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Animal biotechnology > Stem Cells > Flashcards

Stem Cells Flashcards

1
Q

Why do cells on the outer surface of the developing embryo give rise to the trophectoderm?

A

Because they have receptors which are not stimulated due but cells inside form the ICM because there full cell-contact areas so they remain pluripotent.

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2
Q

Within the egg, what controls cell division during early embryo development?

A

Maternal factors containing mRNAs and proteins. They can become capped and translated into cyclins and pyrimidines as well as various enzymes for the production of metabolites for new cells.

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3
Q

Where can stem cells be extracted from?

A

ICM

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4
Q

What are the four types of Embryonic Stem Cells?

A
  1. ICM
  2. Trophectoderm
  3. Epiblast
  4. PGCs
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5
Q

Two types of Fetal Stem Cells?

A
  1. Cord blood SC
  2. Amniotic fluid SC
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6
Q

Where are Adult Stem Cells derived from?

A

Tissue-specific:
1. Neural SC
2. Haematopoietic SC
3. Spermatogonial SC
4. Mesenchymal SC
5. Liver SC
6. Epidermal SC
etc

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7
Q

Reprogrammed cells - iPS cells (induced pluripotent stem cells)

A
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8
Q

Two products of ICM

A
  1. All embryonic tissues
  2. Yolk sac
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9
Q

What gives rise to the yolk sac?

A

Primitive endoderm

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10
Q

Why can’t we grow human embryos past day 14?

A

Primitive streak forms and this gives rise to the neural tissues.

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11
Q

What two components are needed to culture mouse ESCs (from blastocyst ICM) and what are their functions to maintain pluripotency?

A
  1. LIF (Leukemia Inhibitory Factor) = cytokine growth factor which means they never differentiate
  2. Feeder cells = feed the cells nutrients

The feeder cells produce the LIF

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12
Q

Definition of pluripotency

A

The ability of cells to differentiate to any somatic cell type and to germ cells.

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13
Q

Stem cells have specfic markers, what are these (genes key for pluripotency)?

A

Oct4, Sox2, Nanog, Alkaline Phosphatase, telomerase actvity, SSEA, and Tra1-81

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14
Q

What is meant by telomerase activity?

A

Stem cells have long telomeres, somatic cells do not. Telomere shortening is a sign of aging.

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15
Q

How does LIF work?

A
  1. LIF binds to its LIFR receptor which induces heterodimerisation of the LIF receptor and gp130 receptor
  2. This triggers a cascade inside the cell to activate STAT3 signalling
  3. This targets many genes to keep their expression high
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16
Q

Which other molecule is involved in maintenance of pluripotency in mouse ESCs?

A

BMP4 (and Wnt) activate inhibitor differentiation (Id) proteins (SMAD, B-catenins) for Oct4 & Sox2 activity.

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17
Q

What is diapause and why do some animals undergo it?

A

Arrest of embryonic development. Diapause enables mating to occur and young to be born at times of the year optimal for that species

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18
Q

In the compact morula, what do the cells on the outside activate and why?

A

Cdx2 gene is activated which enables the formation of the trophectoderm.

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19
Q

Which cells form the hypoblast due to polarity?

A

Primitive endoderm cells. The hypoblast cells are the layer below the eipblast cells.

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20
Q

All of the genes involved in maintenance of pluripotency are transcription factors.

A
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21
Q

Where is Oct4 expressed and then expressed within the embryo?

A

During development, it is expressed maternally in the oocytes and is restricted to the cells within the blastocyst which are going to give rise to the ICM.

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22
Q

What gene does Oct4 have an antagonistic relationship with and why?

A

Cdx2. Oct4 expression enables formation of the ICM but Cdx2 enables trophectoderm formation.

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23
Q

Where is Nanog expressed and what does it give rise to?

A

ICM to produce the epiblast (future three germ layers)

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24
Q

Where are human ESCs derived from?

A

Preimplantation blastocysts

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25
Q

Human ESCs are LIF-independent, what do they require instead?

A

Activin & FGF, like mEpiSC. HESCs do not express LIFR and gp130.

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26
Q

What do Actvivin and FGF ativate in HESCs?

A

Activin = SMADs 2/3
FGF = MAPK & P1RK

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27
Q

What is a differene between HESC and mESC?

