5. 2 Stem cells & stroke: cell therapy and activation of endogenous stem cells

Why is the brain a complex organ?

All connections between the neurons, synapses, and glia need to be right, so all parts of the brain can execute their function properly.

What are 4 types of different cells involved/present in the CNS?

• Neurons;

• Astrocytes;

• Microglia;

• Oligodendrocytes.

Via what do the new (and old) cell types in the CNS form connections?

The connectome

What gives the brain its form?

Neuronal stem cells

What is the thin layer in the development in the cortex named and what is its function?

The ventricular zone; a lot of proliferation takes place here. From here, different cell types will start to form, that will later on be present in the newly formed layers.

Can brain damage be repaired?

Not fully, because there is no regeneration.

What are the 2 major types of stroke?

• Hemorrhagic stroke: bleeding

• Ischemic stroke: infarct

What are the therapeutic options for a hemorrhagic stroke?

Control bleeding/lower pressure in brain/ lower blood pressure / clipping / coiling.

What are the therapeutic options for an ischemic stroke?

Drugs that remove blood clot/tissue plasminogen activator, large clots can be removed with surgical device in a catheter.

Is there an option for recovery in the core where there is no to minimal blood flow?

No

Is there an option for recovery in the core where there is an area with mild to moderate

reduction in cerebral blood flow?

Yes

Why is the immune system activated during a stroke?

Because the dying cells will secrete cytokines etc.

What happens when the immune system gets activated during a stroke?

Microglia will go to the damaged area and become activated and start to clean up debris. Different immune cells, such as macrophages, will also go to the damaged area. These macrophages can pass the BBB and will also start to clean up debris. This process happens very quickly. If it takes too long for the other immune cells to arrive to the area, this can lead to even more damage. The immune cells do not only secrete protective factors, but also toxins.

What are stem cells?

Stem cells are self-renewal, can differentiate in different celtypes and longevity.

What are 3 types of stem cells?

• Embryonic – toti/omni/pluripotent (can turn into any type of tissue)

• Tissue specific – multipotent

• Induced pluripotent – toti/omnipotent

What is the earliest stem cell and where is it located?

The embryonic stem cell in the inner cell mass of a blastocyt.

What can tissue specific stem cells form and how is it done?

Neural stem cells can be isolated and cultured. These stem cells can then form several neurons, astrocytes and oligodendrocytes and no other body cells. From these you can form an IPS cell, which you can form into any cell you want.

What are induced pluripotent stem cells?

Differentiated cells that were differentiated into stem cells, that look like embryonic stem cells.

How can you differentiate induced pluripotent stem cells?

Skin fibroblast → add substrates (myc,oct3/4,sox2and Kif4) → stem cell → different cell types

What are 2 limitations of induced pluripotent stem cells?

• Sometimes you still have epigenetic marks, that cannot be differentiated away;

• There are mutations in the fibroblast (this needs to be kept in mind).

What are advantages of induced pluripotent stem cells?

• Formation of stem cells that do not get rejected (body’s own);

• The same genetic information;

• Less ethical issues;

• Patient specific models for personalised treatment.

What does the ventricular zone contain?

The progenitors of neurons and glia. The neurons to be generated establish different layers. At the end, six cortical layers are formed.

What do neural precursors in the subventricular zone (SVZ) continue to generate?

Neurons that migrate rostrally into the olfactory bulb, even during postnatal life.

What is an advantage of the neural precursors in the SVZ?

They will divide less quickly than in the embryonic phase, which lowers the risk at mutations/stroke.

Where is the VZ still located in the human brain?

In the hippocampus and along the ventricles. The stem cells will differentiate here and will then go towards the olfactory bulb.

What are multipotent cells?

Neurons, astrocytes and oligodendrocytes.

Do multipotent stem cells proliferate quicker or slower than the transit amplifying stem cells?

Slower

What will the transit amplifying cells form?

Neuroblasts that will start to proliferate quickly and will then differentiate.

What are 3 ways to prove that stem cells are present in the hippocampus and along the ventricles?

