Flashcards in SC8 Deck (35):
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
what are tetrocarcinomas
tumour containing cells from many different lineages (muscle, bone etc)
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
how can embryoid bodies give rise to a mous
if they are put back into the blastocyst
what is a golden assay
standard assay for ES cells: proof of stemness
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
what is an important factor in ES self-renewal
what is LIF
leukemia inhibitory facto interleukin 6 cytokine
where is LIF normally expressed, what does this mean for culturing ESCs
in the trophectoderm, so removing ES from ICM removes source of LIF
what does LIF do
inhibits differentiation and allows self renewal
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.
what happens to STAT3 deficient mouse
embryos cannot develop beyond embryonic day 7, when gastrulation begins
how can ESCs be grown without LIF
can be genetically engineered to be LIF independent, via overexpression of Nanog`
where does the earliest sign of mammalian differentiation occur
in the blastocyst stage, where ICM differentiates from the trophectoderm.
what are important TFs in development of mammals
*work via antagonising or cooperating
why is OCT3/4 important
for development of the ICM and anatagonising development of the trophectoderm
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
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
how can ESCs be made from adullt cells
fuse somatic cell to ES cell, resulting tetraploidy cells are like ES.
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
how can iPSCs be isolated from somatic cells
via morphological expression and OCT4 reporter expression
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.
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
how does iPSC chromatin differ from ES
not identical, several iPSCs have mRNA and microRNA abnormalities
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.
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
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.
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.
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)
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.
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.
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
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.
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