Quiz hPSC Flashcards
Why is the epigenetic landscape of a PSC important for maintenance of pluripotency?
Allows expression of pluripotency-associated genes
Silences the expression of differentiation-associated genes
What are the common epigenetic features of pluripotency-associated genes in undifferentiated PSCs?
Open chromatin structure (euchromatin)
Low levels of DNA methylation
Nucelosomes contain histones that are aceylated
Nucleosomes contain histones that have H3K4Me3 modifications
What are bivalent domains and why are they important epigenetic features of PSCs?
Function to silence key developmental genes in PSCs while keeping them poised for activation later in development
Consist of large regions of chromatin, which feature inhibitory H3K27Me3 marks along with activating H3K4Me3 marks.
As PSCs differentiate bivalent domainsare resolved and genes either become activated or definitively repressed = retain only one type of methylation
What are the two different kinds of PSCs?
Embryonic stem cells
Induced pluripotent stem cells
What tissues are ESCs derived from?
Inner cell mass of pre-implantation blastocysts
Which cytokines support the undifferentiated proliferation of mESCs in feeder-free culture?
LIF
BMP-4
What culture conditions support the undifferentiated proliferation of monkey ESCs when they were first derived by Thomson et al?
First dervied on feeder cells with serum and LIF
If was found LIF was not needed for successful derivation
Undifferentiated non-human primate ESCs could not be maintained in feeder-free culture in medium with serum and LIF.
Therefore they have different cytokine requirements from mESCs
Which cell surface markers have been used to characterise PSC, how does their expression differ between mouse and non-human primate species?
mESCs express SSEA-1
Non-human primates do NOT express SSEA-1
They express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 = same as human embryonal carcinoma cells
How did Thomson et al demonstrate pluripotency of non-human primate ESCs?
ESCs were transplanted into immune deficient mice and allowered to form tumours
These tumours were then examined hisolofically to show that they contained cell/tissue type tha trepresents the 3 embryonic germ layers
What is the source of tissue for the derivation of hESCs?
Hyman pre-implantation embryos generated through assisted reproductive technologyies = source of tissue for derivation of hESCs
Good quality spare embryos and poor quality embryos that were being discarded (fresh and frozen) have been successfully used
What are the essential characteristics of primate ESCs set our by Thompson et al?
Derived from pre-implantaion or peri-implantation embryos
Prolonged undifferentiated proliferation in vitro
Stable developmental potential
Maintaining a normal (diploid) karyotype in culture
What is somatic cell nuclear transfer?
Technology in which the isolated nucleus of a somatic cell is transffered into an enucleated oocyte
Factors present in oocyte reprogram the somatic cell nucleus back to toipotency allowing reproductive cloning of animals
What are ntESCs and how are they made?
ntESCs are PSC lines that are produced from blastocysts made by SCNT.
They have the potential to create donor-specific PSCs
What are the limitations of human ntESCs?
Technically challening so only some labs have been successful at making human ntESCs
Technology requires use of human oocytes, which are difficult to obtain and raise theical issues around their use
What is an iPSC and how are they made?
Pluripotent stem cell that has been reprogrammed from a normal somatic cell by the process of transcription factor-mediated reprogramming
What different cell types have been used to make hiPSCs, how has this enable the more wide spread use of hiPSC versus hESCs?
A range of easily accessible somatic cells have been used to produce hiPSCs including, dermal fibroblasts, keratinocytes, peripheral blood mononuclear cells, shed urinary bladder epithelial cells
What changes in gene expression take place when a somatic cell is reprogrammed to pluripotency?
Activation of pluripotency-associated genes
Silencing of differentiation-associated genes
Silencing of transgenes for reprogramming factors
What changes occur in promoter methylation of pluripotency-associated factors i.e. OCT4 and NANOG takes place when somatic cells are reprogrammed to pluripotency?
DNA methylation of promoters of pluripotency-associated genes is erased during the process, allowing the endogenous genes to become switched on
What happens to the expression of the transgenes when somatic cells are reprogrammed to pluripotency?
When the reprogramming factors are introduced with recombinant retroviruses they will be switched off during the process and the expression of endogenous genes will take over to maintain pluripotency in iPSCs
‘When the reprogramming factors are introduced using non-integrating vectors (Sendai virus, self replicating mRNAs), the vectors will be progressively lost from the cells as they divide and the endogenous factors will take over
What are the main advantages of using hiPSCs over hESCs?
Do not need to use human embryos
Reprogramming is straightforward and many laboratories have now made hiPSCs
Can produce hiPSCs from patients with specific diseases
Can produce donor-specific hiPSCs
What are telomeres, how do they function to maintain genome integrity?
Telomeres are repeating DNA sequences at the end of chromosomes that can reach a length of 15kb
Telomeres act as insulators and function to prevent chromosomes from losing DNA from their ends when a cell divides
Telomeres stop chromosomes from fusing with each other preventing genomic re-arrangement
What are the components of the telomerase complex, how does the expression of the subunits change during development?
Telomerase is a ribonucleoprotein (RNP) complex that comprises the RNA template and telomerase reverse transcriptase
In humans the RNA template (hTR) is ubiquitously expressed
In human the reverse transcriptase (hTERT) is highly expressed in the early embryo and stem cells but is lost in most somatic cells
What happens to the telomeres of normal somatic cells with each cell division, how does this differ from hESCs?
Every time a normal somatic cell divides it’s telomeres shorten, this limits their replicative lifespan
hESCs have high levels of telomerase activity, their telomeres do not shorten with each cell division, and they can be maintained indefinitely in culture
10.What happens to hTERT and telomerase during transcription factor-mediated reprogramming
Drug discovery using hiPSC-derived cells e.g. cardiomyocytes, neurones
Disease modelling using hiPSCs from patients with specific diseases or through gene editing to make relevant genetic alterations
Regenerative medicine using a patient’s own hiPSCs to make the required cell type(s) for transplantation into affected tissues
11.What are the potential applications of hiPSCs?
hiPSCs offer a potentially limitless source of disease-relevant human cells
Potential to make cells from hiPSCs that would be difficult to obtain patients i.e. neurones from the brain
Capture the genotype of the patient, this is particularly relevant where the cause of the disease is poorly understood or if it is multifactorial
There may be differences in the genetics or pathophysiology of the disease between humans and animals, hiPSCs may therefore provide more relevant models
12.What advantages could hiPSCs offer for drug discovery and disease modelling over animal models?
hiPSCs offer a potentially limitless source of disease-relevant human cells
Potential to make cells from hiPSCs that would be difficult to obtain patients i.e. neurones from the brain
Capture the genotype of the patient, this is particularly relevant where the cause of the disease is poorly understood or if it is multifactorial
There may be differences in the genetics or pathophysiology of the disease between humans and animals, hiPSCs may therefore provide more relevant models
13.Contrast and compare the origins, potency and potential usability of hESCs, human ntESCs, and hiPSCs
hESCs are derived from pre-implantation blastocysts
Human ntESCs are derived from somatic cell nuclei reprogrammed using enucleated human oocytes
hiPSCs are made from normal somatic cells
All cell lines are pluripotent
The creation of hESC lines requires the destruction of human embryos and therefore ethical issues have limited their use
human ntESCs are made using human oocytes which has ethical issues associated and it is technically very challenging to make them. Both of these features has limited their use
human ntESC technology creates donor-specific PSCs
hiPSC technology creates ethically acceptable donor-specific PSCs
