2 hPSC and Reprogramming Flashcards
What are the two different kinds of pluripotent stem cells?
ESCs and iPSCs
What do ESCs and iPSCs have in common and different?
Similar properties
Different cellular origins, which affects their potential uses
How are iPSCs derived?
Derived from normal somatic cells by transcription factor-mediated reprogramming
What 4 transcription factors are highly expressed in ESCs?
KLF4, OCT4, SOX2, c-MYC
What do OKSM transcription factors have the ability to do and where are they upregulated?
Highly expressed in ESCs
Ability to reprogramme a somatic cell to pluripotency = make iPSC
What properties of ESCs do iPSCs display?
Morphology
Growth properties
ESC marker genes
What happened when subcutaneous transplantation of iPSCs into nude mice?
Resulted in tumors containing a variety of tissues from all three germ layers
What happened when iPSC was injected into blastocyst?
iPS cells contributed to mouse embryonic development
What is the role of OCT-4, SOX2, and Nanog in pluripotency?
Maintenance of pluripotency in BOTH early embryos & ESCs
What is the function of c-MYC, KLF-4, E-Ras, and Stat3 in pluripotency?
Long-term maintenance of ESC phenotype and rapid prolifation of ESCs in culture
These factors are requently upregulated in tumours
What is G418 selection?
When gene of interest is expressed then the promoter also transcribes cassette with resistance gene
These cells then survive concentrations of the antibiotic G418
Induction of the pluripotent state could be detected as resistance to G418
How were each of the 24 candidate genes introduced into mouse embryonic fibroblasts (MEFs) from Fbx15βgeo/βgeo embryos?
Retroviral infection
What did resistance to G418 say about the cells?
They were in pluripotent state
They did not obtain drug-resistant colonies with any single factor, what does this indicate?
No single candidate gene was sufficient to activate the Fbx15 locus
What two genes are essential for reprogramming to pluripotency?
OCT4 and Klf4
We know this because when these genes are individually removed no G418-resistant colonies form
What happened when Sox2 was removed?
Very few G418-resistant colonies formed
What happened when c-MYC was removed?
G418-resistance colonies did form but with flatter, non-ESC-like morphology
What are Yamanaka factors vs Thomson factors?
Klf-4, c-MYC, OCT4, SOX2
Lin28, Nanog, OCT4, SOX2
What are the benefits and disadvantages of useign recombinant retroviral vectors to deliver the reprogramming factors?
Stable integration into host cell genome and sustained expression of transgene
RISK OF INESRTIONAL MUTAGENESIS
How are recombinant retroviral vectors used to deliver the reprogramming factors? ***
Envelope protein binds to LDL receptor expressed on most cells
Receptor mediated endocytosis
What do hiPSCs have in common with hESCs?
Cell morphology
Proliferation
Surface antigens
Gene expression
Epigenetic status of plripotent cell-specific genes
Telomerase activity
Compare HDF with iPSC methylation
HDF pluripotent genes have highly methylated CpG = silenced compared to iPSCs
Are the expression profiles of hiPSCs the same as hESCs?
They are similar but not the same
What is c-MYC function?
Cell cycle regulator controlling numerous genes involved in cell cycle control and metabolism
NOT a pluripotency associated gene
What is the advantage of transcription factor-mediated reprogramming?
Generation of ethically acceptable, donor specific PSC wihtout use of human embryos or oocytes
What are the limitations of transcription factor-mediated reprogramming?
Process if slow and inefficient
Only small % of cells that express reprogramming factors becomes iPSCs
But it is simpler than SCNT for reprogramming
What epigenetic remodelling occurs in TF-reprogramming?
Chromatin structure change
DNA methylation change
Histone post-translational modifications
What may be a rate limiting step in conversion of somatic cells to iPSCs?
Demethylation of promoters of pluripotency-associated genes
How does DNA methylation change?
Demethylation of pluripotency-associated genes
Methylation of differentiation associated genes = silencing
What is the role of the TET enzyme?
Initiates demethylation
By catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine
What might cause partially reprogrammed cells to fail to reactivate endogenous genes?
Insuffient DNA demethylation at promoter regions of pluripotency-associated genes by TET1
This leads to failure to reactivate the endogenous genes leading to the process stalling
What is the global change in acetylation during reprogramming?
Global increase because histone activator
What is the global change in H3K4 methlyation during reprogramming?
Widespread acquisition at promoters
Enhancer of pluripotency genes
Histone activator
What is the global change in H3K9 methlyation during reprogramming?
