1 hPSC and Reprogramming Flashcards
What makes a pluripotent stem cell?
An undifferentiated cells that can self-renew and differentiate
Define self-renewal
When stem cells divides to produce 2 identical undifferentiated daughter cells
Define how stem cells can differentiate
Can differentiate to produce all the different cell types that make up the body
What are the 3 germ layers?
Ectoderm, mesoderm & endoderm
They form during gastrulation
What is gastrulation?
Gastrulation occurs during week 3 of human development.
The process of gastrulation generates the three primary germ layers ectoderm, endoderm, and mesoderm.
Gastrulation primes the system for organogenesis and is one of the most critical steps of development
Describe the epigenetic landscape of pluripotent stem cells
Allows expression of pluripotency-associated genes
Silencing of differentiation-associated genes
Maintenance of pluripotent state
What are the common features of pluripotency-associated genes?
Open chromatin (euchromatin)
Low levels of DNA methylation (since open structure)
Histones enriched with active marks (acetylation, H3K4 methylation)
Histones depleted of inhibitory marks (H3K9 + H3K27 methylation)
What epigenetic landscape changes occur during stem cell differentiation?
Chromatin structure
Nucleosome position
DNA methylation
Histone post translational-modification
What are bivalent domains?
A novel epigenetic signature = featured in many developmental genes in PSCs
What do bivalent domains do?
Proposed to function to silence genes in undifferentiated cells while keeping them poised for activation later in development
Describe a bivalent domain
Consists of large regions of chromatin w inhibitory H3K27Me3 along with activating H3K4Me3 marks
What happens to bivalent domains as PSCs differentiate?
Domains are resolved and developmental genes become marked with either K4 or K27 methylation
This gives two possible outcomes:
Removal of H3K27Me3 = gene activation
Removal of H3K4Me3 = gene suppression
Describe the pluripotent stem cell niche (and what this means)
Locally stable; Globally unstable
Locally stable = small pertubations can be accommodated
Large pertubations = trigger cell differentiation
What is a large perturbation that can trigger cell differentiation?
Removal or additions of different (growth) factors
What is different between ESCs and iPSCs, and what effect does this have?
They have different cellular origins
This affects their potential uses
What is a teratocarcinoma?
Malignant germ cell tumour = that comprises an undifferentiated embryonal carcinoma component + differentiated component that can include cell types that are representative of the 3 embyonic germ layers
Describe primate embryonal carcinomas
Highly aneuploidy
Have restricted ability to differentiate into wide range of somatic cells in vitro
Limits their use
Why are hPSC of interest to researchers?
Provide limitless source of undifferentiated cells to study (pathwyas/mechanisms regulating pluripotency, lineage commitment, differentiation in the early human embryo)
Can be differentiated to produce cell type of interest for diverse application (disease modelling, drug discover, regenrative medicine)
What are ESCs derived from?
Pre-implantation embryos
What pluripotency-associated factors do ESCs express and their function?
Transcription factors OCT4, SOX2, NANOG
Function to maintain self-renewal of undifferentiated cells
How long does the self-renewing state last?
ESCs can be stably maintain in self-renewal IN VITRO
Self-renewal only exists TRANSIENTLY in vivo in early embryo = lost upon specification of the epiblast
When do cells transition from totipotent to pluripotent?
Zygote to 8-cells (totipotent)
Becomes MORULA
After morula, early blastocysts (pluripotent)
What epigenetic change occurs when pluripotency comes about?
DNA methylation increases after blastocyst
When does zygotic genome activation occur and what does it do?***
Activation occurs at 4-cell stage
It is the switch from maternal RNA to zygotic RNA
What do mESCs need for undifferentiated growth?
LIF and BMP4
What happened when rhESCs were grown without feeder cells but with LIF? What does this tell us?
Without MEF feeder cells, the rhESC died or differentiated
Shows LIF failed to support UNDIFFERENTIATED growth of rhESCs on gelatin in serum-containing medium
Indicates they require different culture conditions than mESCs
What happend when rhESCs were maintained in culture for more than 1 year?
They remained undifferentiated in continuous passage for more than one year
With stable, normal XY karyotype
What does SCID stand for?
Severe Combined Immune Deficient
What happened when rhESCs were injected into SCID mice? And what does this mean?
rhESC formed teratomas, which contained cell types representative of the 3 embryonic germ layers
Confirming pluripotency
What do in vitro experiments show rhESCs can differentiate into? And what does it secrete?
Trophoectoderm, that secretes chorionic gonadotropin
mESC do not generally differentiate into trophoectoderm
What is the trophoectoderm?***
It’s the outer layer of the blastocyst, and it’s essential for the embryo’s development
Trophoectroderm becomes trophoblast
Trophoblasts are cells that develop into the placenta
What is the difference between rhESC and mESC cell-surface marker expression?
rhESC express alkaline phosphatase and SSEA-3/4 and TRA-1-60/81
No expression of SSEA-1 (same as human embyonal carcinoma cells)
When rhESCs differentiate = lose expression of other factors and gain expression of SSEA-1
mESCs express SSEA-1 and do NOT express the others
Fundamental difference between ESCs
What cell does rhESC have similar cell-surface marker expression to?
Human embryonal carcinoma cells
Could Marmoset monkey ESCs be maintained in undifferentiated state for more than 1 year?
