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Cell Biology > Lecture 16 - Stem cells and therapy > Flashcards

Lecture 16 - Stem cells and therapy Flashcards

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1
Q

How can stem cells be generated in vitro?

A
  1. Fertilisation then grow egg to the blastocyst stage
  2. Isolate ICM to get a population of pluripotent stem cells
  3. Grow these stem cells on a feeder layer (normally mouse fibroblasts
  4. Remove feeder layer to form embryoid bodies (requires non-adherant conditions)
  5. Expose embryoid bodies to a differentiation stimulus
    - can also get SC from cancers, these dont have feeder layer
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2
Q

What are teh benefits and disadvantages of not using a feeder layer when generating stem cells in vitro?

A
  • lack possible contamination

- but expensive

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3
Q

What are the features of the differentiation stimulus that a stem cell must be exposed to in vitro?

A
  • 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
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4
Q

What are the therapeutic potentials of ESC?

A

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
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5
Q

What are the features of using ESM to regenerate whole organs?

A
  • 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
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6
Q

What are the two ways we can direct ESC differentiation?

A

Manipulate growth conditions

Genetic manipulation

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7
Q

How can you direct ESC differentiation by manipulating growth conditions?

A
  • 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
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8
Q

How can you direct ESC differentiation by genetic manipulation?

A
  • 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
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9
Q

What are the risks of directing ESC differentiation by genetic manipulation?

A

with the delivery mechanism, don’t know the full effects

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10
Q

Give an example of the therapeutic use of ESC differentiation by genetic manipulation?

A

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
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11
Q

What experiment demonstrated the differentiation of ESC to dopamine producing neurons? (Parkinsons)

A

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

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12
Q

What does an in vivo functioning assay demonstate about ESM that have been cultured with FGF8, Shh and Nurr1? (parkinsons)

A

once differentiated in the lab and implanted into the CNS, these restore dopamine function

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13
Q

What are the two methods for differentiating ESCs produced by via manipulating growth conditions?

A

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

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14
Q

Outline paralysis therapy in mice

A
  • 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
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15
Q

What process is used for therapeutic cloning?

A

Somatic cell nuclear transfer

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16
Q

What is somatic cell nuclear transfer?

A
  • ‘reproductive cloning’
  • fusion of a somatic cell nucleus with an empty egg
  • quite inefficient for producing embryo
17
Q

What are the potential uses of somatic cell nuclear transfer?

A
  • 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
18
Q

What are the advatages of the therapeutic potential uses of adult stem cells?

A
  • allows use of patients own adult stem cells

- fewer ethical and safety concerns, less prone to tumour genesis

19
Q

Give some examples of the uses of adult stem cells in therapy?

A
  • Haematopoietic stem cells
  • Mesenchymal stem cells
  • Epidermal stem cells
  • Neural stem cells
  • Limbal stem cells
20
Q

Outline the production and use of haematopoietic stem cells via adult stem cells?

A
  • 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
21
Q

Outline the production and use of mesenchymal stem cells via adult stem cells?

A
  • 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
22
Q

Outline the production and use of Limbal stem cells via adult stem cells?

A
  • 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
23
Q

What are induced pluripotent stem cells?

A

when genes are introduced to somatic cells e.g. fibroblasts, that are associated with ESC pluripotency to reprogram somatic cells to pluripotent stem cells

24
Q

What are teh possible combination of factors to induce pluripotent stem cells?

A

2 possible combinations

  • Oct4, Sox2, Nanog, Lin28
  • Oct4, Sox2, KIF4, cMyC
25
Q

What is the result of introducing factors to induce pluripotency in somatic cells?

A
  • 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
26
Q

How can you test that induced pluripotent stem cells have pluripotency?

A

Inject into mice and they will form a teratoma

27
Q

What are the differences between cells produced by induced pluripotent stem cells and SCNT?

A

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
28
Q

How can induced pluripotent stem cell therapy be used to treat sickle cell anaemia in mice?

A

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
29
Q

What is the risk of teratoma formation in induced pluripotent stem cells?

A

Still a risk, but it is identical to that of the patient

30
Q

Aside from therapy to treat genetic diseases (e.g. sickle cell anaemia) what can induced pluripotent stem cells be be used for?

A

In vivtro screening of drugs on healthy and unhealthy cells

-or full transplants

Cell Biology (15 decks)

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