Global decrease because histone repressor
Specifically decreased at promoters of pluripotency genes
What is the global change in H3K27methlyation during reprogramming?
Global decrease because histone reperssor
Retained at developmental genes = leading to re-establishment of bivalent domains
What are bivalent domains?***
Bivalent domains are regions of chromatin in a cell’s DNA that simultaneously carry two different, and often opposing, histone modifications: H3K4me3 and H3K27me3.
These modifications are typically associated with gene activation (H3K4me3) and repression (H3K27me3), respectively.
In embryonic stem cells and other stem cells, bivalent domains are found at the promoters of developmental genes and help to maintain these genes in a poised, but inactive state, ready for rapid activation when needed during cell differentiation.
What are the net effect of epigenetic changes in reprogramming?
Silence genes associated with differentiated state
Re-establish bivalent domains
Activation of endogenous pluripotency-associated genes = that maintain pluripotent state of iPSC
Silencing exogenous reprogramming factors
What happens to telomerase in reprogramming?
It is reactivated so that telomeres can be extended = allowing division
What are the components of telomerase?
hTERT = telomerase reverse transcriptase
hTR = RNA template
Why is telomerase activity lost in most somatic cells?
Because telomerae reverse transcriptase expression is switched off during development
hTR (RNA template) is ubiquitously expressed
What is the Hayflick limit?
Number of cell division a cell can divide before becoming senescent
What triggers senescence?
Aka mortality stage 1 (M1) = triggered when telomeres typically reach 4-6kbp in length
How do cells fall into crisis or mortality stage 2 (M2)?
If cell escapes replicative senescence (M1) by inactivating cell cycle checkpoint (p53)
Continue to suffer telomere loss until they reach M2
What occurs in crisis?
Massive cell death triggered by critically short and dysfunctional telomeres
What occurs if a cell escapes crisis?
Able to maintain telomere length = in most cases by reactivation of hTERT gene expression
This leads to unlimited cellular proliferation = spontaneous immortalization
What are 3 aplications of hiPSCs?
Drug discorvery
Disease modelling
Autologous cell therapy
How are iPSCs useful in drug discovery?
Source of differentiated cell types of interest that may normally be dificult to access or may have limited potential to proliferate
Better than using animal models, which may differ in pathophysiology
How can CRISPR/Cas9 be used?
Make insertions/deletions at specific genetic loci = disrupt gene leads to loss of function
Knock-in a donor DNA seq to tag a gene of interest or introduce a specific mutation
Name the repair methods after double-stranded break
Non-homologous end joining = disrupts gene of interest
Homology-directed repair = corrects gene of interest
How can gene editing of iPSCs help us?
Make a pateint-derived iPSC
Correct a mutation in the patient iPSC
Study effects of the mutation by compareing fixed and mutated iPSCs
Then put it back into patient
What is the benefit of useing gene editing to creat a cell line with specific mutations in a gene of interest?
Can make a series of mutation using a single hiPSC line
Posible to study the consequence of those mutations without having to consider other genetic differences arising from multiple donors
What are hiPSC good manufacturing practice standards?
HiPSCs need to be dervied, maintained and differentiated into relevant cell types using XENOFREE reagents
No products of animal origin = minimise risk of introducing and pathogens of animal origins into patients
Avoid insertional mutagenesis
Extensive characterization of the cells to be transplanted = ensuring purity and functionality
Purity = no risk of tumourigenicity from contaminating undifferentiated hiPSCs
How can we prevent the possiblity of insertional mutagenesis when making iPSCs?
Reprogramme using integration free method
Self-replicating mRNA
Sendai virus
How does self-replicating RNA prevent insertional mutagenesis?***
It doesn’t integrate into the host genome
Uses RNA polymerase to be translated and expressed
How does Sendai virus prevent insertional mutagenesis?***
They enter the host cell and release thier RNA into the cytoplasm of the cell
They do not possess reverse transcriptase
The RNA is translated using its own RNA-dependent RNA polymerase
What are ntESCs?
Nuclear transfer embryonic stem cells (ntESCs) are pluripotent stem cells (PSCs) that are generated from somatic cells using the process of somatic cell nuclear transfer (SCNT). The process of generating ntESCs is also called therapeutic cloning
What is the difference between ntESC and iPSC?
ntESC are derived from embryos/oocytes and reprogrammed in hours (fast) to totipotency during SCNT
iPSCs are created from reprogramming comatic cells using Yamanaka factors which takes weeks (slow)
Both are donor specific, which ESCs are NOT
