measure and comparison
What are three forms of possible genetic manipulation?
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
What are the steps involved in a knockout?
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..
What is the concept behind CRISPR/Cas9-mediated knock-out?
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
Following cleavage of the DNA, what are the possible outcomes?
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
How is transient genetic manipulation achieved through CRISPR/Cas-9?
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)
How can you achieve genetic analysis of cell culture through “Non-Manipulation” means?
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.
What are two problems with patient-derived cells?
Lack of isogenic control
Not always accessible
What are two possibilities for model generation?
2D cell culture:
Primary neurons
Differentiated Stem Cells (ESCs or iPSCs)
3D cell culture:
Spheroids
Organoids
Human vs Non-Human (?)
How could you obtain a 2D cell culture of mouse neural cells?
Take an E15.5 mouse embryo, disect the cortex and dissassociate and isolate neural cells
What are some downsides to this mouse neural primary cell culture method?
Mainly applicable to Animal models
Short life in culture
Not suitable to study complex interactions
What is an alternative method for 2D cell culture? Describe the steps
Differentiated Stem Cells:
human iPSC dual SMAD inhibition protocol or NGN2 direct differentiation:
- hiPSC maintenance
- Neural induction
- LDN+SD or NGN2 lentivirus
- Neural expansion
- FGF2 or BDNF
- Neuronal culture maintenance
What are some pros and cons to the differentiation stem cell 2D culture?
- Both human and mouse
- Short life in culture
- Not suitable to study complex interactions
Elaborate on three cons of 2D cell culture
- 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
Contrast the two methods of 3D cell culture
- Organoid
- Spheroid
- Derived from stem cells
- Derived from cell line monoculture
- Multiple cell lineages
- Represent single/ partial tissue compartments
- Recaptulate organ psyiological parameters
- Transiently resemble cell organisation
- Long term culture
- Difficult to maintaim long term
How do these differences between organoids and spheroids arise aside from their derivation?
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.
Describe two methods regarding neural organoids
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
What are cortical organoids best suited for?
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.
What does cortical organoids allow for when compared to other cell culture models?
Allow to discover human-specific phenotypes
Describe the cortical organoids structure
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
Give four advantages of 3D cell culture
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.
Give three ways of analysing organoids
Immunostaining
RNA-sequencing
Electrophysiology
Describe the cencept of immunostaining organoids
- Fixation with PFA
- Cryostat slicing (16-20 um)
- IMMUNOFLUORESCENCE (IF) with
specific antibodies marking different cell types
Describe the concept of RNA sequencing
- 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)
Describe the concept behind electrophysiology
- 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
