Differentiation: driving stem cells into specific cell types Flashcards
What are the progeny of stem cells?
1) Further stem cells
2) Cells destined to differentiate
What causes ES cells to differentiate?
Example?
Anything that disturbs the stable, perfectly balanced and perfectly controlled process that keeps the cells pluripotent
Example:
- Removal of extrinsic factors for self-renewal (LIF)
What happens to ES cells when LIF is removed?
Triggers differentiation:
- Cells clump together and form heterogenous AGGREGATES called ‘embyroid bodies’
What are embryoid bodies?
Cell aggregates that resemble 1) Gastrulation
2) Early embryonic development in vivo (resemble the early organised structure of the embryo)
- Heterogenous
What cells were embryoid bodies originally formed from?
What cells can they also be formed from?
Embryonic carcinoma cells
Can also be formed from:
- hES cells
- mES cells
What does Wnt signalling do in EB?
How is this seen?
Mediates:
1) Self-organisation
2) Axis formation
Seen using a LacZ under the influence of the Axin 2 promoter:
- Shows Wnt activity as Ant activates the Axin promoter and switches on the LacZ reporter
–> Can see where Wnt is active in the embyro
–> Shows similar organisation of Wnt in the EB as you do in the embryo
What is the formation of EBs an assay for?
Pluripotency
How does Wnt activity affect LacZ expression?
What can this change in activity also been seen with?
What are the conclusions of this experiment?
Place Wnt into the media:
- Earlier than normal activation of lacZ and earlier formation of Wnt signalling centres int he EB
Addition of DKK1 (inhibitor of Wnt):
- Delay in the formation of Wnt signalling centres in the EB and activity of LacZ
No extracellular Wnt added:
- EB still form with Wnt signalling centres
Can also be seen with a GFP reporter driven by an alternative Wnt sensitive enhancer (Dcf)
Conclusions:
- Formation of the EB can be externally controlled by Wnt
- Localised Wnt signalling centres form in the EB –> organises the embry
- Ultimately get the formation of an axis and a domain of Wnt activity that you would also get in the human embryo
What are the pros of embyroid bodies?
- Cheap and easy to produce
- Generates the 3 germ layers
What are the cons of embyroid bodies?
- Difficult to control aggregation in a REPRODUCIBLE way (shape and size)
- Different shapes and sizes have an impact on the different derivatives that the EB can produce
- Different outcome of the EB depending on how long they are left for
What are the 3 types of EB?
Describe their properties
Vary in nature and size:
1) Cystic - thin outside layer, large balloon of fluid
2) Very light (bright cavity) - no cystic cavity
3) Dense (dark cavity) - cells are very compacted together
What cells are the cystic EBs better at producing?
Endoderm
What cells are the very light EBs better at producing?
Good production of the 3 germ layers
What cells are the dense EBs better at producing?
Good production of the 3 germ layers
What type of EB are the closest to real embyros?
Why?
The bright cavity EBs
As they are the best organised
What are the 2 ways of controlling the variability of EB (to make them more reliable in shape and size):
1) Hanging drop method
2) Controlled aggregation method
Describe the hanging drop method of producing EB
1) Make small bodies (with a fixed number of cells)
2) Plate the droplet on top of a Petri dish lid and turn the lid upside down
Due to superficial tension:
–> Hold the droplets from the lid
–> One cell in the centre growing in the small droplet of the media
–> Create a single EB per drop of a reasonably comparable size and shape
What cells is the hanging drop method used for?
Mouse ES cells
Describe the controlled aggregation method of producing EB
In tissue culture plates with special geometry:
1) Multi-well plates each with a fixed number of cells with a known composition
- Normally get one EB per well with comparable size and shape
What must be done to the stem cells if want to produce large number of a particular cell lineage/cell type?
Need to force the cells down a specific lineage using other tools (directed differentiation)
What are examples of the tools that can be used to direct differentiation?
- Using EB or plating cells as monolayers
- Growth factors
- Substrate that the cells are grown on
Why does using EB or plating cells as monolayers direct differentiation?
Have more control over the formation of the lineage you want
How do growth factors help to direct differentiation?
- Applied externally to control the environment
- Used in combination
When using growth factors to direct differentiation, why must be considered?
How can these be predicted?
- WHICH GF to use
- What CONCENTRATION to use
- What COMBINATIONS to use
- The TIMING of the addition of GF
Can be predicted based on the developmental biology of a system - BUT not always right (need to establish in vitro)
Why does the substrate that the cells are grown on be considered when trying to direct differentiation?
