Stem cells in neurobiology
18 important questions on Stem cells in neurobiology
To which manifestations leads lower motor neuron degeneration?
To which manifestations leads upper motor neuron degeneration?
Which two types of ALS are there?
- Sporadic (90%) --> no familial history
- Familial (10%)
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Which classes of genes can be affected in ALS?
In which 2 ways can iPCSs derived from FUS-ALS patients and healthy controls be generated?
- Skin punch biopsy of dermis
- Fibroblasts from dermis --> dedifferentiation/reprogramming into iPSCs, by adding OKSM/Yamanaka factors (delivered using a retro- or lentiviral vector
Method 2:
- Plucked human hair
- Convert into keratinocyte culture
- Add Yamanaka factors to keratinocytes --> iPSCs
How can iPSCs be characterized?
- Karyotyping --> euploidy verification via microarray profiling of SNPs --> checking whether all 46 chromosomes (human) are present
- Genotyping
- Immunostaining for pluripotency markers (e.g. Nanog, Oct4, Sox2, SSEA4, TRA-1-60)
- Pluripotent differentiation potential: formation of embryoid bodies and differentiation into the three different germ layers + verification by immunostainings for markers
How is motor neuron differentiation verified?
What are the pro's of iPSC-derived models of diseases?
- They recapitulate the genetic situation in patients
- They are human cells --> no evolutionary distance
- the disease-relevant cell type(s) can be generated
- Key hallmarks of the disease can be recapitulated
- E.g (neuro)pathological hallmarks, e.g loss of neuronal populations, formation of cytoplasmic protein aggregates (in the case of FUS)
- Electrophysiological deficits
What are the cons of iPSC-derived models of disease?
- They are cultured cells --> the in vivo cellular context is missing, and behavior cannot be evaluated
- However: clinical data of the patient from whom cells were derived is usually available, including data from imgaging, diagnostic tests, EMG (electomyography), etc.
- The human genome is variable (outbred background) -->
- Two sources of variation when comparing patients to controls:
- The disease-causing mutation
- The genetic background
- BUT: it is possible to generate isogenic controls
What are isogenic controls?
What are the functional domains of FUS mutations?
What is a mutation within NLS causing?
What is the difference in the classical NLS and PY-NLS mechanism?
What are the two pathways that influence FUS nucleocytoplasmic shuttling?
- Transportin (TNR)-mediated nuclear import of FUS
--> modulated by arginine methylation of the FUS RGG3 domain: inhibition of protein N-argininemethyltransferase1 (PRMT1) restores TRN-mediated nuclear import of FUS PY-NLS mutants
- DNA-PK mediated nuclear export of FUS
--> this is mediated by phosphorylation of the FUS N-terminus
How can FUS-ALS motor neurons be characterized?
- Subcellular localization and cytoplasmic aggregation of FUS
- Electophysiology --> hypoexcitability of FUS-ALS motor neurons
- Age-dependent distal axonal degeneration
- Age-dependent motor neuron apoptosis
How does the culturing of hiPSC-derived motor neurons in microfluidic chambers work and why is this done?
Motor axons will grow towards higher gradient of growth factors (which is higher in distal compartment). Then, by imaging, differences between proximal and distal axons can be analyzed.
In ALS: distal axons degenerate as 1st, all the way back to the cell body --> 'dying back' mechanism.
What are the advantages of compartmentalized cultures?
- Gradients of e.g growth factors can be established
- cell bodies and promixal/distal axons can be imaged separately, including in vivo imaging
- cell bodies and or axons can be harvested separately for biochemical or omics approaches
- Compounds can be selectively added to axons or cell bodies
- By adapting the relative volume of the medium in proximal vs distal wells, the direct of the medium flow can be determined.
In which 3 ways can axonal transport be studied?
- Labeling of mitochondria or lysosomes in live cells --> by mitotracker or lysotracker
- in vivo imaging
- analysis of movies (time-lapse) to determine displacement and speed
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