L10: Stem Cell Technologies Flashcards
1
Q
How are hES cells useful in therapies?
A
- Diabetes
- Parkinson’s
- Arthritis
- Regenerative medicine
- Toxicology
2
Q
How are hES cells useful in regeneration?
A
- Regenerative medicine
- Replacing tissues/cell types e.g. beta cells in T1DM
3
Q
What is the basic definition of a stem cell?
A
- ‘Primitive’ cell that can self-renew, is potent (i.e. can make a range of cell types), and can differentiate
- This allows the development of embryos and repair of tissues later in life
4
Q
Types of pluripotent stem cells:
A
- Blastocysts (mES, hESC, EpiSC, human naive etc) -> Pre-implantation
- Epiblast (hESC, EpiSC) -> Post-implantation
5
Q
Outline the stages in the use of human pluripotent SCs in medicine:
A
- Originally derived from teratocarcinoma (EC)
- Can also be derived from surplus IVF embryos and induced pluripotent stem cells
- SCs are cultured in vitro
- They are then differentiated for various functions
- e.g. B cells for diabetes
- e.g. Neurons for Parkinsons
- e.g. Cartilage cells for arthritis
6
Q
Procedure for obtaining Human ES cells:
A
- Obtain SCs (usually spare embryo from IVF), culture in vitro
- Remove trophectoderm from hatched blastocyst, typically by exposure to an anti-trophectoderm antibody followed by complement-mediated killing
- ICM cellls re-plated into feeder cells (originally mouse fibroblasts)
- Examples of medium used: Thomson, Reubinoff
7
Q
How do we characterise human pluripotent stem cells?
A
- Flow cytometry / in-situ staining
- Genetic/epigenetic testing
- Gene expression via microarrays/qPCR
- Functional tests e.g. clonogenic assays (ability to form colonies) , teratoma test (can it differentiate into the 3 germ layers in this model?)
8
Q
Key differences between human and mouse ESCs:
A
- Phenotypic (i.e. density of colonies, cytosol density) -> although overall they are relatively similar
- Signalling (e.g. SSEA1 + mice, - humans in early development, type of cell-surface marker)
9
Q
What is the issue with long term culture of stem cells? (including example mutation)
A
- Over time, liekly to acquire genetic abnormalities
- Selective for colony survival and proliferation (e.g. Chr 12, 17 shown to have a high distribution of cytogenetic abnormalities in culture adapted hES cells)
10
Q
What is the consequence of non-optimal conditions in SC culture:
A
- Cells either self-renew, die or differentiate
- If the conditions are too harsh, selection pressures occur for adaptation -> genetic abnormality
- In extreme cases, human embryonal carcinoma can arise due to lack of differentiation
11
Q
Why is epigenetic testing important in iPCs?
A
- Induced pluripotent -> need to epigenetically reprogramme
- Must display required methylation pattern for success
12
Q
How can we test for pluripotency?
A
- Embryoid body (allowing cells to coagulate into 3D structure, triggering differentiation) -> not very reproducible
- Reproducibility can be improved by adding spin step
- Can add GFs to this protocol for specific lineages
- 2D Monolayer (most common): mimicking developmental biology by sequential application of GFs -> issue in lack of 3D structure
- Teratoma -> adding to immunocompromised mice and testing ability to form different germ layers
- Organoids (variation on embryoid body with polarisation step)
13
Q
How are SCs maintained in a pluripotent state?
A
- Suppress BMP signalling
- Activate TGFB and MEK/ERK and PI3K
- Net effect: Supress differentiation, promote self-renewal
14
Q
How are iPS cells induced?
A
- Differentiated somatic cells in culture
- Switch off known markers for cell type to resume pluripotency
- Originally doing using viral vectors, where mouse line used selection via drug morphology (very low efficiency)
15
Q
Methods for non-integrating delivery of reprogramming factors:
A
- Transient transfection using mRNA (‘Gold standard’)
- TAT-fusion
- Adenoviral vectors
- Chemical based reprogramming
- ‘Floxed’ vectors (Cre-Lox system which uses cre to remove DNA via loxp sites)
- Using a transposon to integrate the DNA then remove it again
16
Q
Protein expression markers for iPCs?
