Lecture 16 - Stem cells and therapy Flashcards
How can stem cells be generated in vitro?
- Fertilisation then grow egg to the blastocyst stage
- Isolate ICM to get a population of pluripotent stem cells
- Grow these stem cells on a feeder layer (normally mouse fibroblasts
- Remove feeder layer to form embryoid bodies (requires non-adherant conditions)
- Expose embryoid bodies to a differentiation stimulus
- can also get SC from cancers, these dont have feeder layer
What are teh benefits and disadvantages of not using a feeder layer when generating stem cells in vitro?
- lack possible contamination
- but expensive
What are the features of the differentiation stimulus that a stem cell must be exposed to in vitro?
- diff stimuli drives cells down different lineages
- must identify stimulus from the body and when exposed
- if don’t do this effectively may result in teratoma forming ESCs
What are the therapeutic potentials of ESC?
Could in theory generate any cell in the body and therefore:
- could replace worn or diseased body parts in Neurodegenerative diseases, Diabetes, Corneal defects, Cardivascular disease, Musculoskeletal disease
- Use instead of animals in toxicity testing
- use in gene therapy
- use in model systems e.g. exmaine how they grow in vitro
What are the features of using ESM to regenerate whole organs?
- hard to do as many cell types make them up
- to dictate this must modify SC growth conditions
- also use gene expression modification by over expressing certain signals
What are the two ways we can direct ESC differentiation?
Manipulate growth conditions
Genetic manipulation
How can you direct ESC differentiation by manipulating growth conditions?
- cells respond to cues in the extracellular environment
- adhesion to different ECM substrates can cause changes in cell phenotype
- through integrin mediated interactions changes how cell behaves
- or expose to growth factors (external stimuli) to generate different germ layers and lineages
How can you direct ESC differentiation by genetic manipulation?
- over/express genes known to be present in the differentiated cell
- only possible if specific cues are identified
- genes must be activated at particular times
- genes control regulation of differentiation
What are the risks of directing ESC differentiation by genetic manipulation?
with the delivery mechanism, don’t know the full effects
Give an example of the therapeutic use of ESC differentiation by genetic manipulation?
Therapeutic use in parkinsons
- in parkinsons dopamine producing neurons in the CNS dies off, other neurons consequently fire out of control
- TF Nuclear-receptor-related-1 (Nurr1)induced in differentiation of neuron precursor cells into dopamine producing neurons
- in normal midbrain, Dopamine neurons require FGF8 and Shh
What experiment demonstrated the differentiation of ESC to dopamine producing neurons? (Parkinsons)
Experiment
-ESC expose to FGF8 and Shh and overexpress Nurr1 (via viral delivery) in their system then culture cells where the introduction has been successful
Result
-get expression of dopamine produing neuronal markers
-view by western blot
-but this does not demonstrate functional differentiation, just suggest differentiating as planned
-must then do an in vivo functioning assay
What does an in vivo functioning assay demonstate about ESM that have been cultured with FGF8, Shh and Nurr1? (parkinsons)
once differentiated in the lab and implanted into the CNS, these restore dopamine function
What are the two methods for differentiating ESCs produced by via manipulating growth conditions?
Dont differentiation in vitro
-could implant into CNS and let host tissue provide the correct cues
-riskier, chance some ESC will retain pluripotency and form teratomas
OR
Differentiation in vitrro
-expose ESC in a petri dish to supportive cell types (i.e the feeder layer)
-this is less selective than the method definining the growth factors the cells are exposed to
-less reliant on specific biology
Outline paralysis therapy in mice
- transplant neuronal cells derived from tumour pluripotent cells directly into the spine of mice whose legs were paralysed
- results in improved function
- relies on host tissue cells to induce differentiation
- could also use adult neuronal stem cells
What process is used for therapeutic cloning?
Somatic cell nuclear transfer
What is somatic cell nuclear transfer?
- ‘reproductive cloning’
- fusion of a somatic cell nucleus with an empty egg
- quite inefficient for producing embryo
What are the potential uses of somatic cell nuclear transfer?
- can be used to generate stem cells via a blastocyst with ICM that can be isolated and expanded in vitro
- for full organism production
- possibly for generating immunocompatible tissues for transplant (take the nucleus from the somatic cell of a patient
What are the advatages of the therapeutic potential uses of adult stem cells?
- allows use of patients own adult stem cells
- fewer ethical and safety concerns, less prone to tumour genesis
Give some examples of the uses of adult stem cells in therapy?
- Haematopoietic stem cells
- Mesenchymal stem cells
- Epidermal stem cells
- Neural stem cells
- Limbal stem cells
Outline the production and use of haematopoietic stem cells via adult stem cells?
- can harvest bone marrow or get it from blood transfusions
- can store and match marrow
- treat leukemia, where there is a lack of haematopoietic stem cells
Outline the production and use of mesenchymal stem cells via adult stem cells?
- less well characterised than haematopoietic stem cells
- used to treat osteogenesis imperfecta, which is caused by mutations in α1 or α2 (collagen) genes
- mesenchymal stem cells give rise to osteoblasts
- must take donor MSCs (patient’s usually have problems, OI is a genetic disease)
- must match the donor MSC
- can restore functionallity
Outline the production and use of Limbal stem cells via adult stem cells?
- epithelial
- restore the cornea
- can migrate out of its niche
- requires substrate material to support its growth (e.g. amniotic membrane) to minic the cornea in the body
- do biopsy of limbal tissue on host or donor, digest tissue to remove LSCs anad culture on the amniotic membrane
What are induced pluripotent stem cells?
when genes are introduced to somatic cells e.g. fibroblasts, that are associated with ESC pluripotency to reprogram somatic cells to pluripotent stem cells
What are teh possible combination of factors to induce pluripotent stem cells?
2 possible combinations
- Oct4, Sox2, Nanog, Lin28
- Oct4, Sox2, KIF4, cMyC
What is the result of introducing factors to induce pluripotency in somatic cells?
- increased telomerase activity
- increased ESC surface markers
- induce the ability to differentiate into 3 germ layers (ie pluripotency)
- express SSEA-4, SSEA-3 (human ESC markers), but not SSEA-1 (from mice, this is good)
- can differentiation in to specific somatic cells
How can you test that induced pluripotent stem cells have pluripotency?
Inject into mice and they will form a teratoma
What are the differences between cells produced by induced pluripotent stem cells and SCNT?
induced pluripotent stem cells
- v low efficency, v few somatic cells actually formed, possiblity of p53 regulation (ESC don’t have p53)
- epigenetic ‘memory’ is noticibly close to the cells they’re derived from ‘retain marks’, SCNT had more robust genetic similarity
How can induced pluripotent stem cell therapy be used to treat sickle cell anaemia in mice?
Sickle cell anaemia in mice due to a defective β globin gene
- harvest fibroblasts from the mice tail and expose to a combination of the four factors
- grow clones, get colonies, confirm by doing teratoma test
- these cells will now form induced pluripotent stem cells but still have a genetic defect
- now must do genome editing
- get embryoid body and produce functional HSC progenitors and inject
What is the risk of teratoma formation in induced pluripotent stem cells?
Still a risk, but it is identical to that of the patient
Aside from therapy to treat genetic diseases (e.g. sickle cell anaemia) what can induced pluripotent stem cells be be used for?
In vivtro screening of drugs on healthy and unhealthy cells
-or full transplants
