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A Stem cell biology > Differentiation: driving stem cells into specific cell types > Flashcards

Differentiation: driving stem cells into specific cell types Flashcards

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1
Q

What are the progeny of stem cells?

A

1) Further stem cells

2) Cells destined to differentiate

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2
Q

What causes ES cells to differentiate?

Example?

A

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)

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3
Q

What happens to ES cells when LIF is removed?

A

Triggers differentiation:

  • Cells clump together and form heterogenous AGGREGATES called ‘embyroid bodies’
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4
Q

What are embryoid bodies?

A

Cell aggregates that resemble 1) Gastrulation

2) Early embryonic development in vivo (resemble the early organised structure of the embryo)
- Heterogenous

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5
Q

What cells were embryoid bodies originally formed from?

What cells can they also be formed from?

A

Embryonic carcinoma cells

Can also be formed from:

  • hES cells
  • mES cells
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6
Q

What does Wnt signalling do in EB?

How is this seen?

A

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

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7
Q

What is the formation of EBs an assay for?

A

Pluripotency

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8
Q

How does Wnt activity affect LacZ expression?

What can this change in activity also been seen with?

What are the conclusions of this experiment?

A

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
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9
Q

What are the pros of embyroid bodies?

A
  • Cheap and easy to produce

- Generates the 3 germ layers

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10
Q

What are the cons of embyroid bodies?

A
  • 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
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11
Q

What are the 3 types of EB?

Describe their properties

A

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

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12
Q

What cells are the cystic EBs better at producing?

A

Endoderm

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13
Q

What cells are the very light EBs better at producing?

A

Good production of the 3 germ layers

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14
Q

What cells are the dense EBs better at producing?

A

Good production of the 3 germ layers

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15
Q

What type of EB are the closest to real embyros?

Why?

A

The bright cavity EBs

As they are the best organised

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16
Q

What are the 2 ways of controlling the variability of EB (to make them more reliable in shape and size):

A

1) Hanging drop method

2) Controlled aggregation method

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17
Q

Describe the hanging drop method of producing EB

A

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

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18
Q

What cells is the hanging drop method used for?

A

Mouse ES cells

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19
Q

Describe the controlled aggregation method of producing EB

A

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

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20
Q

What must be done to the stem cells if want to produce large number of a particular cell lineage/cell type?

A

Need to force the cells down a specific lineage using other tools (directed differentiation)

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21
Q

What are examples of the tools that can be used to direct differentiation?

A
  • Using EB or plating cells as monolayers
  • Growth factors
  • Substrate that the cells are grown on
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22
Q

Why does using EB or plating cells as monolayers direct differentiation?

A

Have more control over the formation of the lineage you want

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23
Q

How do growth factors help to direct differentiation?

A
  • Applied externally to control the environment

- Used in combination

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24
Q

When using growth factors to direct differentiation, why must be considered?

How can these be predicted?

A
  • 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)

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25
Q

Why does the substrate that the cells are grown on be considered when trying to direct differentiation?

A

The substrate that the cells differentiate on pushes the cells into a particular direction

Eg. plastic vs laminin

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26
Q

Why do we need to isolate the cell of interest?

A

Culture conditions tend to generate a mixture of cells (with the desired cell type being contaminated by other cell types)

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27
Q

How can we purify the cell type of interest from the mixture? (3)

A

1) FACS to sort for specific cell markers
2) Density gradients
3) Insertion (through genetic recombination) of selectable markers

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28
Q

What must be known prior to FACS?

A

Cell markers for a particular lineage:
- Markers that define an intermediate progenitor stage

  • Markers that define the final cell type (fully differentiated cell type)
29
Q

How can density gradients be used to purify the cell of interest?

A

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

30
Q

How can the insertion (through genetic recombination) of selectable markers be used to purify the cell of interest?

A

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

31
Q

How can we differentiate ES cells to get endoderm or mesoderm?

A

Block the formation of ectoderm by upregulating:

  • Activin
  • Wnt
  • BMP4

(that usually inhibit the formation of ectoderm and promote the formation of mesoderm/endoderm)

32
Q

What happens if apply Wnt to ES cells at a very EARLY stage?

A

Get mesoendoderm

Block mesoderm

33
Q

What happens when apply activin to ES cells at a later stage?