A
- Alkaline phosphatase
- Pluripotency factors e.g. Nanog, Oct4
- Surface markers e.g. Tra1-81, SSEA3
17
Q
What can induced pluripotent SCs be used for?
A
- They can ‘capture’ the genotype of a person or a population
- Can then be used in drug discovery, comparing response of patients somatic cells to a normal individual
- Drug screens for the desired effect and other potential side effects throughout the body (pharmacogenomics)
18
Q
What diseases have been modelled with iPSC methods?
A
- Cardiac….
- Long QT (KCNQ1 and 2)
- Timothy syndrome
- LEOPARD syndrome
- Other…
- Schizophrenia
- Alzheimer’s
- Retinitis pigmentosa
19
Q
How have iPSCs been useful in modelling Long QT type II?
A
- Cardiomyocytes from LQT patients show action potential lengthening
- It has been proposed that calcium-influx through L-type calcium channels contribute to action potential duration and ‘early after depolarisations’
- Therefore, inhibition of this current may be anti-arrythmic
- -> Calcium blocker nifedipine stops EADs
- Potassium blocker pinacidil?
- Can model whether arrhthymia is likely using ‘ECG’ of cardiomyocytes
20
Q
iPSC studies into Parkinson’s disease:
A
- Alongside brain-wide changes like the presence of Lewey bodies, there is a lack of dopamine-secreting neurons in substantia nigra -> interference with control pathways -> difficulty initiating movement
- Around 2% of the population over 70 display signs
- Attempted to transplant foetal neural cells -> sometimes successful
- As an alternative, they hoped to use iPSCs to differentiate dopamine-secreting neuron and transplant them back into the substantia nigra
21
Q
iPSCs in the study/treatment of DMI:
A
- Gold standard: Edmonsen protocol using islet transplantation; limited by donor availability
- Regenerative prospects: Triggering differentiation of beta cells through mimicking pancreatic environment in iPSCs (has successfully been developed in vitro; first transplants into patients in 2021 with improvement shown in islet cell function)
22
Q
iPSCs in the study/treatment of age-related macular degeneration:
A
- Retinal pigment epithelium cells degenerate (responsible for phacoytosing debris from photoreceptors)
- More common with age, in its 2 forms (wet and dry AMD)
- Dry AMD is untreatable (majority) -> blindness
- HESC-derived RPE cells have been obtained
23
Q
Give 3 tools for directing hPSC differentiation:
A
- Small molecule and recombinant proteins -> inhibit or activate key signalling pathways like WNT, BMP to mimic developmental programmes
- Matrices and self patterning (organoid/co-culture/hydrogel)
- Transcriptional programming
24
Q
What are the principles behind differentiation approaches using hPSCs?
A
- Directing lineages by mimicking temporal and spatial conditions that embryonic tissue would be exposed to during real organ development
- Signalling information should first programme the correct germ layer, then encode A - P and D - V coordinates
- This will result in terminal fate specification
25
Q
How are terminally differentiated cells isolated from hPSC population?
A
- FACS
- MACS
- Functional assays
26
Q
3 methods for quality control of differentiated progeny:
A
- Immunocytochemistry (i.e. cell fate markers) and electron microscopy (ultrastructure)
- Genetic and epigenetic profiling (mRNA, protein, DNA, histone methylation can all be assayed in bulk populations as well as single cells)
- Biochemical and physiological assays (calcium imaging, electrophysiology, hormone/NT release)
27
Q
Types of pluripotent SCs in mice vs human: (Include corresponding epiblast stage for each pluripotent stem cell type)
A
- Naive PSCs: Undergo capacitation to formative state (and form trophectoderm in humans) -> Pre-implantation epiblast
- Formative PSCs: Form PGCs and somatic tissue (as well as amnion in humans) -> early post-implantation
- Primed PSCs: Form somatic tissue (both tissues) -> late post-implantation
28
Q
+ Advancement in protocol for differentiating mDA lineages (Parksinon’s)
A
- Transitioning through ‘floor plate’ instead of epithelial cell fate -> this strategy resulted in more authentic dopamine neuronal population due to its closer resemblance to actual neuronal development
- Canonical WNT signalling was particularly important in this approach (CHIR small molecule)
- Successfully tested in various animal models for Parkinson’s disease
