Stem cells and organoids

Define properties of a stem cell

Self-renewal and ability to differentiate into specialized cell types

During grastrulation, the primitive streak forms and the embryo is re-organized into

The tree germ layers: mesoderm, endoderm, and ectoderm

Embryonic stem cells are derived from this structure

From the blastocyst inner cell mass

Describe John Gurdon experiment

He transplanted the nucleus of a differentiated intestinal epithelial cell from a tradpole into an enucleated frog egg, which developed into a normal tradpole

Gurdon’s experiment demostrated that …

The nucleous of a differentiated cel, still contains all the genetic information needed to genérate an entire organism

Name the 4 yamanaka transcription factors

Sox2 Oct3/4 Klf4 c-Myc

Yamanaka factors are used for …

Reprogramming adult somatic cells: generating iPSCs (induced pluripotent stem cells)

Explain what the teratoma assay tests

It tests pluripotent by injecting candidate stem cells into a mouse and checking whether the generated tumor contains differentiated derivates of all tree germ layers

Endoderm

Internal layer, gives rise to digestive tube

mesoderm

Mid layer, gives rise to musscle, bones, blood and connective tissue

ectoderm

External layer, gives rise to skin and nervious system

What happens before gastrulization

Implantation of the embryo

The chimera assay is considered the most stringent functional test of pluripotency because it shows that injected stem cells can

Contribute to multiple tissues of developing embryo, including the germline

A commonly used molecular marker to confirm that a cell line is pluripotent

Expression of yamanaka transcription factors: SOX2 OCT4 and NANOG

Totipotent cell type that can give rise to both embryo and extra-embryonic tissues

Zygote

Dolly the ship was significant because

She was the first mammal cloned from the nucleous of an adult differentiated somatic cell

T/F Mature, differentiated cells can be reprogrammed tombecome pluripotent

True

T/F commited progenitor cells can selfrenew and divide rapidly

F: they do divide rapidly but can not self renew

Hemaopoietic stem cells, wich give rise to blood cell líneages, reside primarily in the

Bone marrow

Embryonic stem cells are pluripotent because:

They can give rise to all body cell types but not extra-embryonic tissues such as the placenta

Tissue (adult) stem cells (such as hematopoietic stem cells) are best described as:

Multipotent cells, able to genérate múltiple but not all cell types

Spermatogonial stem cells, which give rise to sperm cells, are an example of which potency category?

Unipotent

Name the potency categories seen in class

Totipotent, pluripotent, multipotent, oligopotent and unipotent

What is the result of stem cell asimeric divission

2 dauughter stem cells, expanding the stem cell pool

What is the Hayflick limit?

The stablished limit of how many times most differentiated human somatic cells in culture can divide before entering to senescence due to loss of telomers (50 times aprox)

Why can differeniated cells not divide indefinitely, while stem cells can?

Hayflick limit: stem cells still have telomerase activity

Describe types of Stem Cell divisions

Asymetric: One stem cell and one differentiated cell, Symetric self renewal: Two stem cells, Symetric differentiation div: two differetiated cells

It is also known as consumptive division

Symetric differentiation division (two differentiated cells)

T/F: iPSC are pluripotent

T: iPSC Pluripotent Stem Cells

T/F: Stem Cells produce Commited progenitores

True

T/F: Commited progenitores have nule or limited self-renewal

True

2007 Nobel price in physiology or medicine awarded to Capecchi, Evans and Smithies

For discovering the principles for introducing specific gene modifications in mice by the use of ESC with viruses

T/F: iPSC need embryonic stem cells to be generated

F

T/F Reprogramming is a slow and inefficient process: Only a very small fraction of the starting cells give rise to iPS colonies

T

T/F: Epigenetic marks and DNA methilation are good molecular tests for cell pluripotency

T

Describe Chimera Assay for funcional test of PSC

Starting with GPF + PSC, that are injected in a blastocyst and transfer to a pseudo pregnant female: expected to generate chimera offspring and GPF observation in al cell types (from the 3 layers: ectoderm, mesoderm and endoderm), even in germline cells

Describe Yamanaka experiment

It started with mouse specialized cells (mouse embryonic fibroblast from conective tissue), which were forced to express defined transcriptional factors through retroviral infection, resulting in iPS.

