Les epigenetics

it can self-renew and differentiate

two ‘super-powers’ of stem cells

EpiSC, EG, EC and iPS cells.

What are examples of pluripotent stem cell types besides ES cells?

From the epiblast, a later stage than the inner cell mass.

From which developmental stage are EpiSC derived?

From primordial germ cells.

From which cells are EG cells derived?

Pluripotent cells derived from teratocarcinomas (tumors).

What are EC cells?

Hematopoietic, intestinal epithelial and spermatogonial stem cells.

Give examples of adult stem cells.

Homeostasis, tissue maintenance and repair.

What is the main function of adult stem cells?

Oogenesis is a complex multistep process starting during embryonic life.

Why is producing mature oocytes biologically difficult?

Only about 500.

How many oocytes typically mature during a woman's lifetime?

• CM = apolar
  → cellen zitten als een compacte, minder georganiseerde celmassa in de blastocyst

• Epiblast = polar
  → cellen krijgen een duidelijke oriëntatie: een “boven- en onderkant”

• Die polariteit hoort bij verdere organisatie van het embryo
  → epiblastcellen vormen een meer geordende laag/structuur

Principe: de ICM is nog een compacte apolaire celgroep, terwijl de epiblast al meer georganiseerd en gepolariseerd is tijdens verdere embryonale ontwikkeling

What is the polarity difference between ICM and epiblast?

Naïve pluripotency.

What pluripotent state do mESC represent?

Primed pluripotency.

What pluripotent state do mEpiSC represent?

mEpiSC.

Which mouse stem cell type is most similar to conventional hESC?

Primed.

Are conventional hESC usually naïve or primed?

Human PSCs are less stable in the naïve state and need specific culture conditions.

Why are naïve human PSCs harder to maintain?

mESC can integrate into a blastocyst and contribute to chimeras, while primed EpiSC cannot.

What functional property distinguishes mESC from primed EpiSC?

To gain stronger blastocyst integration potential.

Why might hESC need conversion to a naïve state?

Retention of marks from the original donor cell that biases differentiation toward related lineages and can reduce efficiency toward unrelated cell fates.

What is epigenetic memory in iPSCs?

Disease modelling, drug screening, gene correction and generation of transplantable healthy cells.

What can patient-specific hiPSCs be used for in personalized medicine?

Different drugs can be tested in vitro on patient-derived cells to predict response.

Why can patient-specific hiPSCs help choose treatments?

Derived cells often resemble immature rather than fully adult cell types.

What is a major limitation of iPSC-derived disease models?

It may fail to accurately reproduce adult disease characteristics.

Why is immature phenotype a problem in disease modelling?

Correct the mutation in patient-derived hiPSCs, differentiate them into healthy cells, then transplant them.

How can genetic diseases potentially be treated using hiPSCs?

They are more restricted: mESCs mainly form epiblast-derived embryonic tissues and normally do not generate trophectoderm.

How do lineage boundaries differ in mouse mESCs?

Amnion.

What extra-embryonic lineage can primed human PSCs generate more readily than trophectoderm?

Naïve hPSCs have broader developmental potential, while primed hPSCs are more lineage-restricted.

What is a key difference between naïve and primed human PSCs?

In vitro differentiation

teratoma formation

chimera formation

tetraploid complementation.

What are common ways to test pluripotency?

Whether they can generate multiple cell lineages.

What does in vitro differentiation test in pluripotent stem cells?

That pluripotent stem cells can form tissues from different germ layers in vivo.

What does teratoma formation demonstrate?

Whether donor stem cells can integrate into an embryo and contribute to multiple tissues.

What is chimera formation used to test?

Tetraploid complementation.

What is considered the most stringent test of pluripotency?

• Een tetraploid embryo heeft 4n-cellen
  → die kunnen vooral extra-embryonale weefsels vormen, zoals placenta

• De toegevoegde diploid PSCs hebben 2n-cellen
  → die moeten dan bijna volledig het embryo proper vormen

• Als er een levende embryo/foetus ontstaat, betekent dit dat de PSCs alle embryonale celtypes kunnen maken

• Daarom is dit een veel strengere test dan bv. teratoma formation of in vitro differentiation

Principe: tetraploid complementation bewijst dat PSCs niet alleen losse celtypes kunnen vormen, maar functioneel een volledig embryo proper kunnen opbouwen.

