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Stem Cell > 1 hPSC and Reprogramming > Flashcards

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 mouse 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

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25
Q

What do mESCs need for undifferentiated growth?

A

LIF and BMP4

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26
Q

What happened when rhESCs were grown without feeder cells but with LIF? What does this tell us?

A

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

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27
Q

What happend when rhESCs were maintained in culture for more than 1 year?

A

They remained undifferentiated in continuous passage for more than one year

With stable, normal XY karyotype

28
Q

What does SCID stand for?

A

Secere Combined Immune Deficient

29
Q

What happened when rhESCs were injected into SCID mice? And what does this mean?

A

rhESC formed teratomas, which contained cell types representative of the 3 embryonic germ layers

Confirming pluripotency

30
Q

What do in vitro experiments show rhESCs can differentiate into? And what does it secrete?

A

Trophoectoderm, that secretes chorionic gonadotropin

mESC do not generally differentiate into trophoectoderm

31
Q

What is the trophoectoderm?***

A

The first tissue to differentiate in a placental mammal’s embryo

It’s the outer layer of the blastocyst, and it’s essential for the embryo’s development

32
Q

What is the difference between rhESC and mESC cell-surface marker expression?

A

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 facts and gain expression of SSEA-1

mESCs express SSEA-1 and do NOT express the others

Fundamental difference between ESCs

33
Q

What cell does rhESC have similar cell-surface marker expression to?

A

Human embryonal carcinoma cells

34
Q

Could Marmoset monkey ESCs be maintained in undifferentiated state for more than 1 year?

A

Yes, like the rhesus monkey ESCs

35
Q

What happened when marmost monkey ESC were put on gelatin medium-containing serum and LIF?

A

Without the MEF feeder cells and LIF alone = mmESCs could not be maintained in undifferentiated state

36
Q

How was pluripotency confirmed in marmoset monkeys ESCs?

A

Same as rhESC

Transplanting cells into SCID mice = formed teratomas, which contained cell types representative of the 3 embryonic germ layers

37
Q

What cell surface markers did marmoset monkeys ESCs have?

A

Same as rhesus monkey ESCs

38
Q

What two things need to removed from the blastocyst to isolate the ICM?

A

Zona pelucida

Trophoectoderm

39
Q

What treatment is used to remove the zona pelucida?

A

Acid Tyrode’s solution

OR

Pronase

40
Q

What treatment is used to remove the trophoectoderm?

A

Immunosurgery

41
Q

What idid hESCs have high levels of?

A

Telomerase activity

42
Q

What are the essential characteristics of PRIMATE ESCs?

A

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
Q

What is not a testable property of many primate species ESCs?

A

Ability of ESCs to contribute to germ line = for ethical and practical reasons

44
Q

What growth factors do hESC have receptors for?

A

Stem cell factor (SCF)

Fetal liver tyrosine kinase-3 ligand (Flt3L)

Basic fibroblast growth factors (bFGFB)

45
Q

What did they discover about basic FGF?

A

Necessary and sufficient for maintenance of undifferentiated hESCs in ABSENCE of feeder cells

46
Q

What are the cell of ICM that differentiate to pluripotent state similat to?

A

Similar to pluripotent cells of the epiblast of POST-implantation blastocysts

47
Q

What do naive hESC lines more closely resemble?

A

Pluripotent cells present in ICM of PRE-implantation blastocysts

48
Q

What state are conventional hESC regarded to be in?

A

‘primed’ pluripotent state

49
Q

What are the limitations of hESCs?

A

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
Q

How do we overcome limitations of using hESCs?

A

Find alternative methods of producing pluripotent stem cells

To enable production of donor-specific stem cell lines

51
Q

What is somatic cell nuclear transfer (SCNT)?

A

Reprogramming somatic cells to pluripotency

52
Q

What did SCNT prove?

A

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
Q

What is the process of somatic cell nuclear transfer?

A

Enucleate an oocyte

Take out somatic cell nucleus and implant it into enucleated oocyte

Reprogramme the nucleus with signals from the oocyte

54
Q

What has somatic cell nuclear transfer been used to do?

A

Reproductive cloning

AND

production of ESCs from blastocysts = donor-specific nuclear transfer ESCs (ntESCs)

55
Q

What phase is the enucleated occyte in?

A

Metaphase II

56
Q

After nuclear transfer, what do metaphase promoting factors do?***

A

Lead to the breakdown of the somatic nucleus’ membrane

and premature chromosome condensation

Metaphase promotin factors = already present in ooplasm

57
Q

After you get a reconstituted oocyte, what needs to be done and why?

A

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
Q

How is degradation of metaphase promoting factors mimicked in SCNT?***

A

DMAP?

59
Q

What are donor-specific nuclear transfer ESCs?

A

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
Q

What is the process of making ntESCs?

A

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
Q

What is the use of inactivated HVJ-E envelope?***

A

The use of envelope from inactivated hemagglutinating virus of Japan (HVJ-E) = to fuse nuclear donor cells with enucleated MII oocytes while maintaining cytoplasts in meiosis

62
Q

What stage are somatic cell arrest in?

A

G0/G1

63
Q

What are the advanced techniques for therapeutic cloning using ntESCs?

A

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
Q

What is the role of DMAP and trichostatin A?***

A

DMAP = inhibitor of metaphase promoting factors (acts like PLC-zeta would)

Trichostatin A = histone deacetylase inhibitor (keeps chromatin in open conformation)

65
Q

What are the limitations of ntESCs?***

A

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

Stem Cell (7 decks)

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