The substrate that the cells differentiate on pushes the cells into a particular direction
Eg. plastic vs laminin
Why do we need to isolate the cell of interest?
Culture conditions tend to generate a mixture of cells (with the desired cell type being contaminated by other cell types)
How can we purify the cell type of interest from the mixture? (3)
1) FACS to sort for specific cell markers
2) Density gradients
3) Insertion (through genetic recombination) of selectable markers
What must be known prior to FACS?
Cell markers for a particular lineage:
- Markers that define an intermediate progenitor stage
- Markers that define the final cell type (fully differentiated cell type)
How can density gradients be used to purify the cell of interest?
1) Cells in a tube with a viscous media with a density gradient
2) Put the cells in the density gradient
- Cells with particular shapes and properties will separate themselves at the interface between the gradients
How can the insertion (through genetic recombination) of selectable markers be used to purify the cell of interest?
1) Gene into the cell that gives the cell a selectable drug resistance (eg. to antibiotics)
2) OR use of a REPORTER GENE - to make the cells fluoresce –> select for fluorescence
How can we differentiate ES cells to get endoderm or mesoderm?
Block the formation of ectoderm by upregulating:
- Activin
- Wnt
- BMP4
(that usually inhibit the formation of ectoderm and promote the formation of mesoderm/endoderm)
What happens if apply Wnt to ES cells at a very EARLY stage?
Get mesoendoderm
Block mesoderm
What happens when apply activin to ES cells at a later stage?
Get the formation of endoderm and mesoderm
How did Li et al purify neural precursors from ES cells?
By selecting for neural precursors during the differentiation of mouse ES cells:
1) Replacing one of the alleles of the SOX2 GENE with a cassette that has:
1) A LacZ reporter
2) A neomycin resistant gene
2) Made the cells differentiate (produces some neural and some non-neural progenitors)
3) Selected for the SOX2 expressing cells (neural progenitors) - SOX2 expressing cells will be RESISTANT to the exposure of the antibiotic and these cells will survive
- Using FACS
What happens to SOX2 in non-neural progenitors?
It is switched off
What is SOX2 important in?
It is a pluripotency gene that is important in driving ECTODERMAL differentiation
And therefore NEUROECTODERMAL derivatives an NEURONS
What antibiotic is use for the selection of the SOX2+ progenitors (neural progenitors)?
G418
What is a marker of neurons?
Beta-tubulin (TUJ1)
What can be done after the formation of a pure culture of neurons?
How?
Can GROW the culture into a larger neuronal population
Using FGF2
What does FGF2 in relation to SOX2+ cells?
Enhances their proliferation
What did the first transplantation done in human patients of pancreatic islet show?
How?
That patients can become insulin INDEPENDENT
As following transplantation (of donated adult pancreatic islets)
Treatment of the patients with insulin became UNNECESSARY
What is the first step in order to produce pancreatic derivatives?
Why?
Need to explore the NORMAL development of the pancreas and the Islet cells
In order to know the needed GF and combinations of GF that direct the differentiation of cells into pancreatic derivatives
How can we follow developmental stage of the formation of the islet cells and the pancreas?
Identification of specific markers that are indicative of each stage
What did D’Amour et al do? (2006)
Produced pancreatic hormone-expressing endocrine cell from hES cells:
- Created a protocol exposing the cells to a series of CHANGING growth factors
- See how gene expression/markers change though the intermediate steps that are described during the normal development of the mouse
What stage of development did D’Amour et al focus on?
Why?
Stage 4 of development (the pancreatic endoderm and endocrine precursor)
As in this stage - not COMPLETELY mature but is mature enough to be of potential use
How is insulin produced?
What happens during this?
As an IMMATURE protein that is cleaved
Release the C-peptide outside of the cell
What is the measurement of the C-peptide a measure of?
Indirect measurement of the PROCESSING of insulin (measuring the EFFICIENCY of the cells)
Were the cells that D’Amour et al produces efficient?
Why?
NO
Were not very efficient at processing insulin (low levels of the C peptide produced):
- Only a SMALL percentage of insulin-expressing cells obtained
- Not glucose responsive
- Do not process proinsulin well
- Did not maintain expression of the key beta cell markers
- More like FETAL beta cells (not getting the fully differentiated phenotype)
Problem with the maturation of the cells in vitro
- Difficult to make mature cells
- Problem with the last bit of differentiation
What did Kroon et al do? (2008)
Improved the protocol that was first laid down by D’Amour et al –> generated GLUCOSE-RESPONSIVE insulin secreting cells in vivo from pancreatic endoderm derived from hES cells
What are the markers of the pancreatic endoderm cells (stage 4)?
