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Flashcards in SC8 Deck (35):
1

Describe ESCs

1. dervied from ICM of blastocyst.
2. pluripotent: give rise to all embryonic tissue and generates chimeric mice
3. Proliferate idefinitely if kept in undifferentiating conditions

2

what are tetrocarcinomas

tumour containing cells from many different lineages (muscle, bone etc)

3

how are tetrocarcinomas form

ICM taken from blastocyst and cultured in vitro to produce embryoid bodies which are then injected into mice tissue, forming tetrocarcinomas

4

how can embryoid bodies give rise to a mous

if they are put back into the blastocyst

5

what is a golden assay

standard assay for ES cells: proof of stemness

6

what are the three conditions the golden assay must satisfy

1. can differentiate in vitro to produce multiple lineages
2. produce tetrocarcinoma when injected into mouse
3. if injected into blastocyst gives rise to chimeric mouse (most notable in black white chimera)
*** must use multiple cell lines to validate, not unique to a particularly line/individual

7

what is an important factor in ES self-renewal

LIF

8

what is LIF

leukemia inhibitory facto interleukin 6 cytokine

9

where is LIF normally expressed, what does this mean for culturing ESCs

in the trophectoderm, so removing ES from ICM removes source of LIF

10

what does LIF do

inhibits differentiation and allows self renewal

11

what is the mechanism of action of LIF

1. LIF binds receptors triggering signalling cascade of IL-6 family of cytokines
2. This leads to activation of JAK/STAT, specifically STAT3
3. STAT3 is an important TF of self renewal genes.

12

what happens to STAT3 deficient mouse

embryos cannot develop beyond embryonic day 7, when gastrulation begins

13

how can ESCs be grown without LIF

can be genetically engineered to be LIF independent, via overexpression of Nanog`

14

where does the earliest sign of mammalian differentiation occur

in the blastocyst stage, where ICM differentiates from the trophectoderm.

15

what are important TFs in development of mammals

OCT3/4
Nanog
STAT3
*work via antagonising or cooperating

16

why is OCT3/4 important

for development of the ICM and anatagonising development of the trophectoderm

17

What does Nanog do in the developing embryo

mediates transition from ICM to epiblast (1 of 2 distinct layers arising from the ICM) and blocks transition into primitive endoderm
therefore mediating self-renewal and blocking differentiation

18

What is a nuclear transfer experiment

nuclei removed from somatic cell or blastocyst and put into frog egg.
*higher efficiency of development if from blastocyst stage

19

how can ESCs be made from adullt cells

fuse somatic cell to ES cell, resulting tetraploidy cells are like ES.

20

Describe how iPSCs were made from mouse embryonic and adult fibroblast cultures

Differentiated cells reprogrammed to ESC state by transfer of nuclear contents into oocysts or fusion with ES
Whittled down factors needed based on growth of cell colony after removal of factor.
Four factors necessary for growth: OCT4, c-myc, Sox2 and Klf4.
Made iPSCs from fibroblasts with factors under ESC conditions: chimeric mouse proved SC

21

how can iPSCs be isolated from somatic cells

via morphological expression and OCT4 reporter expression

22

What are the 6 main milestones in iPSC research

1. four factors needed
2. hand picking colonies increases effiicicent of iPSC
3. iPSC colony formation requires several days due to extensive chromatin modifications. Using HDAC (histone deacetylates) inhibitors increases efficiency
4. NSCs have more Sox2, c-myc and can be reprogrammed with OCt4 and Klf4
5. somatic cells obtained from reprogramming can be reprogrammed again.
6. SCs easier to reprogramme than differentiated cells, however even B-cells can be reprogrammed to iPSCs.

23

what are four considerations to take into account for iPSC

1. Differences in iPSC chromatin
2. Low efficiency of reprogramming
3. Tradefoof between efficiency and cancer
4. Incomplete reprogramming

24

how does iPSC chromatin differ from ES

not identical, several iPSCs have mRNA and microRNA abnormalities

25

what is iPSC reprogramming efficiency like

quite low, often no colonies are formed.
may reflect the need for precise timing, balance and absolute levels of expression of reprogrammed genes.

26

what is the trade-off in iPSC between efficiency and cancer

mice generated from iPSCs are more susceptible to tumour formation. Oncogene expression if viruses are used to alter expression.
Inactivation of p53 shown to significantly increase efficiency - obviously more likely to lead to cancer

27

what are the problems of incomplete reprogramming in iPSC

particularly challenging due to reformation of epigenetic code.
However, three seperate groups were able to find mouse embryonic fibroblast (MEF) derived iPSC that could be injected into blastocyst resulting in live birth.

28

give an example of a diference between ESC and iPSCs

found SSEA-1 a carbohydrate antigen is found in ES but not iPSCs
* plays an important role in migration and adhesion in the pre-implantation embryo.

29

What was the 2014 attempt to target iPSC efficiency

1. pw for efficiency remodelling required down regulation of nucleosome remodelling and deacetylaation complex (NuRD).
2. over expression of Mbd3 a subunit of NuRD was found to inhibit iPSC induction.
3. Depletion was therefore shown to improve reprogramming efficiency resulting in deterministic and synchronised iPSC reprogramming. (near 100% in humans and mouse)

30

what was the 2007 attempt to target oncogenes in iPSCs

'Stem cells this time without cancer' paper by Yamanka who won nobel prize.
- reported iPSCs can be made without oncogene c-Myc.
However the progress was less efficienct and took longer, but resulting chimeras didn't develop cancer.

31

what was the 2008 attempt to target oncogenes in iPSCs

found way to remove viruses with oncgenes once expression was no longer needed.
viruses carrying reprogramming genes flanked by flow sequences so addition of protein allowed removal.

32

what did 2012 study on single cell expression analysis during cellular reprogramming reveal

an early stochastic and late heirachic phase
- the early stage of reprogramming shows considerable variation in gene expression (compared to late)
- expression of Esrrb, Utf1, Lin28 and Dppa2 found to be best markers for progress to iPSCs.
- downstream factors derived from late phase (not Oct/sox/kif/c-myce) can activate pluripotency

33

what is STAP

stimulus triggered acquisition of pluripotency
* 2 articles claimed a type of iPSC can be generated by subjecting cells to certain stress types: bacteria, low pH, physical squeezing, however papers retracted.

34

2009 paper on promotion of iPSC using ESC microRNA

microRNA miR-291 enhanced efficiency of iPSCs by acting downstream of c-Myc.
** hypothesized that this particular microRNA may block the expression of repressors of Yamankas factors

35

how do iPSC compare with ESCSs

1. simialr morphology: round, large nucleolus; similar colonies.
2. Growth properties: self-renewal, proliferation and divide at same rates
3. SC markers: iPSC express atleast some ESC cell surface markers: SSEA4
4. SC genes: iPSCs express genes expressed in undifferentiated ESCs - OCT3 etc.
5. Teleomerase activity: allows cell division to be sustained unrestricted by Hayflick limit. (expressed highly in both)
6. pluripotency
7. both can form embryoid bodies