ESC Differentiation

Question 1

Define differentiation

Answer 1

Stable change in gene expression

These changes are heritable = changes will persist beyond the cell cycle

Question 2

What are embyroid bodies?

Answer 2

Three-dimensional cell cluster formed in a LAB by culturing ESCs under certain conditions

Essentially mimicking the early stages of embryo development

Question 3

What happens when retinoic acid is added to embryoid bodies?

Answer 3

Causes a high proportion of the cells to express multiple neuronal properties

Question 4

What happened when they changed conditions and removed serum from EBs?

Answer 4

Many cells died = causing high selectivity

Question 5

Why is removing serum beneficial?

Answer 5

Because serum has many factors that need to be taken into account

Question 6

What is SDIA and its function?

Answer 6

Stroma cell-derived inducing activity (neural induction)

The ability to induce neural differentiaiton of mESCs into dopaminergic neurons

SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies

Question 7

What is the role of BMP?

Answer 7

Inhibits neural differentiation

(Suppresses SDIA-induced neuralization and promotes epidermal differentiation)

Question 8

What are the downsides of inducing neural cells via retanoic acid from embryoid bodies?

Answer 8

First, it is difficult to analyze and control each regulatory step of differentiation in this method because EBs contain many different kinds of cells, including mesodermal and endodermal cells.

Second, RA, a strong teratogen, is supposed to perturb neural patterning and neuronal identities in EBs as it does in vivo.For instance, RA treatment of early embryos causes suppression of forebrain development

Precise specification of a particular neuronal characteristic, such as neurotransmitter choice, is crucial when induced neurons are to be used for therapeutic applications or basic neuroscience research. It is therefore preferable to avoid RA treatment unless RA induces the particular type of neurons of one’s interest.

Question 9

What type of neurones are produced from SDIA?

Answer 9

dopaminergic neurons

Question 10

What are PA6 cells?

Answer 10

Stromal cells derived from skull bone marrow

Question 11

What can PA6 stromal cells do?

Answer 11

Induce differentiation of ESCs into dopaminergic neurons

Even once they are killed (PFA fixed) = which suggests there is a cell surface protein on them that induces differentiation

Question 12

What technique did they use to kill PA6 cells?

Answer 12

Paraformaldehyde (PFA) fixation

Question 13

What does paraformaldehyde fixation do?

Answer 13

Chemically crosslinks proteins within the sample, essentially “gluing” them in place to preserve its structure and morphology for further analysis, like microscopy or staining, by preventing degradation and maintaining the spatial arrangement of cellular components

Question 14

Is it only PA6 cells that have neural-inducing activity?

Answer 14

No

MEF, OP9, and NIH3T3 cells without fixation = showed weak neural-inducing activity if any at all

Question 15

Is direct physical contact between ES cells and PA6 cells was essential for neural induction?

Answer 15

ES cell colonies were cultured on gelatin-coated dishes and separated from cocultured PA6 cells by a 0.4 μm filter membrane.

In the absence of physical contact, PA6 cells were still able to induce significant neural differentiation of ES cells cultured on gelatin = indicating that PA6 cells produce soluble inducing factor(s).

However, PA6-conditioned medium could not elicit significant induction

Question 16

What are the two posibilities for neural induction mechanism?

Answer 16

One is that SDIA consists of two different neural-inducing factors, a cell surface-anchored factor and a labile soluble factor.

Another scenario might be that SDIA is mediated by secreted factors, which are secondarily tethered to the cell surface, as exemplified by Wnts and FGFs (16, 5).

At present, we cannot exclude either possibility, but the latter appears to be supported by our preliminary data that preincubation of PA6 with heparin before fixation removes the inducing activity from the cell surface, as reported for Wnts

Question 17

What is the default mechanism of neural specification?

Answer 17

The idea that embryonic stem (ES) cells can directly acquire neural stem cell identity without external influences

Question 18

What happens during vertebrate gastrulation?

Answer 18

Cells derived from ectoderm segragate into either NEURAL or EPIDERMAL primordia

Via process of NEURAL INDUCTION

Question 19

What is the role of the organizer?

Answer 19

Organizer (aka mesendoderm) sends postivie signals to induce ectoderm cells to adopt neural fate

Without this signal, the ectoderm cells differentiate into epidermis

Question 20

Define the primitive NSC proposed

Answer 20

This term has been used previously to describe a stem cell that is primarily tissue-specific, but that retains a certain degree of pluripotency during a restricted early period of development

Question 21

How does TGFβ-related signalling affect primative NSCs?

Answer 21

Negatively regulates the transition from ESC to primitive NSC

Example = BMP4 (type of TGFβ)

Question 22

What is adherent monoculture?

Answer 22

Cell culture where a single cell type is grown on a solid surface = adhering to it

Question 23

What gene is earliest known specific marker of neuroectoderm in mouse embryo?

Answer 23

Sox1 gene

First expressed in neural plate and subsequently maintained in neuroepithelial cells through entire neuraxis

Question 24

When is Sox1 gene downreagulated?

Answer 24

During neuronal and glial differentiation

Question 25

Why did they make GFP knock-in reporter ESC line?

Answer 25

To examine process by which ESCs acquire neural identity

Question 26

How did they make GFP knock-in reporter ESC line?

Answer 26

Replaced open reading frame of Sox1 gene with coding seq for GFP and a resistance gene

Question 27

How does the Sox1-GFP reporter work?

Answer 27

GFP not detectable in undifferentiated ESCs But becomes evident in significant proportion of cell after induction of neural differentiation by aggregation and treatment with retinoic acid

Question 28

What conditions are needed to make Sox1-GFP positive cells?

Answer 28

ESCs plated on gelatin-coated tissue culture plastic Differentiation triggered by withdrawal of LIF in ABSENCE of serum

Question 29

Why was the TK23 ESC line made?

Answer 29

To monitor production of neurones during monolayer differentiation GFP integrated into Tau locus and is therefore ONLY expressed in neurones

Question 30

What are the cells that were Sox1-GFP positive and their properties?

Answer 30

Neural precursors They can be directed into particular neuronal fates

Question 31

What signalling factor is needed for neural specification?

Answer 31

FGF

Question 32

What were some problems with neural differentiation?

Answer 32

Cells do things in their own time due to heterogeneity ESC come through and if they are not removed will react to anything and make result interpreation difficult Most make GABA neurones but can change this using growth factors Full mechanism still unkown

Question 33

What is the endoderm and what does it differentiate into?

Answer 33

The endoderm is the innermost germ layer of an embryo, Differentiates into the epithelial lining of the digestive tract, including the stomach, intestines, liver, pancreas As well as the lining of the respiratory tract, thyroid, and bladder Essentially forming the internal organs involved in digestion and respiration; making it crucial for nutrient absorption and gas exchange

Question 34

What are the 3 steps for differentiating mESC to insulin-producing cells?

Answer 34

(i) the formation of embryoid bodies (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage

Question 35

Approximately how many days does it take to differentiate mESCs to insulin-producing cells?

Answer 35

33 days