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Neurosurgery > Stem cells for movement disorders > Flashcards

Stem cells for movement disorders Flashcards

1
Q

2 important characteristics for stem cells in general

A
  1. They are unspecialized and able of own renewal (immortal in a culture)
  2. They can differentiate into different cell types
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2
Q

Levels of potency of stem cells

A

totipotent, pluripotent, multipotent

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3
Q

totipotent cells

A

Can form a living organism: placenta, the 3 germ layers and the germ line

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4
Q

pluripotent cells

A

Can form the 3 germ layers and the germ line - less potent than totipotent cells but more than multipotent

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5
Q

multipotent cells

A

Can form cell from one germ layer - these can be progenitor cells

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6
Q

How stem cell potency match the difference in totipotent and pluripotent cells

A

At fertilization, be have 1/2 cells (they’re toti-potent), once these cells have formed the blastocytes, you have a outer cell layer and inner cell mass (outer cell layer will be placenta and inner will form the organism –> inner cells are pluripotent)
Conclusion: pluripotent cells can’t form placenta

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7
Q

How do we make embryonic stem cells differnetiation (2 methods)?

A

1) We can look at if they spontaneous differentiate. PSC need certain factors to be pluripotent (e.g. Fibroblast growth factor) and if they don’t have these, they will differentiate to different cell types
2) We can use specific growth factors to guide differentiation into the direction we want

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8
Q

How do we ensure that our embryonic stem cells are pluripotent (2 methods)?

A

First option is to inject into immunodeficient mice –> will differentiate into different types, but because they stay for longer and the surroundings are better, you will get mature looking tissue and will form a tumor with different cell types (teratoma).

The real gold standard to demonstrate pluripotency would be to inject them into an embryo where the inner cell mass is forming –> will add to inner cell mass –> put into the mother –> off-spring will have body cells from the injected stem cells (mosaic). If they’re transmitted to the next generation, you know they’re able to form the germline are pluripotent.

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9
Q

How can embryonic and iP stem cells help?

A

We can use them to create disease models. iPSCs might also be used in regenerative medicine to “repair” lost cells.

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10
Q

Process of creating cells through iPSC

A

skin biopsy –> fibroblasts –> overexpress Y4F (Oct4, Sox2, Klf4 and cMyc) –> 4 weeks –> iPSCs –> differentiation into specific cells

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11
Q

How do you show that your iPSC are pluripotent?

A

Markers
You have different markers associated with the different stages –> immunofluorescent staining. Markers have to be those not overexpressed (generally we use surface markers)

You can inject them into a immunosuppressed animal. The promoter regions of pluripotency associated factors are de-methylated(usually a semi-quantitative issue), you can see human derma-fibroblasts they are still methylated, but in pluripotent stem cells most are de-methylated, which is a sign that the promotor regions are highly active

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12
Q

Process for disease modeling with iPSCs

A

patient cell –> iPSC –> differentiation (whatever you want) –> drug/genetic screening, toxicology, screening –> drug development

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13
Q

Neuro-degeneration of the cortex is cause by which disorders?

A

ALS & dementias

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14
Q

Neuro-degeneration of the striatal projections is cause by which disorders?

A

Huntington’s & mixed-chromosome dystonia Parkinson’s

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15
Q

Neuro-degeneration of the forebrain interneurons is cause by which disorders?

A

dystonia, schizophrenia & some type of dementia

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16
Q

Why does modeling of a disorder require that we work with the affect cell-type?

A

Because we need to look at the degeneration and how they are affected

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17
Q

Patterning

A

The process of regional specification. It leads neural stem cells to acquire the regional identity they will later have. The use of it experimentally utilizes what we know about how the cells develop (e.g. that interneuorns are from the medial ganglionic eminence)

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18
Q

Neural induction

A

the process of deriving neural cells from pluripotent stem cells (giving them the identity of neural stem cells)

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19
Q

Differentiation

A

is about moving from a less defined and more proliferative cell type into to a more defined cell type (maturing)

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20
Q

What is the intermediate stage between having a neural stem cell and a neuron/glia?

A

The precursor cells which are derived from the stem cells. They have a specific fate, but are still proliferating and divide.

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21
Q

What’s the neural induction protocol from 2009?

A

1) you have your stem cells in high density
2) Start differentiation by taking out the factors for pluripotency
3) you add factors that only let them go into a certain direction

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22
Q

The most important decision on cell fate before day?

A

6

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23
Q

Development: the CNS is derived from?

A

the neural tube

24
Q

4 regions of the CNS in development (prosencephalon is devided)

A

telencephalon, diencepalon, mesencephalon and the rhombencephalon

25
Q

Dorsal side of telencephalic development

A

becomes the cortex. Only the cortical projection neurons are formed here

26
Q

Subdivision of the ventral part in telencephalic development

A

medial and lateral ganglionic eminence (MGE and LGE)

27
Q

the lateral ganglionic eminence produce which neurons?

