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
