Lecture 13- Adult neural stem cells Flashcards
What 2 functions are adult stem cells involved in?
- smell (SVZ= subventricular zone)
- memory (dentate gyrus)
How did they work out that there were stem cells present in the adult brain?
- took tissue from different parts of the brain and grew it in culture to see if any of the cells were capable of proliferation and differentiation
- slice cultures containing rostral subventricular zone tissue were grown in culture and upon migration out of the explant, precursor cells within these cultures had the capacity to proliferate and differentiate
- proliferating cells from the SVZ could differentiate into neurons and glia, cells from other tissue such as striatum or cortex couldn’t
When does the SVZ form and is it present in the adult brain?
- the SVZ forms during embryonic development and is most prominent in the ganglionic eminences (LGE and MGE)
- it gets significantly smaller as the brain matures but even in an adult there are remnants of the SVZ
picture: A= E15, B= Adult
What are the two sites of adult neurogenesis?
- The SVZ of the lateral ventricle
- The subgranular zone (SGZ) of the dentate gyrus in the hippocampus (involved in memory/learning and anxiety)
picture: the two squares point out the sites of neurogenesis
Can neural stem cells be isolated from any other part of the CNS in the adult brain?
-yes, can be isolated from most areas of the CNS but in much lower numbers
Where is the SVZ and what is its structure?
- in the lateral ventricle just below the corpus callosum
- structure: several types of cells
NSC-neural stem cell
NPC- neural precursor cell
OB neuron= olfactory bulb neuron (differentiated cell/neuroblast)
What region of the brain does the adult SVZ provide neurons for?
- the olfactory bulb
- the neurons that project to the epithelium of the nose (for sense of smell) are constantly replaced throughout life
- the precursors for new neurons in the olfactory bulb arise in the SVZ several mms away from their final destination, thus they have to migrate quite far
-
How do neurons from the SVZ migrate to the olfactroy bulb?
-cells migrate in chains along the rostral migratory stream (RMS) into the core of the OB where they differentiate into interneurons (in the granule and glomerular layer of the OB)
What are the 4 major cell types in the SVZ?
- Type A cells: small and non-proliferative, these are the migrating neuroblasts going to the olfactory bulb
- Type B cells: GFAP+ astrocytes- enclose A cells, slowly proliferative adult neural stem cells
- Type C cells: immature precursor cells, highly proliferative, amplify the number of cells that can proliferate
- Type E cells: ependymal cells, line the ventricle, usually non-proliferative
What is the adult neural stem cell (the cell type)?
- the Type B cell from the SVZ, it transietntly expresses the astrocyte marker Glial Fibrillary Acidic Protein
- it is really an astrocyte
Why are the Type B cells classified as stem cells?
it has:
1) the potential of self-renewal and
2) the ability to give rise to multiple distinct cell types (in this case it can give rise to neurons, astrocytes and oligodendrocytes)
- it is classified as multipotent
What is the relationship between Type A, B and C cells?
- The “type-1”(dentate gyrus) cells (or ‘B’ cells in the SVZ) are similar to the radial glial cells observed during development, and have a morphology and physiology similar to mature astrocytes. Although they reside in the SGZ, they extend processes up into the molecular layer.Type-1 and B cells are relatively quiescent.
- In contrast “Type-2” cells (or ‘C’ cells in the SVZ),have a high proliferative activity but have a small roundish morphology.
- The current hypothesis is that Type-2 cells (or ‘C’ cells) give rise to Type-3 (or A cells) representing neuronally committed neuroblasts.
What are the immature precursors (type C cells in the SVZ) also called?
- transit-amplifying cells
- type B cells give rise to them every now and then to amplify the number of neuroblasts migrating out as they proliferate quickly
What is are neurospheres and what are they good for?
- a culture system composed of free-floating clusters of neural stem cells
- neural stem cells cannot be studied in vivo, neurospheres provide a method to investigate neural precursor cells in vitro
- Putative neural stem cells are suspended in a medium lacking adherent substrates but containing necessary growth factors, such as epidermal growth factor(EGF) and fibroblast growth factor(FGF2).
- neural stem cells form into characteristic 3-D clusters. However, neurospheres are not identical to stem cells; rather, they only contain a small percent of neural stem cells
- they are capable of proliferating in culture and producing more of themselves
- can dissociate the neurospheres to isolate the neural stem cells
Can neurospheres be grown from adults?
- yes
- but get much less from adults than from embryos
What is the neurosphere assay for? (cell culture studies)
- way to test if the cells from the SVZ or SGZ are capable of self renewal and if they are multipotent (the requirements to be considered a stem cell)
- get tissue from the SVZ or SGZ and put into medium with growth factors such as EGF and FGF2, the cells will self-renew
- to test for multipotency (the ability to produce more than one type of neural cell) you tranfer the cells into a medium with factors promoting cell differentiation, the cells will differentiate into neurons, astrocytes and oligodendrocytes (not microglia!)
