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Pharmacology of Regenerative Medicine > Pluripotent stem cells > Flashcards

Pluripotent stem cells Flashcards

1
Q

What are stem cells?

A

They are unspecialised cells that can turn into many other cell types.

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2
Q

What is the functional definition of a stem cell? (3)

A
  1. Cell able to self-renew, generate a daughter cell identical to mother cell.
  2. Be clonal - a single cell can generate more stem cells and differentiated progeny.
  3. Produce progeny with more restricted potential i.e. differentiate.
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3
Q

Where are stell cells found? (5)

A
  1. Fertilised eggs
  2. Early embryos
  3. Umbilical cord
  4. Adult tissues and organs
  5. Cancers
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4
Q

What is a totipotent cell?

A

Able to form all tissues needed for reproduction, example: fertilised egg.

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5
Q

What is a pluripotent cell?

A

able to give rise to all cells of a embryo and adult e.g. embryonic stem cells.

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6
Q

What is a multipotent cell?

A

Able to form a limited number of cell types e.g. most adult stem cells, HSC, NSC.

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7
Q

What is a unipotent stem cell?

A

Can only form one type of cell, e.g. committed progenitor cells.

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8
Q

How is ES cell pluripotency maintained?

A

(a) promotion of proliferation

(b) suppression of differentiation

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9
Q

How is differentiation of ES cells suppressed?

A

Extrinsic signals: growth factors and cytokines, signalling pathways, intrinsic factors: transcription factors.

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10
Q

What type of stem cell can only form one type of cell?

A

Unipotent.

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11
Q

What is LIF?

A

Leukaemia inhibitory factor - LIF - is an extrinsic factor that can maintain mESC in undifferentiated, pluripotent state.

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12
Q

What type of stem cell can only form a limited number of cell types?

A

Multipotent

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13
Q

What type of stem cell can form all tissues required for reproduction?

A

Totipotent

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14
Q

What type of stem cell can form any cell type?

A

Pluripotent.

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15
Q

What impact does LIF have on STAT3?

A

Activates stat3 which results in mESc self-renewal/maintainence of pluripotency.

Dominant negative forms of STAT3 reduce self-renewal.

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16
Q

What other factors other than LIF control mouse ESC pluripotency?

A

Bone morphogenetic proteins 2+4.

BMP2/4 work togther with LIF to maintain pluripotency.

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17
Q

How is Wnt signalling related to mouse ESC self-renewal?

A

Wnt signalling = glycogen synthase 3 is inhibited.
B-catenin stabilises and accumulates in nuclues.
B-catenin relieves TCF-3 repression of transcription and reinforces self-renewal.

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18
Q

What impact do small molecule inhibitors of GSK-3 have on mESC pluripotency?

A

Small molecule inhibitors of GSK-3 maintain pluripotency of mESCs.

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19
Q

Dual inhibition of MEK and GSK-3 is referred to as _____

A

2i conditions.

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20
Q

Mouse ESCs cultured in 2i conditions enter a so-called _______

A

Ground state

21
Q

mESCs cultured in 2i are characterised by______

A

mESCs cultured in 2i are characterised by uniform expression of key pluripotency transcription factors.

22
Q

What are the intrinsic factors controlling mouse ES cell pluripotency? (3)

A

Oct-4
Sox-2
Nanog
‘Master Regulators’

23
Q

What is Oct-4

A

Involved with control of mouse pluripotency.
Binds an octamer sequence of DNA (5’ATGCAAAT 3’).
Repress or activate transcription of genes

24
Q

What impact do levels of Oct-4 have on pluripotency of mESC?

A

High: primitive endoderm-like cells.
Moderate: pluripotent mESC
Low: trophectoderm (placenta)

25
Q

What is Sox-2?

A

SRY-related high mobility group-box protein 2.
Implicated in regulation of transcription and chromatin.
Expressed by mouse and human ESC (and neural stem cells).

26
Q

What does knock-out of sox-3 cause?

A

Multi-lineage differentiation of stem cells.

27
Q

How do Oct-4 and Sox-2 interact?

