stem cells in vivo and vitro Flashcards
adult stem cell properties
most are specialised and tend not to/slowly divide
but some tissues need constant renewal (cornea surface, skin, intestinal lining…)
so these are situations where stem cells are mehtod for renewing constantly lost cells
stem cell basic properties
self renewal
differentiation into specialised cell types
make a choice between self renewal and differentiation
hierarchy of lineage restriction
branching differentiation
cells that gradually lose potency arising from a cell that couls potentially give rise to anything
eg epiblast cells that give rise to any embryo cell type
to lateral mesoderm cells that only give rise to lateral mesodderm cell types
waddington’s picture of cell lineage diversification
going down one way preludes it from going down a different differentiation pathway
restriction of potency
use for stem cells in embryo??
single cell embryo needs to produce a free living organism able to reproduce
why would embryo need stem cells for this?
stem cell type in mouse embryo
epiblast cells i think
gives rise to all the germ layers
i think this referring to the progenitors during axis elongation stuff
how does mouse embryo elongate?
through cell proliferation and movement
cell movements around the primitive streak
p streak at posterior end
cells in epiblast move towards p streak
undergo ETM transitions
then go down and out to become mesoderm
cells that have dome this will exit from the p streak region and begin to differentiate
if they fail to get caught in p streak they become ectoderm
then neurulation forming neural tube
end up with mesoderm tissue flanking the neural tissue along the embryo
because the mesenchymal mesoderm cells have condensed into somite blocks flanking the somites
-neural tube
-flanked by somites
-notochord underneath
-endoderm beneath that
mouse embryo growth until midgestation
process of axis elongation
E7.5 - cup shaped epiblast
E8 - progenitors at posterior end
progenitors here proliferating and generating more and more posterior - elongating the embryo
flat representation of the phylotypic stage in mouse
head
trunk w limbs
tail
neural tube along length
somites flanking it
progenitors at posterior end that generate the neural tube and the somites of the axis
somites are not segmented at first - generates clump of it which buds off the separate somites - determines no. of vertebrae the animal will have
axial elongation mechanism
these posterior progenitors proceed axial elongation by addition of cells from the posterior end
primitive streak?
ends up as tail bud at end?
Tail bud and P streak derived from same cell type
does the tail bud contain one multiotent progenitor cell type of many unipotent restricted ones
testing the potency of the axial/tail bud stem cells by self renewal
dissect parts of labelled primitive streak
transplant these into unlabelled embryo
can culture this for 48hrs (1/2 of elongation)
at this point there will still be tail bud cells left in the embryo
can test if any of these are stem cells by taking them and grafting them into the primitive streak of another earlier embryo
do this in a loop of grafting and growing
after grafting these cells - they were labelled in the differentiated parts of the tail bud
these progenitors can make the same axial region SEVERAL TIMES
-so have potential to act like stem cells
but when these cells were grafted had to graft groups of them to work
so cant say anything about self renewal capabilities yet
Rosa26laacZ approach for tail bud cells
for clonal analysis of tail bud cells
lacZ construct which has a duplicated sequence that interrupts the stop codon part of the ORF
this duplication can be recognised as a mistake by host recombination machinery
in a rare event can do homologous intragenic recombination
is spontaneous and random so happens in low enough frequency to be good clonal label
-can use this to look at clonal history of cells of interest
progenitor type in tail bud
a bi-potent
clones in the neural tube and somites from Rosa26laacZ transgenic tail bud cells
began in anterior limit in brain
and went back to (and includes) the TB progenitor region
show that these cells act like stem cells
resident in the primitive streak and tail bud
continuously producing neural tube AND somites
data from the LaacZ and grafting experiments suggest they are bi-potent and self renewing
tail bud cells compared to an adult stem cell
are not exactly equivalent to adult stem cells
TB cells give rise to:
somites in earlier embryo - repeated segments w v similar structure
BUT in later embryo - diff somites go on to make v different parts of the axis
-somites have now become vertebrae and each vertebra is DIFFERENT from its neighbour
does this happen:
-After the cells have differentiated and EXITED from the axial progenitor compartment?
-or before?
progenitors in the axial progenitor region changing over time
there are quite profound changes in gene expression profile of these progenitors over time
BUT when grafting these cells from an older embryo to a younger one (where the expression profile will be the different earlier one)
the grafted cells can adjust to the new environment
eg older one expressing chick Hox10 when JUST grafted
but 1hr later - no longer expressing markers for the older posterior progenitor region
readjust themselces to express similar to neighbours
evidence that the older grafted cells can rest their gene expression to match a younger cell type environment
neuromesodermal blurring with the TB progenitors
since the axial progenitors are Bi-fated neuromesodermal progenitors that act like stem cells
can give rise to neural tube cells AND somite mesoderm (paraxial mesoderm) cells
-this messes with the idea of the three germ layers being completely distinctly separated at the top of the heirarchy
-there is persistance of a cell type after initial germ layer formation that has not yet made the decision between neurectoderm of somite mesoderm
or is constantly doing so at least
other putative stem cells in the embryo
neural crest
neural stem cell
myotome stem cell
mesoangioblast
Haematopoietic stem cell/haemangioblast
these cells also mature over time
have a maturing embryo with stem cell like cells that are progenitors for tissues being made over time
