6. Spinal cord regeneration Flashcards
Why can salamanders and zebrafish regenerate spinal cord but not mice?
Salamander and zebrafish have different tissue stem cells - better at regeneration - ependymal cells upon injury can produce different cell types needed to repair the injury
Which animals are know to regenerate spinal cord well?
Salamanders (axolotl) and zebrafish can regenerate the central nervous system after different types of injuries
Central nervous system: brain + spinal cord -> will be focusing on spinal cord in this lecture
Mice and humans can’t - mice only short after brith can but not later
What are ependymal cells?
Ependymal cells - adult spinal cord stem cells
Final fate of embryonic neural stem cells
How do ependymal cells differ in axolotl and mice?
- Differentiation capacity: in axolotl ependymal cells upon injury can produce neurons, glial cells, and regenerate axons - allows full spinal cord regeneration
In mice ependymal cells can only produce glial cells - not enough to regenerate spinal cord injury
- Morphology: ependymal cells have different morphology in mouse and axolotls
- Axolotl ependymal cells retain neural stem cell capacity all throughout life but in mice the stem cell capacity decreases with age - only newborn mice can regenerate spinal cord injury - but overal even simple glial cell generation become inefficient in mice
How are ependymal cells organised in the mouse brain?
In the brain (not spinal cord) ependymal cells are located relatively close to the surface, just below ciliated cells that push spinal fluid
How do ependymal cells in axolotl gain the ability to regenerate spinal cord?
Axolotl ependymal cells revert to an embyronic-like state to regenerate - upon injury reporogramming - gain capacity to regenerate
Where are ependymal cells located in the spinal cord?
In the centre of the spinal cord - little region
Foxj1 - ependymal marker
What is a common characteristic of ependymal populations in spinal cords?
Ependymal populations are morphologically heetrogenous - have different looking cells with different properties
What does the heterpgeneity of ependymal cell population represent?
Was not known, studied in Rodrigo Albors et al. 2023 paper
Conclusions:
- used scRNaseq to establish different populations within ependymal cells - found their specific expression profiles
- did in vitro and in vivo spinal cord injury assays - identified immature cells as potential spinal cord stem cells
- uncovered cell maturation and altered identities in human spinal cord tissue samples
How was the ependymal heterogeneity studied by Rodrigo Albors et al. 2023?
scRNAseq - a cell map of spinal cord central canal cells expression - done by two methods:
- 10x genomics - got all cells
- Smart-seq2 - explored ependymal cells in detail
What are the advantages and disadvantages of the two scRNAseq methods?
What were the results of scRNAseq analysis of central canal sample?
Found several central canal cell clsuters - one of them ependymal cell cluster
What was found when spinal chord scRNAseq ependymal cell cluster was investigated further?
Ependymal cell cluster consists of ependymal cell subtypes whihc have different expression profiles:
- mature
- immature
- proliferating
- lateral
- dorsal
- ventral
What was found when these ependymal subtypes were investigated in situ?
When these ependymal subtypes were investigated in situ in tissue sections - tried to validate observed marker genes of each subtype - found how they arranged within the central canal:
- dorsal
- ventral
- lateral mature
- lateral immature
-> heterogeneity in ependymal cell population
What was found in scRNAseq dataset that was not validated in situ?
In scRNAseq dataset Fos gene upregulation was observed - Fos is a tissue damage gene - not observed when investigated in situ tissue sections -> probably was upregulated in cells upon cll dissasociation for scRNAseq and not in actual intact ependymal cell population
Explain what is RNAscope
RNAscope - single molecule fluorescence in situ hybridisation - method to visualise individual RNA transcripts in situ -> can quantify in situ
- Tissue section and permeabilisation
- Probe hybridisation onto the tissue
- Hybridisation of signal amplification moelcules bound to RNA
- Hybridisation of labelled probes for visualisation
- Confocal imaging
How were age-related changes in spinal cord ependymal cells studied?
Again scRNAseq performed comparing young and old mice - their spinal cord cell population changes
Observed changes in astrocytes, microglia but ependymal showed minor change
What specific gene expression was observed to have changed within ependymal cells in young vs old mice?
Although ependymal cell populations showed only minor changes, Sntn expression increases significantly with each within ependymal cell population - Sntn mature ependymal cell marker - observed increase in Sntn with age
Ependymal cell numbers don’t change but they transform more into mature ependymal cells
How was Sntn expression further investigated in vitro?
Sntn expression was studied in spinal injuries in vitro and in vivo, in vitro - spinal injury: spinal cord dissected out of a mouse - vibratome slicing - slice culture in vitro - fixed and processed on day 0, day 3, day 5 =>
- change in Sntn observed (downregulate - cells go back to immature - but then again upregulated - cells go back to mature w/o regenerating the tissue)
- change in Ki67 observed - cell cycle entry gene - upregulated and then downregulated
=> ependymal cells in mice enter immature ependymal cell stage but smth prevents them from driving regeneration - go back to being mature
How was Sntn expression further investigated in vivo?
IN vivo spinal injury - same Sntn and Ki67 expression pattern observed as in vitro
How were the ependymal cell identities studied in human spinal cord?
Had some frozen human spinal cord tissue sections of different age patients - stained for cell-specific markers - observed that with age human tissue got disorganised - heterogenous results
IN other stains observed that two ependymal cell domains started to merge into one (??) with age
How was human human embryonic spinal cord studied?
had some human embryonic tissue - sections trhough developing spinal cord - observed separated domains like in mice - ZIC1 (dorsal ependymal), ARX (ventral ependymal), PAX6 (lateral ependymal)
Schematic key findings of the paper
In mice spinal cord ependymal cells have stem cell identity which declines with age - they respond to spinal cord injury byentering more immature proliferative state, but then go back to being mature without regeneration (similar mechanism used in salamanders to regenerate but in their case the immature actually regenerates injury) => something is preventing mouse ependymal cells from regeneration ability when they become more immature in injury sites
Paper discussion questions
Read paper Rodrigo Abors et al. 2023
What are the further questions to answer beyonf the paper?
Study if ependymal cell maturation in mice limit spinal cord regeneration:
- try KO maturation - have only immature ependymal cells - see if can regenerate spinal cord
- label immature vs mature cells in lineage tracing - see how develop and why different
- use cross-species genetic analysis via scRNAseq to discover similarities vs differences that mught drive ability to regenerate
What is another organism that can be included in cross-species genetic study for regeneration genes?
Spiny mouse