• Molecular marker;

• Take a biopt of the brain and make a culture from it. This will start to grow and divide;

• Adding a radioactive/readable substance.

What is the rostrale migratory system (MRS)?

It is a specialised migration route of neural stem cells towards the bulbus olfactorius from their origin (the SVZ).

What is shown in controversial RMS?

There is no evidence for chain migration.

What shows that RMS is present?

There is more staining present.

What is commonly used to study cell proliferation in living tissues?

BrdU

What is GFAP-δ?

A marker that makes stem cells visual.

What happens with the number of stem cells when you get older?

The number of stem cells becomes smaller.

Apart from the RMS towards the occipital bulb, where does the other stream go towards and what is it called?

To the frontal cortex (medial migratory stream)

Where do the stem cells from the 2 streams now migrate to and what do the researchers want to discover?

Towards a specific place; the researches want to know if they will go to the area of the stroke.

How can you prove if the cells are really stem cells?

By isolating the cells from the brain tissue and bring them into culture. If the cells can then still grow, possibly into other cell types, than you know these are stem cells.

What was formed from stem cells isolated from adult human brains post mortem?

Neurospheres

Are isolated cells stem cells?

Yes

For which 2 forms of transplantation does stem cell treatment work?

• Skin transplantation;

• Bone marrow transplantation.

What was discovered in 1981?

That embryo’s are built from stem cells.

What is totipotent?

That the isolated stem cells cab differentiate into many different types of stem cells.

What is still an issue when using embryonic stem cells?

The cells can still be rejected.

What models were used to test embryonic stem cells as a therapeutic option?

Mouse models

What is a major obstacle when using embryonic stem cells?

The host immunological response to transplanted foreign tissue.

What was the old technology for using embryonic stem cells as a therapeutic options?

Make person specific embryonic stem cells for potential therapy:

• Nucleus of the skin fibroblast → oocyte → blastocyte → stem cells

• Ethical issues

What is the new technology for using embryonic stem cells as a therapeutic options?

Induced pluripotent stem cells derived from patient’s skin:

• Prevent the cells from getting erejected

What are 3 advantages of developing stem cells from the skin?

• No ethical problems;

• Cells of own body, don’t need the fetus;

• For therapy and model for diseases.

What are the factors found in mouse embryonic / adult fibroblasts?

Oct3/4, Sox2, C-Myc (oncogene), KIf4

What factors were found in 2007 / 2008 in human skin fibroblasts?

Same factors or OCT4, SOX2, NANOG, LIN28

What is a problem of the induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors?

Tranduce pluripotency genes for only a short time “hit and run” strategy => to prevent tumors or gene deregulation.

What did the study “human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery“ show?

They show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intra-cortical implantation in a rat stroke model.

What did the study “activity in grafted human iPS cell-derived
cortical neurons integrated in stroke-injured rat brain regulates motor behavior” show?

That human IPS-cells that are injected into a new specific part of the brain leads to behavioral/motor changes. These new neurons derived from IPS are also not really active. These IPS-cells thus seem to have a high potency, mbut we aren’t there yet.

Is the area around the ventricles more active before or after a stroke and why?

After; the stem cells will try to repair the damage, even though they are not fully capable of doing this and a certain amount of damage will be remained.

• Cytokines an chemokines activate the stem cells and attract them.

Where do SVZ-derived neuroblasts turn into in the post-stroke adult striatum?

Mature neurons

How can you stimulate stem cells to divide and repair more of the damage after a stroke?

By injecting a growth factor (into the active stem cells).

What are 4 reasons as to why stem cell therapy for brain disease is still not succesful?

• Ethical problems;

• High chance of rejection of tissue;

• Induced pluripotent stem cells → no rejection because from own body;

• There might always be a chance that the stem cells develop into tumours.

How can we stimulate stem cells to replace a specific cell type in the brain?

At this moment it is still unsure how to do this in vivo. In vitro this is possible, but very difficult. If you were to inject random stem cells, you cannot control what kind of cells they will form.

How can we arrange that the right connections (between stem cells) are formed?

At this moment it is not possible yet, but it is needed to use stem cells as a therapeutic option in the clinic.