What is the waddington landscape?

A representation of the epigennetic landscape, representing the celluar decision-making.

What is plasticity?

The capacity of a cell to change to a different cell identity

T/F Plasticity and potency are the same

F

T/F: Plasticity and potency are correlated but even differentiated or low-petency cells can be reprogramed to change identity

True

How can we define cell identity?

Through the expression marker genes (transcriptome of a cell)

Transdiferentiation process

Directed reprogramming to a apecific cell identity, it changes the gene expression profile.

What are master regulators?

These are genes, usually transcription factors that participates in the specification of a cell lineage by regulating multiple downstream genes (by activating or repressing them).

T/F: Transcription factors can be used as identity identifiers

true

T/F: TFs bind to specific DNA motifs

True

T/F: Binding motifs are usually kb long

False, they are usually 6-15 bp

T/F: TFs can recognize similar DNA sequences, so the same motif can be bound by different proteins

True

T/F: TFs are modulated by morfogenes

True

Morfogen

Is a signaling molecule that are key in development, they generate spatial and temporal differences in signaling.

T/F: Morphogens bind to DNA

F, morphogens are just signalling molecules that can modulate TFs

T/F: Signaling molecules can drive the differentiation of pluripotent cells, they can even induce fate conversion (transdiferentiation)

False: Signaling molecules can drive the differentiation of pluripotent cells but do not induce fate conversion (transdiferentiation)

Transdiferentiation process depends on:

developmental age of the system, enviroment: where are the cells (physically)?, relatedness (how related are the cell identities?, it is often easer to do transdiferentiation within the same germlayer

Impact of environment on cell fate: experiment

Isolation of thymic epithelial cells and its transplantation into skin subsequenlty produce its transdiferentation to skin cells. Proving the impact of enviroment on cell fate

T/F: acetilation epigenetic marks are always activation marks

True

T/F: methylation epigenetic marks are always repressing marks

False: it depends

T/F: transcription factors interact with epigenetic regulators

T

How do TFs activate or repress genes

TFs bind to DNA and recuit co-activators or co-repressors that subsequently recuit histone-modifiying enzimes to add activation or represion marks.

What is an insulatior?

A DNA sequence that protects genes from unwanted neighborn regulation signals, it prevents enhancers to activate wrong genes

Types of heterocromatin

Facultative: developmentally regulated and reversible, constitutive: stably repressed.

Euchromatin

Open chromatin

T/F: The epigenetic marks stablished through TFs is mantained even when the TFs are no longer present

True

T/F: Stablishing and mantaining histone modifications requires enzimes: readers, writers and erasers

True

T/F: Cell fate changes require large-scale chromatin reorganization, which is difficult because cells have more compact chromatin and fewer chromatin remodelers

True

T/F: Pluripotent stem cells have a higher proportion of heterochromatin

False: Pluripotent stem cells have a higher proportion of euchromatin

T/F: Differentiated cells have a more compact chromatin (heterochromatin)

True

Pluripotent stem cells have higher levels of chromatin remodelers and differentiated cells have low ones

True

T/F: Removing epigenetic marks and perturbing chromatin regulators facilates cell fate reprogramming (transdifferentation)

True

T/F: Chromatin landscape impacts plasticity

True

T/F: Early embrionic chromatin is transcriptionally active

F: Early embrionic chromatin is transcriptionally silent

What is zigotic genome activation?

Zygotic genome activation (ZGA) is the vital developmental stage when an embryo's new combined DNA begins to transcribe its own RNA

T/F: Until zygotic genome activation, early development relies entirely on stored maternal RNA and proteins inside the egg cell.

True

Why the yamanaka factors work?

Because they are pioneer factors that are able to open close chromatine to start transcriptions

T/F: Pioneer factors can iniciate new gene expression programs even on closed chromatin

True

Why is reprograming an inneficient process?