A

striatal projection neurons, the medium spiny neurons

28
Q

the medial ganglionic eminence produce which neurons?

A

forebrain GABG-based interneurons and cholinergic interneurons

29
Q

GABAergic cells can ONLY be produced where during telencephalic development?

A

In the ventral part

30
Q

Neurons in these parts secrete signal proteins that influence identity of the cells in the CNS. What are they called and what are the examples?

A

They’re called morphogens. Examples are Sonic hedgehog (shh) and Wnt

31
Q

Sonic hedgehog (Shh) in neural development

A

Shh is in the ventral part and is induced by the notochord and ventral identity is mainly derived on the presence of this factor.

32
Q

Wnt in neural development

A

Wnt is important for dorsal/ventral patterning (whether it’s should become part of the dorsal or ventral part). Important in defining caudal phenotypes in the midbrain.

33
Q

What helps with differentiating pluripotent cells into striatal interneurons?

A

Sonic hedgehog receptor activation from day 1 and antagonists to Wnt receptors

34
Q

What do we lose when adding shh agonist and wnt antagonist

A

The ability for the cells to become part of the cortex through dorsal development

35
Q

When is patterning over?

A

20 days

36
Q

How long does it take for interneurons to mature?

A

50 days

37
Q

MAP2

A

a microtubules associated protein (present in all neurons)

38
Q

the 2 stages of X-linked dystonia-parkinsonism

A

Stage 1: muscle cramps leading to involuntary movements

Stage 2: more classical Parkinson’s symptoms (rigidity, hypokinesia)

39
Q

What’s the gene we think might be relevant for X-linked dystonia-parkinsonism?

A

the TAF gene. Its a trans activator factor and therefore not super relevant for neurons (it plays a role in transcription activation). You find a an insertion that is quite long in patients

40
Q

How does neuronal degeneration work in X-linked dystonia-parkinsonism?

A

In the first stage, you loose cells in the indirect pathway of the basal ganglia (why we have more movement) and later we see degeneration in the rest of the striatum, we get the hyper movement and parkinsonian symptoms

41
Q

What do we use for genome editing and how?

A

CRISPER/cas9
you take the parent cell line and change individual aspects and see how it affects function. We have many options where we can cut out parts of the genome. You can destroy genes by deleting it or you can make homology related repair or add things into the genome

42
Q

isogenic cell lines

A

Cell lines with almost identical genes.
We can either use genome editing in healthy cells to insert a mutation and compare them or delete a mutation from a diseased cell and compare them. The last option is more popular as you’re sure you capture all disease modifying genes this way.

43
Q

Is TAF1 expression generally decreased in X-linked dystonia-parkinsonism patients?

A

No, it is in iPSCs and medium spiny projection neurons, but not in cortical neurons

44
Q

Does deleting the retro transposon in X-linked dystonia-parkinsonism patients increase TAF1 expression?

A

iPSCs yes, but not in the actual spiny projection neurons (not a good methods for treatment then)

45
Q

A father has almost no symptoms and the son had a lot, though they had the same genetic mutation. What does the example show us?

A

The important role of modifying factors in genes

46
Q

Explain inducible over-expression models

A

These are models that work as their own controls. The mutation in question can only be activated when tetracycline/doxycycline is added, so the tet component expresses the gene (turns transcription on).

47
Q

Does over-expression of a gene affect the number of number of progenitor cells or neurons we have?

A

Over-expression generally doesn’t affect this, but if the over-expression is induced during differentiation, it causes differentiation to fail, and we won’t get any neurons.

48
Q

iPS cells can be derived from patient‘s fibroblasts using which 4 factors?

A

Oct4, Sox2, Klf4 and cMyc

49
Q

iPS cells can be differentiated into cells of the three embryonic germ layers - what are they called?

A

endoderm (inner layer), the ectoderm (outer layer), and the mesoderm (middle layer)

50
Q

Development of the cns requires which 3 processes?

A

neural induction, patterning and differentiation

51
Q

What mechanisms do the experimental regional patterning exploit?

A

The mechanisms that happen during embryogenesis

52
Q

What is the most important ventralizing factor in the cns?

A

Sonic Hedgehog

53
Q

What plays an an important role in rostro/caudal-patterning?

A

The WNT-cascade

54
Q

How can the ventral telencephalic phenotype be promoted in vitro?

A

by Hedgehog agonists (SHH, Pumo) and WNT-Inhibition (DKK)

55
Q

How do we create isogenic lines?

A

With CRISPR/Cas9

56
Q

Which model has it’s own internal control?

A

The inducible overexpression model

Neurosurgery (10 decks)

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