A

Sox2 and Oct 4 form a complex and cooperate.

Sox-2 and Oct-4 share many of the same binding sites on DNA.

28
Q

What is Nanog?

A

Homeodomain-containing transcription factor.

29
Q

How does Nanog influence differentiation?

A

Nanog is expressed highly in undifferentiated ESC.

Nanog can maintain mESC in an undifferentiated state in the absence of LIF.

30
Q

Where does the name for Nanog come from?

A

Tir nan Og - land of the ‘ever young’

31
Q

How do Oct-4, Sox2 and Nanog cooperate to maintain pluripotency?

A

PGRN: pluripotent gene regulatory network.
Platform for recruiting other factors from signalling pathways, transcriptional circuits, regulatory RNAs, epigenetic mechanisms.

32
Q

In the UK, what is the only source of hESCs?

A

Spare IVF eggs.

However, they have to be destroyed after 5 years if not implanted.

33
Q

Why is it more difficult to culture hESCs than mESCs?

A
  1. They dislike being separated into single cells and grow more slow.
  2. They need a layer of ‘feeder’ cells for best growth.
  3. They cannot be maintained in LIF
34
Q

What is the best combination of factors for the growth of hESCs?

A

Activin A + FGF-2.

For mESCs it is simply LIF.

35
Q

Induced pluripotent stems cells (iPS cells) have been generated from ________

A

Mouse embryonic fibroblasts.

36
Q

Where are mouse embryonic fibroblasts found?

A

Embryo

Tail tip

37
Q

How was reprogramming of mouse fibroblasts to pluripotency achieved?

A 
The use of 4 transcription factors:
Oct-4
Sox-2
c-myc
Klf-4
38
Q

How can human iPS be generated?

A

Infect adult human dermal fibroblasts with retroviruses which encoding different transcription factors.

39
Q

What transcription factors do the retroviruses used in generating human iPS encode for?

A

Oct-4, Sox-2, c-MYC, Klf-4

OR

Oct-4, Sox-2, Lin28, Nanog

40
Q

What occurs after 8x10^5 human fibroblast cells per dish are infected with retroviruses?

A

They are incubated for 6 days and then replated into hES culture conditions.

41
Q

What is the efficiency rate of human induced pluripotency stem cells using retrovirus conversion?

A

Crap.

Approx 1 in 5000 cells are reprogrammed to ES-like cells (0.0002%)

42
Q

What four transcription factors are used to reprogramme mouse fibroblasts to pluripotency?

A

Oct-4
Sox-2
c-myc
Klf-4

43
Q

In converting human fibroblast cells into induced pluripotency stem cells, why do we need to seed at low density?

A

At high density, non-ES colonies predominate and because the conversion rate is so low already (0.0002%) we need as favourable conditions as possible.

44
Q

What are the 7 steps involved in iPSC derivation?

A
  1. Choice of appropriate factors
  2. Methods of factor delivery
  3. Choice of cell type.
  4. Parameters of factor expression
  5. Derivation conditions
  6. Identification of iPSC colonies
  7. Expansion and characterisation
45
Q

What are the potential applications of iPSCs? [4]

A
  1. Possible to generate patient and disease specific iPSCs.
  2. Use of iPSCs for ‘Disease in a dish’ models.
  3. Use of iPSCs to generate 3-dimensional ‘organoids’
  4. Use in cell replacement therapies.
46
Q

What are 3-dimensional organoids?

A

Emerging novel system to study tissue development, organogenesis and stem cell behaviour ‘ex vivo’.

The cells spontaneously self-organise into functional cell types and progenitors.

47
Q

What organoids have been successfully created?

A 
Gastric 
Intestinal 
Liver bud
Thyroid
Lung 
Optic cup
Stratified cortical organoid
Pituitary 
inner ear
48
Q

How could 3d organoids be used for modelling human disease?

A

Intestinal organoids from a CF patient could be used for drug discovery.

Optic cups could be used for studying retinal disease.

Pharmacology of Regenerative Medicine (14 decks)

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