Yes, like the rhesus monkey ESCs
What happened when marmost monkey ESC were put on gelatin medium-containing serum and LIF?
Without the MEF feeder cells and LIF alone = mmESCs could not be maintained in undifferentiated state
How was pluripotency confirmed in marmoset monkeys ESCs?
Same as rhESC
Transplanting cells into SCID mice = formed teratomas, which contained cell types representative of the 3 embryonic germ layers
What cell surface markers did marmoset monkeys ESCs have?
Same as rhesus monkey ESCs
What two things need to removed from the blastocyst to isolate the ICM?
Zona pelucida
Trophoectoderm
What treatment is used to remove the zona pelucida?
Acid Tyrode’s solution
OR
Pronase
What treatment is used to remove the trophoectoderm?
Immunosurgery
What enzyme do hESCs have high levels of?
Telomerase activity
What are the essential characteristics of PRIMATE ESCs?
Dervied from pre-implantation or post-implantation embryos
Prolonged undifferentiated proliferation in vitro (maintaining diploid normal karyotype)
Stable developmental potential even after prolonged culture
What is not a testable property of many primate species ESCs?
Ability of ESCs to contribute to germ line = for ethical and practical reasons
What growth factors do hESC have receptors for?
Stem cell factor (SCF)
Fetal liver tyrosine kinase-3 ligand (Flt3L)
Basic fibroblast growth factors (bFGFB)
What did they discover about basic FGF?
Necessary and sufficient for maintenance of undifferentiated hESCs in ABSENCE of feeder cells
What are the cell of ICM that differentiate to pluripotent state similat to?
Similar to pluripotent cells of the epiblast of POST-implantation blastocysts
What do naive hESC lines more closely resemble?
Pluripotent cells present in ICM of PRE-implantation blastocysts
What state are conventional hESC regarded to be in?
‘primed’ pluripotent state
What are the limitations of hESCs?
Require the use of human embryos = ethical implicatoins
Their use is controlled by legal framework
Resulting in limited number of hESC lines produced = limited genetic diversit
How do we overcome limitations of using hESCs?
Find alternative methods of producing pluripotent stem cells
To enable production of donor-specific stem cell lines
What is somatic cell nuclear transfer (SCNT)?
Reprogramming somatic cells to pluripotency
What did SCNT prove?
Differentiated state of cell could be reversed
Factors present in oocyte = sufficient to reprogram a somatic nucleus back to pluripotency
(able to reverse the epigenetic remodelling that occurs in differentiation)
What is the process of somatic cell nuclear transfer?
Enucleate an oocyte
Take out somatic cell nucleus and implant it into enucleated oocyte
Reprogramme the nucleus with signals from the oocyte
What has somatic cell nuclear transfer been used to do?
Reproductive cloning
AND
production of ESCs from blastocysts = donor-specific nuclear transfer ESCs (ntESCs)
What phase is the enucleated occyte in?
Metaphase II
What happens once metaphase promoting factors are broken down?
Transition to anaphase
Lead to the breakdown of the somatic nucleus’ membrane
and premature chromosome condensation
Metaphase promoting factors = already present in ooplasm
After you get a reconstituted oocyte, what needs to be done and why?
Degradation of metaphase promoting factors so that cell can shift to anaphase
In fertilization, the spermocyte PLC-seta normally does this = need to mimick it in SCNT
How is degradation of metaphase promoting factors mimicked in SCNT?***
DMAP1 = Decreased Metaphase Arrest Protein 1
Directly promotes cyclin B degradation
MPF = Cyclin B/CDK1
What are donor-specific nuclear transfer ESCs?
Production of ESCs from the somatic cell of the patient themselves = less chance of rejection
Can make blastocysts and harves ICM to make ntESCs
What is the process of making ntESCs?
Hold oocyte in metaphase-II
Somatic cell arrested in G0/G1 + inactivated HVJ-E envelope = inserted into periviteline space
Cell fusion forming reconstructed oocyte
Reconstructed oocyte activated with electroporation = increase the permeability of the cell membrane
DMAP inhibits MPF
Trichostatin A inhibits histone deacetylase
Forms SCNT construct
What is the use of inactivated HVJ-E envelope?***
The use of envelope from inactivated hemagglutinating virus of Japan (HVJ-E)
To fuse donor somatic cell nucleus with enucleated MII oocytes while maintaining the cytoplasmin meiosis
What stage are somatic cell arrest in?
G0/G1
What are the advanced techniques for therapeutic cloning using ntESCs?
Removal of genome from oocyte without compromising it’s reprogramming potential
Cell fusion for nuclear transfer
Use of caffeine to hold oocyte in metaphase-II = to allow efficient premature condensation of the somatic cell chromatin
Oocyte activation and use of DMAP and trichostatin A = to favour formation of pseudo-pronucleus and inifiation of development
What is the role of DMAP and trichostatin A?***
DMAP = inhibitor of metaphase promoting factors (acts like PLC-zeta would)
Trichostatin A = histone deacetylase inhibitor (keeps chromatin in open conformation)
What are the limitations of ntESCs?***
Derivation of human ntESCs requires use of human oocytes
Technique is limited because difficult to obtain oocytes
Use of oocytes and creation of human blastocysts through SCNT = ethical issues
Diffical SCNT procedures = may not be widely transferable between labs