Why is tetraploid complementation such a strong test?

As embryos grow, they need more efficient oxygenation and nutrient exchange.

Why are embryos transferred to a rotating culture system after early static culture?

They undergo large-scale epigenetic resetting. So an exception of the Waddington’s landscape

What is special about germ cells during development?

Through epigenetic differences that switch specific genes on or off, leading to different cell fates.

How can cells with the same DNA develop into different cell types?

• Epigenetic regulation changes how accessible genes are

• It does not change the DNA sequence

• It is relatively stable and can be passed to daughter cells

• This helps maintain cell identity after cell division

Principe: epigenetics fixes gene expression states by heritable changes in chromatin accessibility, not by changing the DNA code.

What distinguishes epigenetic regulation from ordinary gene regulation?

It helps cells maintain their identity after cell division, such as liver cells remaining liver cells.

Why is heritable epigenetic regulation important in tissues?

Cells may lose normal identity and control, increasing the risk of disorders such as cancer.

How can loss of stable epigenetic identity contribute to disease?

DNA methylation, histone modifications/variants, ATP-dependent chromatin remodeling complexes, and non-coding RNAs.

What are four major mechanisms of epigenetic regulation?

Writers add marks, erasers remove marks, and readers recognize marks to influence gene expression.

What do writer, eraser, and reader proteins do in epigenetics?

On the tails of histone proteins.

Where do many histone modifications occur?

ATP-dependent repositioning or restructuring of nucleosomes to change DNA accessibility.

What is chromatin remodeling?

They can recruit chromatin-modifying complexes or help silence/activate specific genes.

How can non-coding RNAs regulate cell fate epigenetically?

Addition of a methyl group to carbon 5 of cytosine, forming 5-methylcytosine.

What is DNA CpG methylation in mammals?

At CpG sites, where a cytosine is followed by a guanine.

At which DNA sequence does CpG methylation usually occur

Because CpG sites are complementary, allowing matching methylation patterns after replication.

Why is CpG methylation often symmetrical on both DNA strands?

Regions with many clustered CpG sites, often located near gene promoters.

What are CpG islands?

Reduced gene expression or gene silencing.

What is a common effect of promoter CpG methylation?

DNMT1, DNMT3A, and DNMT3B (DNMT2 and DNMT3L are not major catalytic methyltransferases).

Which DNA methyltransferases are the main functional enzymes for DNA methylation in mammals?

Copying existing methylation patterns onto the newly synthesized DNA strand after replication, mainly by DNMT1.

What is maintenance DNA methylation?

Gradual loss of methylation through cell divisions when methylation marks are not maintained.

What is passive DNA demethylation?

Enzymatic removal of DNA methylation marks independent of DNA replication.

What is active DNA demethylation?

Because cells generally try to preserve stable epigenetic information.

Why is active demethylation tightly regulated?

Because many different histones, amino acid residues, and modification types can be combined.

Why are histone modifications considered more complex than DNA methylation?

Trimethylation of lysine 27 on histone H3.

What does H3K27me3 mean?

Acetylation of lysine 8 on histone H4.

What does H4K8ac mean?

It precisely identifies the histone, residue, and type of modification.

Why is notation such as H3K27me3 important?

Genetic

environmental

social mechanisms.

What are three major mechanisms of sex determination in animals?

Sex can change depending on social hierarchy or group structure.

How does social sex determination work?

Reproduction without fertilization.

What is parthenogenesis?

Because no Y chromosome is present.

Why does parthenogenesis in XX/XY species usually produce females?

It can produce ZZ or ZW offspring, allowing male or female progeny.

How can parthenogenesis in ZW species differ from XX/XY species?;

A long non-coding RNA that initiates X-chromosome inactivation.

What is XIST?

They add repressive histone marks such as H3K27me3.

What is the role of PRC2/EZH2 during X-inactivation?

To make X-chromosome silencing stable.

Why are multiple silencing mechanisms used during X-inactivation?

In germ cells.

When is the inactive X chromosome mainly reactivated?

It can induce silencing of that chromosome region.

What can happen if XIST is moved to another chromosome?

X-chromosome inactivation.