It needs to overcome epigenetic barriers: closed chromatin

T/F: Transcription factors define cell identity and the directionn of transition, they activate lineage-specific genes

True

T/F: Epigenetics have no inference in making cell fates more or less accesible

F: Epigenetic states make some fates more/less accesible by regulating chromatin packaging

T/F: Yamanaka factors can be used to rejuvenation, improving function in specific tissues, reverting aging markers.

True

Metaplasia

natural process of transdiferentation in vivo: cells that change its cell identity

Cell type that is often used for direct cell fate conversion: transdifferentation

Fibroblast

Chemical reprogramming

Direct cell fate conversion that relies exclusively on small molecules like morphogens, enhancing a cell fate transition that would not occur naturally.

When are the 3 germ-layers formed?

During gasturlation

What OKSM stands for?

Oct4, Klf4, Sox2, cMyc

Euchromatin histone modifications that active enhacers show

H3K4me1 + H3K27ac: H3K4me1 is a enhancer associated mark, associated to both active and innactive enhancers, H3K27ac is a better marker for active enhancers.

The expression of both together show a strong evidence of active enhancers.

Euchromatin histone modifications that active promoters show

H3K4me3

Euchromatin histone modifications that transcribed gene bodies show

H3K36me3

Facultative heterochromatin histone modifications

H3K27me3

Constitutive heterochromatin histone modifications

H3K9me3

Constitutive heterochromatin contains sequences from

Telomeric and pericentromeric repeats

Which steps are part of the standard ChIP-seq workflow used to map where a transcription factor binds across the genome?

Crosslinking the transcription factor to its bound DNA inside living cells

Immunoprecipitating the protein-DNA complex with an antibody specific to the transcription factor of interest

How have methods for mapping gene regulatory networks (GRNs) evolved over time?

Early gene regulatory network (GRN) maps relied heavily on genetic perturbations, such as loss-of-function experiments, to identify regulatory relationships between genes.

Single-cell multiomic profiling now allows transcription factor expression and chromatin accessibility to be measured jointly in the same individual cell

Describe the role of morphogen gradients (e.g. Shh, Wnt, FGF8, RA, BMP) in neural tube patterning

Cells interpret their position along a morphogen concentration gradient and adjust downstream transcription factor activity accordingly

Different morphogens can act along different axes of the neural tube (e.g. dorsal-ventral versus anterior-posterior) to jointly specify a cell's identity

Morphogen concentration can determine which combination of downstream transcription factors is activated in a given cell, thereby helping specify its identity

T/F: H3K4me1 alone, without H3K27ac, is sufficient to define an enhancer as actively transcribed

F: H3K4me1 alone does not establish that an enhancer is actively transcribed; H3K27ac and/or direct evidence of transcription provide stronger evidence

T/F: CTCF is a histone modification that marks active enhancers

CTCF is a DNA-binding protein/transcription factor, not a histone modification.

T/F: H3K9me3 marks constitutive heterochromatin, such as pericentromeric and telomeric repeats

True

T/F: P300 is a chromatin co-activator

True

Which experimental strategies have been used to test whether a histone modification causally instructs gene expression, rather than merely correlating with it (the "cause vs. cog" debate)?

Altering Histones: Mutating the modified histone residue itself so it can no longer carry that modification, then testing the effect on transcription

Altering writer enzymes: Using a catalytically "dead" version of the writer enzyme to uncouple its non-catalytic functions from mark deposition

Which interventions have been experimentally shown to increase the efficiency of transcription-factor-based reprogramming by removing an epigenetic barrier?

Depletion of chromatin remodeling complexes (Ej. Mbd3/NuRD)

Knockdown of methyltransferase (Ej. H3K79 methyltransferase DOT1L)

Small-molecule chemical reprogramming cocktails typically include this compounds

Inhibitors of epigenetic "erasers" such as HDACs or DNMTs

Inhibitors of TGF-beta/Activin-Nodal signaling

Inhibitors of epigenetic “erasers” such as HDACs or DNMTs

HDACs remove acetyl groups from histones. HDAC inhibitors prevent this deacetylation → chromatin tends to become more accessible → genes associated with pluripotency can be activated more easily.

DNMTs add DNA methylation. DNMT inhibitors reduce DNA methylation → some silenced genes become easier to activate.