ESC Differentiation Flashcards
Define differentiation
Stable change in gene expression
These changes are heritable = changes will persist beyond the cell cycle
What are embyroid bodies?
Three-dimensional cell cluster formed in a LAB by culturing ESCs under certain conditions
Essentially mimicking the early stages of embryo development
What happens when retinoic acid is added to embryoid bodies?
Causes a high proportion of the cells to express multiple neuronal properties
What happened when they changed conditions and removed serum from EBs?
Many cells died = causing high selectivity
Why is removing serum beneficial?
Because serum has many factors that need to be taken into account
What is SDIA and its function?
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
What is the role of BMP?
Inhibits neural differentiation
(Suppresses SDIA-induced neuralization and promotes epidermal differentiation)
What are the downsides of inducing neural cells via retanoic acid from embryoid bodies?
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.
What type of neurones are produced from SDIA?
dopaminergic neurons
What are PA6 cells?
Stromal cells derived from skull bone marrow
What can PA6 stromal cells do?
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
What technique did they use to kill PA6 cells?
Paraformaldehyde (PFA) fixation
What does paraformaldehyde fixation do?
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
Is it only PA6 cells that have neural-inducing activity?
No
MEF, OP9, and NIH3T3 cells without fixation = showed weak neural-inducing activity if any at all
Is direct physical contact between ES cells and PA6 cells was essential for neural induction?
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
What are the two posibilities for neural induction mechanism?
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
What is the default mechanism of neural specification?
The idea that embryonic stem (ES) cells can directly acquire neural stem cell identity without external influences
What happens during vertebrate gastrulation?
Cells derived from ectoderm segragate into either NEURAL or EPIDERMAL primordia
Via process of NEURAL INDUCTION
What is the role of the organizer?
Organizer (aka mesendoderm) sends postivie signals to induce ectoderm cells to adopt neural fate
Without this signal, the ectoderm cells differentiate into epidermis
Define the primitive NSC proposed
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
How does TGFβ-related signalling affect primative NSCs?
Negatively regulates the transition from ESC to primitive NSC
Example = BMP4 (type of TGFβ)
What is adherent monoculture?
Cell culture where a single cell type is grown on a solid surface = adhering to it
What gene is earliest known specific marker of neuroectoderm in mouse embryo?
Sox1 gene
First expressed in neural plate and subsequently maintained in neuroepithelial cells through entire neuraxis
When is Sox1 gene downreagulated?
During neuronal and glial differentiation
Why did they make GFP knock-in reporter ESC line?
To examine process by which ESCs acquire neural identity
How did they make GFP knock-in reporter ESC line?
Replaced open reading frame of Sox1 gene with coding seq for GFP and a resistance gene
How does the Sox1-GFP reporter work?
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
What conditions are needed to make Sox1-GFP positive cells?
ESCs plated on gelatin-coated tissue culture plastic
Differentiation triggered by withdrawal of LIF in ABSENCE of serum
Why was the TK23 ESC line made?
To monitor production of neurones during monolayer differentiation
GFP integrated into Tau locus and is therefore ONLY expressed in neurones
What are the cells that were Sox1-GFP positive and their properties?
Neural precursors
They can be directed into particular neuronal fates
What signalling factor is needed for neural specification?
FGF
What were some problems with neural differentiation?
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
What is the endoderm and what does it differentiate into?
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
What are the 3 steps for differentiating mESC to insulin-producing cells?
(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
Approximately how many days does it take to differentiate mESCs to insulin-producing cells?
33 days