What does XCI stand for?

Preferential inactivation of a specific parental X chromosome, usually the paternal X.

What is imprinted X-chromosome inactivation?

Extra-embryonic tissues such as placenta.

In which tissues is imprinted XCI mainly found in mice?

Both X chromosomes become active again.

What happens to X chromosomes in the embryonic lineage before random XCI? (in mice)

Either the maternal or paternal X is inactivated randomly in each cell.

What is random X-chromosome inactivation?

Different cells inactivate different X chromosomes.

Why are female mammals mosaics for X-linked gene expression?

Because X-chromosome inactivation occurs randomly in each embryo.

Why can cloned calico/tortoiseshell cats have different coat patterns?

They resemble the pre-random XCI embryonic state in which both X chromosomes are active.

Why are female mESCs often described as having two active X chromosomes?

They undergo random X-chromosome inactivation.

What happens to X-chromosome status when female mESCs differentiate?

Naïve pluripotent cells often have two active X chromosomes, while differentiated/primed states show X inactivation.

Why is XCI status used as a marker of pluripotent state?

• XX pluripotente stem cells kunnen twee actieve X-chromosomen hebben
  → XaXa, zoals vroege embryonale cellen

• Wanneer deze stem cells differentiëren, wordt één X geïnactiveerd

• Die keuze gebeurt random
  → soms Xm actief en Xp inactief, soms Xp actief en Xm inactief

• Daardoor bootsen ze een normaal embryonaal proces na
  → overgang van pluripotente toestand naar gedifferentieerde somatische cellen

Principe: XX stem cell cultures kunnen embryogenese nabootsen doordat ze tijdens differentiatie van twee actieve X’en naar random X-inactivation gaan.

How can X-inactivation in stem cell culture recapitulate embryogenesis?

It resets X-chromosome status for the next generation.

Why is reactivation of the inactive X important in germ cells?

It helps counting and choosing which X chromosome will be inactivated.

What is the role of transient pairing (“kissing”) of the two X chromosomes during XCI?

One remains active, while the other is selected for inactivation.

What happens after the two X chromosomes separate during XCI initiation?

It is upregulated on the future inactive X and spreads silencing.

What is the function of XIST after XCI choice is made?

By stable epigenetic marks that preserve silencing.

How is the inactive X chromosome maintained long term?

Many histones are replaced by protamines for stronger DNA compaction.

What happens to histones during spermatogenesis?

To support pluripotency and remove stable lineage-specific silencing.

Why do ICM cells undergo strong DNA demethylation?

Genomic regions whose methylation marks are protected and largely maintained during reprogramming.

What are imprinted gDMRs?

Small positively charged DNA-binding proteins that replace many histones in sperm cells to compact and protect the DNA.

What are protamines?

They can affect the mother, the fetus, and the fetus’s developing germ cells.

How can pregnancy exposures affect multiple generations epigenetically?

Chromatin carrying both activating and repressive marks, keeping genes poised for rapid activation.

What is bivalent chromatin?

Because genomic imprinting requires both maternal and paternal parent-specific gene expression.

Why are two maternal genomes normally insufficient for normal embryo development?

By modifying/deleting key imprinting regions so two maternal genomes could support development.

How were bimaternal mice experimentally generated?

The Y chromosome was lost, creating XO cells, followed by X chromosome duplication.

How was an XY cell line converted toward an XX line?

Turner syndrome (single X chromosome).

What does an XO cell state resemble?

• Reversine inhibits the spindle assembly checkpoint
  → cellen controleren chromosoomsegregatie minder streng

• Daardoor ontstaat vaker chromosome mis-segregation
  → cellen kunnen bijvoorbeeld hun Y-chromosoom verliezen

• In deze studie werd dat gebruikt om uit XY-cellen eerst XO-cellen te krijgen

• Daarna kon het X-chromosoom verdubbelen
  → vorming van XX-cellen die richting oocyte fate konden worden gestuurd

Principe: reversine wordt gebruikt om gecontroleerd chromosoomverlies/mis-segregatie uit te lokken, zodat mannelijke XY-cellen kunnen worden omgezet naar een XX-achtige toestand voor oocytgeneratie.

What is reversine used for in the context of generating functional oocytes from male mice in vitro?