A

Get the formation of endoderm and mesoderm

34
Q

How did Li et al purify neural precursors from ES cells?

A

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

35
Q

What happens to SOX2 in non-neural progenitors?

A

It is switched off

36
Q

What is SOX2 important in?

A

It is a pluripotency gene that is important in driving ECTODERMAL differentiation

And therefore NEUROECTODERMAL derivatives an NEURONS

37
Q

What antibiotic is use for the selection of the SOX2+ progenitors (neural progenitors)?

A

G418

38
Q

What is a marker of neurons?

A

Beta-tubulin (TUJ1)

39
Q

What can be done after the formation of a pure culture of neurons?

How?

A

Can GROW the culture into a larger neuronal population

Using FGF2

40
Q

What does FGF2 in relation to SOX2+ cells?

A

Enhances their proliferation

41
Q

What did the first transplantation done in human patients of pancreatic islet show?

How?

A

That patients can become insulin INDEPENDENT

As following transplantation (of donated adult pancreatic islets)
Treatment of the patients with insulin became UNNECESSARY

42
Q

What is the first step in order to produce pancreatic derivatives?

Why?

A

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

43
Q

How can we follow developmental stage of the formation of the islet cells and the pancreas?

A

Identification of specific markers that are indicative of each stage

44
Q

What did D’Amour et al do? (2006)

A

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
45
Q

What stage of development did D’Amour et al focus on?

Why?

A

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

46
Q

How is insulin produced?

What happens during this?

A

As an IMMATURE protein that is cleaved

Release the C-peptide outside of the cell

47
Q

What is the measurement of the C-peptide a measure of?

A

Indirect measurement of the PROCESSING of insulin (measuring the EFFICIENCY of the cells)

48
Q

Were the cells that D’Amour et al produces efficient?

Why?

A

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
49
Q

What did Kroon et al do? (2008)

A

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

50
Q

What are the markers of the pancreatic endoderm cells (stage 4)?

A

NKX6-1

PTF1a

NGN3

NKX2-2

51
Q

What was seen in the cells made by Kroon et al?

What is this indicative of?

A

Co-expression of PDX1 and FOXA2 in the SAME CELL TYPE

Markers of the posterior foregut stage

52
Q

How did Kroon et al test that the cells made work in vivo?

What was seen?

A

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

53
Q

How can it be seen if the grafted cells have the correct morphology?

A

Using electron microscopy:

- Beta cells have a particular density of grains compared to alpha cells

54
Q

After characterising the cells in a normal mouse, what is the next step?

A

Need to show the cells work in a defective animal (diabetes model)

55
Q

How can a diabetic mouse model be made?

A

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)

56
Q

What occurs in a mouse treated with STZ and transplanted with pancreatic endoderm from hES cells?

A

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
57
Q

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?

A

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
58
Q

So, what steps must be done to show the correct differentiation of a particular cell type in vitro?

A

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

59
Q

Although in the experiments by Kroon et al, the transplanted cell were shown to be effective, what was their downfall?

A

Over 15% of the grafts into the animals developed TUMOURS

60
Q

Regarding grafted cells, what are the tumours likely to be a result of?

So, what must be done?

A

Contaminating cells

So, need to try and improve the PURIFICATION of the cells to prevent tumour formation

61
Q

What did Kelly el al do? (2011)

How?

A

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)

62
Q

What is NKX6.1 a marker of?

A

A pancreatic endoderm progenitor

63
Q

What is programming A a marker of?

A

Very differentiated endocrine cells

64
Q

What is CD142 a marker of?

How is this seen?

A

Pancreatic endoderm

  • Very high expression of NKX6.1
  • Very low expression of programming A
65
Q

What is CD200 a marker of?

How is this seen?

A

Endocrine

  • Very high expression of programming A
  • Very low expression of NKX6.1
66
Q

After sorting the cells for transplantation using CD142, what was seen following transplantation?

What does this show?

A

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

67
Q

What did Osafune et al show? (2008)

A

Marked differences in differentiation propensity among hES stem cell lines

  • Some lines are better at producing PDX1+ cells (pancreatic progenitors) compared to other lines
68
Q

What is another way of controlling differentiation?

How this different to using individual cell lines?

A

Organoids: 3D differentiation:

  • Normally the later stages of differentiation
  • Multiple lineages that are more mature and functional

A Stem cell biology (16 decks)

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