Patients derived stem cells Flashcards
Brain cells in a dish
Patient cells
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iPS cells
(neural cells, cardiac cells, hepatocytes, beta cells)
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Cellular studies (diseases in vitro)
Transplantation studies (disease in vivo)
Drug or genetic screens
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Candidate drug?
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Circle back round to patient are cells
Stem cells
Stem cells in the embryo
- make more stem cells
- cells become specialised
Embryonic stem cells (ESC)
- self renewal (—> make more stem cells)
- pluripotency (—> make any cell type of the body)
Cellular reprogramming (cloning)
Development is not one-way
- all genetic information in each cell
- cell specialisation can be reversed
- reprogramming
Induced pluripotent stem cells (iPSCs)
iPSCs
- 4 stem cell genes can reprogram somatic cells
Blastocysts —> Stem cell
Skin biopsy —> Stem cell
(Reprogramming, OCT4, SOX2, KLF4)
iPSCs = ESCs
iPSCs are identical to embryonic stem cells
- Self renewal and pluripotency
- Stable karyotype
- Epigenetic reprogramming (lose marks of speculation and ageing)
- iPSCs are easier to obtain (ethically and practically)
- iPSCs share the same genes as the donor
Individual of interest, fibroblasts (or any cell type), iPSCs
- neurons - ectoderm (3 months)
- chondrocyte - endoderm
- cardiomyocytes - mesoderm
Differentiation
Stem cells can make any somatic cell
(Information from development)
Stem cell differentiation has 3 stages:
1. Neural induction
2. Patterning
3. Maturation
Neural development
The entire CNS starts as a tube
Folds and swells to form the brain and spinal cord
Neural induction
Diffusible signals from the organiser inhibit SMAD signalling (BMP and TGF beta) “be brain” signal
The same signal used for neural induction of stem cells
Patterning
- making neurons is easy
- making specific neurons is hard
More organisers
- signal gradients
- French flag hypothesis
Cellular address (positional identity)
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Cortical neurons
( epilepsy dementia etc)
Different layers of the cortex
Midbrain dopamine neurons
(Parkinson’s)
(Sonic hedgehog signalling, ventral)
Motor neurons
(ALS)
(Wnt (caudal = spine)
(Shh (ventral)
Maturation
Parallel to development
(~ 100 days for functional neurons (electrically active)
Support neurons
(Neurtrophins e.g. BDNF)
Astrocytes help electrical maturation
(Support cells)
Other ways to make aaa neurons
3- dimensional
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- 3D organoids and spheroids (“mini brains”)
- similar protocol (neural induction —> patterning —> maturation)
- self-organising (structural similarities to brain)
- heterogeneous
- limited by diffusion of nutrients
Forward programming
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- overexpression of neuron factors (NGN2)
(Rapid, scalable, easy, homogenous)
(Not physiological)
(Identity of the neuron unclear)
Direct conversion (iNeurons)
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- same principles as iPSCs reprogramming
- scalable? Efficiency is low?
- can you do this in Vivo to replace lost cells?
- iPSC- derived neurons are foetal
- best way to model ageing diseases?
Models of neurodegeneration
- drug or genetic screens
- transplantation studies (diseases in Vivo)
- cellular studies (diseases in vitro)
Disease mechanisms
Example
Familial Alzheimer’s disease
- amyloid plaques made up of amyloid beta
- amyloid beta 40 most abundant
- amyloid beta 42 is a ‘toxicI’ form
Patient heterogeneity
- important for drug trials?
Only possible with physiological model
Understand the disease better?
Drug discovery/ drug screening
Drug discovery
Screening iPSC-derived neurons
- 1 safety of few drugs (neurons and hepatocytes)
- 2 screen for new drugs,
Drug screening
Phosphorylated tau high in AD
Screen for lowering pTau
Screen finds cholesterol pathway
(Drug candidate)
(Understanding of disease)
Cell replacement
Parkinson’s disease mice
- transplantation off iPSC-derived neurons
- reverse disease behaviours
Optogenetics
- light sensitive channels from algae
- Green light —> channel open —> neurons silent
Graft = disease cured
- Green light = disease comes back
(The graft having the effect)
Clinical trials now happenings
The future
Coculture systems - immune system
Neurons, astrocytes, microglia
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Coculture models
(BBB modelling, neurons, astrocytes and endothelial cells)
Assembloids
- dorsal organoid (cortical)
- ventral organoid (interneurons)
- migration of interneurons into circuits
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Genome engineering
- CRISPR-Cas9 technology
Isogenic cells
- well controlled experiment