What can you stain a cell culture with to distingusih neurons, astrocytes and nuclei?
- can stain astrocytes for GFAP (remember that is what the NSC express as they are a special type of astrocyte)
- nuclei for DAPI
- neurons for betaIII-tubulin (part of the cytoskeleton that all neurons have)
What was the experiment which explored the effect of inflammatory cytokines on neurospheres?
- looking at the increase in cell number over time (effect on proliferation) and how many cells die (cytotoxicity=cell death assay)
- have 3 cell cultures with neurospheres:
1. control
2. neurospheres with interferon-gamma (IFNgamma)
3. neurospheres with necrosis factor alpha (TNFalpha)
results:
- control: cells proliferate and some die (normal)
- IFNgamma inhibits neural stem proliferation, it inhibits proliferation, doesn’t kill cells
- TFNalpha inhibits neural stem proliferation, it does so via killing the cells
- so IFNgamma and TFNalpha inhibit proliferation via different processes
What was the experiment with neurospheres exploring the effect of inflammatory cytokines? (continued, here differentiation assay)
- here looking at the effect of IFNgamma and TNFalpha on differentiation of cells
- Interferon-gamma (IFNgamma) promoted neuronal differentiation (betaIII-tubulin expressed) and blocked glial differentiation (GFAP less expressed)
Where is the subgranular zone (SGZ) and what is its structure?
- in the dentate gyrus in the hippocampus
- have same cell types like the SVZ bit instead of being type A,B and C they are Type 1, 2 and 3
- difference to the SVZ neurons is that these don’t migrate very far, they become part of the interbal circuits of the hippocampus so involved in memory/learning and anxiety
What do the proliferating cells of the SGZ of the dentate gyrus give rise to?
-young neurons that migrate a short distance and differentiate into hippocampal neurons in the granular cell layer
How do you label stem cells using BrdU?
- BrdU= Bromo deoxy Uridine, it is thymidine analogue
- proliferating cells (undergoing DNA synthesis, so S phase) can incorporate labelled nucleotides into their DNA
- when cells divide, the DNA remains labelled (but diluted)
- the BrdU can be visualised by using an anti-BrdU antibody. Nuclei of cells derived from proliferative precursors will be stained
- can tell what cell types have developed from the precursor this way
What does the BrdU labelling of stem cells and their progeny in hippocampus look like in real life?
- early all of the stem cells are in the SGZ, laetr see that they’ve moved out as mature neurons
- NeurN= in nuclei of neurons
How does labelling of stem cells with viruses work?
- you label proliferating cells in the hippocampus with a GFP-expressing retrovirus
- the retrovirus only infects dividing cells
- the cells which arise from these proliferating cells are then visioble thanks to the GFP
How does stress, depression and steroids affect neuronal differention in the “normal” brain in the hippocampus?
-negative effects
How does exercise, enriched environment and learning affect neuronal differentiation in the “normal” brain (hippocampus)?
- positive effects
- left column: new neurons, right column: memory/learning
How do the neurons and glia generated in the SVZ normally migrate to te OB? (healthy brain)
- don’t have to know the chemical cues
- normally about 80% of the cells generated by the neural stem cells are neurons, 20% glial cells
What happens when there is an injury in the brain? What do the neural stem cells do?
- more astrocytes and oligodendrocytes are produced and these are attracted to the site of the injury, so they don’t migrate via the RMS as they normally would
- astrocytes seem to help with stopping the bleeding at the site of injury
- there are lot of compounds around the site of the injury including inflammatory cytokines etc.
what must happen to replace damaged neurons:
- the neural stem cells proliferate
- neuroblast and OPC(oligodendrocyte precursor cell) migration occurs
- neuron/oligodendrocyte differentiation= up to here the process goes well
4 neurite extension
- Synaptic connection and remyelination
- most of them die at the site if injury however and don’t seem to do anything useful
How does the transplantation of stem/progenitor cells as a way of repairing the nervous system work? (one of two ways of using stem cells to repair the nervous system)
- can grow neurospheres from almost any part of the embryo (ESC=embryonic stem cells) or can use iPSC (=induced pluripotent stem cells) which can be made from skin neuroblasts that are reverted into an immature stem cell state
- transplant these into the site of injury
- there are many clinical trials underway investigating if this technique works
e. g. 1. Geron’s hESC-Derived Oligodendrocyte Progenitor Cells (GRNOPC1) into SCI injury site (Phase I)
2. ReNeuron’s human embryonic NSCs(CTX0E03) in stroke (Phase I)
3. StemCell Inc’s human adult neural stem cells (HuCNS-SC) into SCI (Phase I/II)
Also a range of other stem cell types: bone marrow, haematopoietic, mesenchymal