A

HESC grow as flat colonies whereas mESC grow as round colonies.

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28
Q

Primitive ectoderm = epiblast
Primitive endoderm = hypoblast

A
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29
Q

Epiblast structure comparison between mice and humans:

A

Mice have a cylindrical structure when inside the embryo, when taken out it forms a disc-like structure. Human embryos have an epiblast with a disc-like structure.

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30
Q

For mice, what day (dpi) do cells derived from the blastocyst become LIF/Feeder cell independent and what do they require instead?

A

5.5-6.25dpi. They no longer have pluripotency and cannot contribute to chimeras when injected into another developing embryo. They require Activin & FGF.

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31
Q

Mouse ES = LIF & BMP
Human ES = Activin & bFGF
Mouse epiblast ES = Activin & bFGF

A
32
Q

What do pig EpiSCs depend on for self renewal?

A

FGF & Activin A

33
Q

How do the pluripotent-linked TFs maintain pluripotency?

A

They target their own promoters to reinforce the expression of their own genes whilst inhibiting other genes which are involved in differentiation. This creates an autoregulatory loop within the stem cell. They can bind to promoters of Hox genes.

34
Q

What do Embryonic Carcinoma Cells contribute to?

A

Germ cell tumours. Embryonic Germ Cells are their physiological counterpart that can be isolated from fetal gonads.

35
Q

What are two negatives with EC cells?

A
  1. Aneuploid (not a normal no. of chromosomes)
  2. Acquire imprinting anomalies
36
Q

What are four applications of embryonic stem cells?

A
  1. Therapeutic cloning
  2. Gene targeting/therapy
  3. Drug screening/toxocological tests
  4. Synthetic embryo development
37
Q

What are two types of Fetal Stem Cells?

A
  1. Cord blood SC which are collected from newborn babies, cells sorted and stored in liquid nitrogen. They’re not ethically controversial.
  2. Amniotic sac SC which can be obtained in large quantities, however there is little information about the growth characteristics and differentiation potential of these cells.
38
Q

Where are Adult SC found?

A

Specific tissues or organs of the body.

39
Q

Adult SC undergo asymetric cell division where a daughter cell is produced and a self-renewed daughter stem cell is produced. They are not as suitable for long term culture and expansion in a lab setting.

A
40
Q

They are multipotent, sometimes unipotent, and are limited to a number of cell types they can differentiate into related to the tissue they’re found in.

A
41
Q

Why is it difficult to maintain a pool of Adult Stem Cells in vitro?

A

They usually reside in a niche which is an environment that maintains SC undifferentiation.

42
Q

Haematopoietic SC form Multipotent Progenitor (MPP) Cells, what do these give rise to?

A

Myeloid or Lymphoid. These then give rise to more specialised cells (multipotent stem cell lineage)

43
Q

Why dont LT-HSC (long term) divide often?

A

This can lead to mutations = cancer

44
Q

Where are Mesenchymal SC found?

A

Bone marrow

45
Q

What do they differentiate into?

A

Osteoblasts, chondrocytes, and adipocytes

46
Q

Mesenchymal SC can aslo be induced to differentiate into muscle and tendon. They have the greatest potential for regenerative medicine. They are currently used for treating extensive wounds.

A
47
Q

Epidermal SC are a type of keratinocyte. What can they give rise to?

A

Interfollicular epidermis, hair follicles, and sebaceous gland.

48
Q

Human spermatagonial SC can differentiate into all germ layers. To be maintained in culture for extensive periods, what two components are needed?

A

LIF & GDNF

49
Q

What potency do Gut SC have and why don’t they divide very often even though the small intestine replenishes its epithelium every 24 hours?

A

Unipotency. They divide once every two weeks to prevent mutations leading to abnormalities or cancer.

50
Q

Which four factors need to be overexpressed to induce pluripotency in mouse/human cells?

A

c-Myc, Sox2, Oct4, klf4

51
Q

What is a major advanatge and major disadvantage of reprogramming cells?

A

+ Overcomes the need of generating embryos derived from pluripotent cell lines

  • Requires genetic modification of retroviral infection of cells encoding the four factors
52
Q

What are the roles of c-Myc and klf4?

