Lecture 6 - iPSCs Flashcards
ESCs are derived from the BLANK of BLANK
ESCs are derived from the inner cell mass (ICM) of pre-implantation embryos
Embryonic stem cells express a network of pluripotency factors, including transcription factors Oct4, Sox2, and Nanog that maintain self-renewal
TRUE OR FALSE
TRUE
iPSCs are derived from BLANK by BLANK
iPSCs are derived from normal somatic cells by transcription factor mediated reprogramming
Which 4 transcription factors are highly expressed in ESCs that reprogram cells to pluripotency?
Klf4, Oct4, Sox2, c-myc
There is also a 2nd group of transcription factors than can reprogram somatic cells to pluripotency: Oct4, Sox2, Nanog, LIN28
iPSCs were first generated using BLANK to deliver the 4 transcription factors
iPSCs were first generated using recombinant retroviral factors to deliver the 4 transcription factors
Retroviruses stably integrated into the host cell genome, giving sustained expression of transgenes:
- Virus taken up by receptor-mediated endocytosis
- Viral RNA genome reverse transcribed to viral DNA which enters the nucleus
- Viral DNA integrated into the host genome and the transgenes are then expressed.
Non-integrating methods of gene delivery for iPSC generation:
[3 methods]
- Plasmid DNA transfection
- Recombinant Sendai virus vectors
- mRNA transfection (Uses synthetic mRNA or self-replicating RNA (srRNA))
Sox2 is dispensable for creation of iPSCs but acts as an enhancer of kinetics and efficiency of reprogramming
TRUE OR FALSE
FALSE
c-myc is dispensable for creation of iPSCs but acts as an enhancer of kinetics and efficiency of reprogramming
Reprogramming to pluripotency is a major epigenetic remodelling process. What general epigenetic changes occur?
Silencing of genes associated with differentiation
Activation of genes associated with pluripotency
Silencing of reprogramming factors
Appropriate transcription of endogenous BLANK, BLANK, and BLANK is required to stabilize a pluripotency gene network that facilitates the reprogramming process
Appropriate transcription of endogenous Oct4, Sox2, and Nanog is required to stabilize a pluripotency gene network that facilitates the reprogramming process
Chromatin remodelling during iPSC reprogramming:
- Chromatin adopts open conformation characterised by sparse dis-organised regions of heterochromatin
- Large number of nucleosomes are disassembled and re-positioned
- Which correlates with changes in gene expression
Histone acetylation during iPSC reprogramming:
There is a global increase in histone acetylation during reprogramming
There are dynamic changes in H3 during reprogramming:
- H3K4me promotes transcriptionally active conformation
- H3K9me and H3K27me promote transcriptionally repressive conformations
- Initial stages of reprogramming there is widespread acquisition of H3K4me3 at the promoters and enhancers of pluripotency genes
- H3K4me primes, but doesn’t trigger expression of pluripotency genes
During reprogramming there is a global decrease in H3K9me and H3K27me:
-H3K27me is retained on a highly selected group of developmental genes that must be silenced for reprogramming to take place
Increase in H3K4, retention of H3K27me, during reprogramming re-establishes bi-valent domains:
TRUE OR FALSE
TRUE
DNA methylation and iPSC reprogramming:
During reprogramming there is a global decrease in DNA methylation
During reprogramming, reactivation of silent-x chromosome in female iPSCs
DNA methylation functions to silence genes involved in differentiation and the reactivation of pluripotency genes
In humans, almost all somatic cells lack telomerase activity.
TRUE OR FALSE
TRUE
hESCs have high telomerase levels, which maintains their telomere length allowing indefinite proliferation in culture
The re-activation of telomerase and the extension of telomeres is a key step in iPSC reprogramming.
TRUE OR FALSE
TRUE