NKX6-1
PTF1a
NGN3
NKX2-2
What was seen in the cells made by Kroon et al?
What is this indicative of?
Co-expression of PDX1 and FOXA2 in the SAME CELL TYPE
Markers of the posterior foregut stage
How did Kroon et al test that the cells made work in vivo?
What was seen?
Made hES cells into pancreatic endoderm and transplanted them into mice
Looked at the phenotype after 3 months to see if the cells were normal
Saw:
- Cells survive
- Produce C-peptides (producing insulin)
- Right levels of markers
- Cells have the correct morphology
- Glucose response (increase glucose –> increases C-peptide)
- ALSO (when measuring insulin directly) saw a DOSE RESPONSE curve with increasing levels of glucose
THEREFORE, cells have the correct morphology and are able to sense glucose in vivo
How can it be seen if the grafted cells have the correct morphology?
Using electron microscopy:
- Beta cells have a particular density of grains compared to alpha cells
After characterising the cells in a normal mouse, what is the next step?
Need to show the cells work in a defective animal (diabetes model)
How can a diabetic mouse model be made?
Using the drug STZ (an aminoglucoside) that in the right doses kills the MOUSE islets but the human cells remain (are not sensitive to the drug)
What occurs in a mouse treated with STZ and transplanted with pancreatic endoderm from hES cells?
Preservation of the response in the STZ UNTREATED mice:
- High levels of glucose –> stimulates insulin secretion
- Insulin then allows the glucose to be cleared from the plasma
After seeing that the transplanted cells give an effect the same as the WT, what can be the next step to PROVE it is these cells?
How is this done?
What was seen in the case of pancreatic endoderm in the mice?
Can remove the cells and see if the phenotype reverts BACK to what it originally was
Implant the cells embedded in a GEL MATRIX (in a node somewhere in the body - not in the proper pancreas)
–> Can surgically explant the cells
SAW:
- Huge increase in the glucose levels
- -> shows the transplanted cells were keeping the glucose levels normal
- -> Right cell type doing the right job in a model of disease
So, what steps must be done to show the correct differentiation of a particular cell type in vitro?
1) Obtain the right molecular profile of the differentiated cells
2) Provide evidence that the cells are functional IN VITRO
3) Evidence that the cells are functional IN VIVO
4) Test in a model of the disease to see if the cells are functional
Although in the experiments by Kroon et al, the transplanted cell were shown to be effective, what was their downfall?
Over 15% of the grafts into the animals developed TUMOURS
Regarding grafted cells, what are the tumours likely to be a result of?
So, what must be done?
Contaminating cells
So, need to try and improve the PURIFICATION of the cells to prevent tumour formation
What did Kelly el al do? (2011)
How?
Used cell surface markers in order to attempt to isolate the pancreatic cell types derived from hES –> make a pure cell culture for transplant and try and prevent the formation of tumours resulting from transplant:
- Screened cells and identified highly concentrated markers: CD142 and C200
- Did isolation based on these markers and looked for other markers to tell us what kind of cells they were
(Looking for the expression of programming A and NKX6.1)
What is NKX6.1 a marker of?
A pancreatic endoderm progenitor
What is programming A a marker of?
Very differentiated endocrine cells
What is CD142 a marker of?
How is this seen?
Pancreatic endoderm
- Very high expression of NKX6.1
- Very low expression of programming A
What is CD200 a marker of?
How is this seen?
Endocrine
- Very high expression of programming A
- Very low expression of NKX6.1
After sorting the cells for transplantation using CD142, what was seen following transplantation?
What does this show?
0/7 engrafted transplants were teratomatous (non of the grafted cells formed tumours)
Compared to 11/24 teratomatous in the NON-SORTED grafts
Shows:
- Purifying cells can give a safety element
What did Osafune et al show? (2008)
Marked differences in differentiation propensity among hES stem cell lines
- Some lines are better at producing PDX1+ cells (pancreatic progenitors) compared to other lines
What is another way of controlling differentiation?
How this different to using individual cell lines?
Organoids: 3D differentiation:
- Normally the later stages of differentiation
- Multiple lineages that are more mature and functional