A

c-Myc = an oncogene that promotes cell division
Klf4 = prevents apoptosis to help cells survive

53
Q

Why is it important that the cells are dividing?

A

Cell division opens up the chromatin which makes it accessible to Oct4 & Sox2. These TFs convert the cell to a pluripotent state so that iPS cells can be produced.

54
Q

Retroviruses/lentiviruses can deliver the factors into the cell, what is positive and negative of these?

A

+ Very effective because they infect almost all cells

  • They integrate randomly into the genome
55
Q

How can this be overcome?

A

Use of adenoviruses

56
Q

Other methods are mRNA reprogramming, episomal vectors, and protein delivery.

A
57
Q

Simply, you would harvest a skin fibroblast and convert to iPS cell, genetically modify the mutation by specific gene targeting, differentiate into embryoid bodies, differentiate into haematopoietic progenitors and transplant the correct haematopoietic progenitors into an irradiated mouse which originally had sick cell anemia (humanised).

A
58
Q

What are some applications for this?

A

Cell therapy, drug screening, disease modelling, reprogramming mechanism, conservation of endagered species.

59
Q

What is a method used to overcome defects in placentation?

A

Production of tetraploid embryos

60
Q

How are tetraploid embryos produced?

A
  1. Fusion of 2N cells from an early developing embryo via electrofusion
  2. Thew newly formed tetraploid cell is allowed to divde and give rise to an early stage embryo 4N
  3. Injection of 2N fESC (foreign) into the tetraploid embryo to contribute to forming various tissues
61
Q

What type of organism is produced by this?

A

Chimeric organism

62
Q

Why is this method used?

A

The 4N cells give rise to extraembryonic tissues like the placenta as they are slower to divide. This makes sure there are no defects in placentation and the 2N cells can give rise to a functional embryo.

Can also modifiy the fESCs used, so you can determine gene functions etc because the developing embryo is 100% ESC-derived.

63
Q

This leads to a 20%-30% success rate.

A
64
Q

iPS cells can give rise to all tissues except extraembryonic tissues because they are not totipotent.

A
65
Q

What are the three levels of gene expression in ESCs?

A

Active
Poised
Silent

66
Q

What is meant by the poised state?

A

Genes are neither fully active or fully repressed and can quickly transition from active to silent.

67
Q

Which two genes have cross antagonism leading to the production of either myeloid or eythroid cells?

A

PU1 - Myeloid
GATA1 - Erythroid

68
Q

How can they antagonistically affect each other?

A

E.g - GATA1 targets its gene to self renew itself
PU1 can bind to the GATA1 TF
PU1 brings methylating machinery with it to suppress expression by changing the histone modifications

69
Q

Why are organoids useful?

A

We can study gene function or disease modellin within a dish as we cannot see directly inside our body.

70
Q

What is interspecies chimeric complementation?

A

Where iPS (stem cells) from a species can be injected into a different species to develop a tissue or organ etc, derived from a different species’ stem cells. The stem cells have the functional gene which may be non-functional in the mutant.

71
Q

What are organoids?

A

Organ-like 3D cell cultures generated from stem cells or dissociated primary tissue.

72
Q

How can they be produced?

A

Blastocyst has pluripotent stem cells extracted from it and undergo signalling pathways or iPS cells obtained from somatic cells via induced pluripotency.

73
Q

What are the applications of organoids?

A
  1. Study the processes of embryonic development
  2. Identify treatment options for individual patients
  3. Disease models
  4. Aid in replacing damaged organs
  5. Drug toxicity and efficacy testing
74
Q

Two models of human disease using organoids:

A
  1. Cystic Fibrosis
  2. Cancer
75
Q

How are organoids effective in understanding Cystic Fibrosis?

A

CFTR can have a mutation in change of F508 and when Forskolin-induced swelling occurs for normal SC, they do not swell. But, you can genetically modify the cells so they do swell (conversion to Wt from mutated F508).

76
Q

Why are organoids useful for drug discovery?

A

2D cell culture is a poor representation of in vivo situation - lack tissue architecture, morphology, cell behaviour so organoids being 3D is a lot more useful. Quick, cheap, high throughput than animal models.

Animal biotechnology (8 decks)

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