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1 hPSC and Reprogramming Flashcards

1
Q

What makes a pluripotent stem cell?

A

An undifferentiated cells that can self-renew and differentiate

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2
Q

Define self-renewal

A

When stem cells divides to produce 2 identical undifferentiated daughter cells

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3
Q

Define how stem cells can differentiate

A

Can differentiate to produce all the different cell types that make up the body

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4
Q

What are the 3 germ layers?

A

Ectoderm, mesoderm & endoderm

They form during gastrulation

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5
Q

What is gastrulation?

A

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

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6
Q

Describe the epigenetic landscape of pluripotent stem cells

A

Allows expression of pluripotency-associated genes

Silencing of differentiation-associated genes

Maintenance of pluripotent state

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7
Q

What are the common features of pluripotency-associated genes?

A

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)

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8
Q

What epigenetic landscape changes occur during stem cell differentiation?

A

Chromatin structure

Nucleosome position

DNA methylation

Histone post translational-modification

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9
Q

What are bivalent domains?

A

A novel epigenetic signature = featured in many developmental genes in PSCs

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10
Q

What do bivalent domains do?

A

Proposed to function to silence genes in undifferentiated cells while keeping them poised for activation later in development

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11
Q

Describe a bivalent domain

A

Consists of large regions of chromatin w inhibitory H3K27Me3 along with activating H3K4Me3 marks

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12
Q

What happens to bivalent domains as PSCs differentiate?

A

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

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13
Q

Describe the pluripotent stem cell niche (and what this means)

A

Locally stable; Globally unstable

Locally stable = small pertubations can be accommodated

Large pertubations = trigger cell differentiation

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14
Q

What is a large perturbation that can trigger cell differentiation?

A

Removal or additions of different (growth) factors

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15
Q

What is different between ESCs and iPSCs, and what effect does this have?

A

They have different cellular origins

This affects their potential uses

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16
Q

What is a teratocarcinoma?

A

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

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17
Q

Describe primate embryonal carcinomas

A

Highly aneuploidy

Have restricted ability to differentiate into wide range of somatic cells in vitro

Limits their use

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18
Q

Why are hPSC of interest to researchers?

A

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)

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19
Q

What are ESCs derived from?

A

Pre-implantation embryos

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20
Q

What pluripotency-associated factors do ESCs express and their function?

A

Transcription factors OCT4, SOX2, NANOG

Function to maintain self-renewal of undifferentiated cells

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21
Q

How long does the self-renewing state last?

A

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

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22
Q

When do cells transition from totipotent to pluripotent?

A

Zygote to 8-cells (totipotent)

Becomes MORULA

After morula, early blastocysts (pluripotent)

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23
Q

What epigenetic change occurs when pluripotency comes about?

A

DNA methylation increases after blastocyst

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24
Q

When does zygotic genome activation occur and what does it do?***

A

Activation occurs at 4-cell stage

It is the switch from maternal RNA to zygotic RNA

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25
What do mESCs need for undifferentiated growth?
LIF and BMP4
26
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
27
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
28
What does SCID stand for?
Severe Combined Immune Deficient
29
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
30
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
31
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
32
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
33
What cell does rhESC have similar cell-surface marker expression to?
Human embryonal carcinoma cells
34
Could Marmoset monkey ESCs be maintained in undifferentiated state for more than 1 year?
Yes, like the rhesus monkey ESCs
35
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
36
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
37
What cell surface markers did marmoset monkeys ESCs have?
Same as rhesus monkey ESCs
38
What two things need to removed from the blastocyst to isolate the ICM?
Zona pelucida Trophoectoderm
39
What treatment is used to remove the zona pelucida?
Acid Tyrode's solution OR Pronase
40
What treatment is used to remove the trophoectoderm?
Immunosurgery
41
What enzyme do hESCs have high levels of?
Telomerase activity
42
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
43
What is not a testable property of many primate species ESCs?
Ability of ESCs to contribute to germ line = for ethical and practical reasons
44
What growth factors do hESC have receptors for?
Stem cell factor (SCF) Fetal liver tyrosine kinase-3 ligand (Flt3L) Basic fibroblast growth factors (bFGFB)
45
What did they discover about basic FGF?
Necessary and sufficient for maintenance of undifferentiated hESCs in ABSENCE of feeder cells
46
What are the cell of ICM that differentiate to pluripotent state similat to?
Similar to pluripotent cells of the epiblast of POST-implantation blastocysts
47
What do naive hESC lines more closely resemble?
Pluripotent cells present in ICM of PRE-implantation blastocysts
48
What state are conventional hESC regarded to be in?
'primed' pluripotent state
49
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
50
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
51
What is somatic cell nuclear transfer (SCNT)?
Reprogramming somatic cells to pluripotency
52
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)
53
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
54
What has somatic cell nuclear transfer been used to do?
Reproductive cloning AND production of ESCs from blastocysts = donor-specific nuclear transfer ESCs (ntESCs)
55
What phase is the enucleated occyte in?
Metaphase II
56
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
57
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
58
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
59
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
60
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
61
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
62
What stage are somatic cell arrest in?
G0/G1
63
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
64
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)
65
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

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