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
difficulty of studying mouse embryos - why go in vitro?
mouse embryos difficult to study
preimplantation mouse embryos are easy to culture
but have small cell numbers, cells themselves are pretty small, and the extent of development that can be studied it limited (as post implantation ones dont grow easily in culture? and this is where most of it happens in utero)
benefits of using stem cells in petri dish
solution to problems of mouse embryo study
BUT
need large enough cell numbers to do the necessary analyses
can try to do micro-scale to see at single cell level
-single cell transcriptomics - possible
-single cell proteomics - difficult
can relevant mammalian development events be modelled in a dish?
how similar are in vitro cells to real development
important events in mammal embryo development
implantation
gastrulation E6/7
axis elongation E8-13(or 19???)
different ES cells at diff stages
preimplantation
and around gastrulation stage
if cultured in vitro - how representative of an in vivo cell type are they?
how do we judge this?
what ES cells from what stages?
ICM ES cells from pre implantation
Epiblast stem cells from post implantation
stem cells relevant basic info
> origin/location: (=niche) what local factors influence self-renewal and differentiation
> molecular markers: eg TFs, cell adhesion/membrane proteins
> descendants: reproducing normal differentiation pathways in vitro should result in normal cells
ICM derived ES cells properties???
in ICM in early blastocyst
after trophoblast has segregated
next segregation is between high and low FGF signalling cells
cells with high FGF signalling become the primitive endoderm
low become epiblast
LIF receptor (signalling) becomes active in blue cells
LIF ligand expressed by trophoblast cells
getting ES cells from the ICM of the blastocyst
- optional - destroy trophectoderm outer layer
2.plate on layer of feeder cells (=irradiated stromal cells -prevents them from dividing)) derived from later embryos which support ES cell growth
- primitive endoderm eliminated by blocking FGF signalling
- once epiblast cells have divided a few times - disaggregate and re-plate
colonies of cells form on the stromal feeder cells - ES cell colonies growing in culture
critical signals for ICM derived ES cells
-LIF - from feeder cells
-BMP - from the serum
-FGF inhibition - preventing differentiation (to PE eg)
Molecular markers of ICM ES cells
in vivo
these cells express
-oct4
-nanog
-sox2
in vitro ES cells grown with LIF, FGF inhib, BMP express these TFs
these factors are key for pluripotency for these stem cell lines - getting rid of any disrupts it
see no expression of characteristic differentiate markers
do descendents of in vitro mouse ICM ES cells act like the normal ones in blastocyst
inject GFP labelled ES cells into blastocoel cavity of mouse embryo
these will integrate into and disperse within the ICM and eventually be able to generate all adult cell types - just like the ICM (=pluripotent)
transplantation of this GFP+ chimeric blastocyts
if this chimeric blastocyst is transplanted into foster mother uterus
implants and grows into liveborn mouse pups
some of them are stripy
(full black genotype of host blastocyst
brown coat id colour of ES cell genotype which have contributed to whole host organism)
so it is chimera of Host and injected ES cells
culture grown ES cells integrate perfectly normally
AND these ES cells can contribute to germline
chimeric mouse bred w WT mouse
chimeric mice can be bred with WT to make entire ES cell derived mice - fully GFP ones in some of progeny
means that these ES cells that haev integrated into the ICM can contribute to the soma and teh Germline
ES cell formed tumours
can form teratocarcinomas
contain wide variety of differentiated cell types and embryonal carcinoma (EC) cells (pluripotent cells that can seed secondary tumours)
non-pluripotent cells:
>when transplanted - dont give v big tumour - not many cell types?
pluripotent cells when transplanted:
give rise to large aggressive tumours - containing cell types from all 3 germ layers
-do this in organised way in embryo but in hectic way in tumour
Epiblast stem cells basic
derived from post implantation epiblast
can dissect it into regions
then dissociated by enzymes into clusters of cells that can be plated
similar to preimplantation
but factors added are different
factors needed to keep these as stem cell line:
-Activin
-FGF
(note// no Wnt)
note on activin/fgf
at normal postimplantation embryos
they are exposed to signalling from TGF beta like molecule - Nodal (most similar to activin which is easier to add to cuclture and act on same receptor as nodal)
FGF signalling in most cells of epiblast at some level
primitive streak is where cells experience WNT signalling - differentiate into different germ layer occuring
so dont want wnt as want them to remain stem cells
and in presence of all nodal fgf and wnt => differentiation
get rid of wnt = stem cells maintained
deriving EpiSCs from ES cells in vitro
culture the ES cells in FGF/activin
molecular markers in epiblast cells
different to ES cells
pluripotency factors Oct4, Sox2 and nanog are coexpressed in preimplantation (E4.5) epiblast
post-implantation (E7.5), Sox2 and nanog partially segregate (arent always expressed together)
gives EpiSC subpopulations?
EpiSC subpopulation markers of differentiation
Brachiary and Foxa2
expressed in part of epiblast in vivo
mark future mesoderm and future endoderm respectively
inhibiting wnt signalling suppresses expression of these markers
nanog and (oct4/sox2??) remain
descendants of Epiblast stem cells
EpiSCs make teratomas
they do NOT form chimeras when injected into blastocyst
may be unable to stick to the ICM of the blastocyst so cannot integrate
because their adhesion differs from preimplantation ES cells
how to make chimera from EpiSCs
generate chimeras when grafted into the post-implantation epiblast
but need to graft them into the correct plase as adhesion differs depending on the location within the epiblast - so need to put in location matching adhesion of the ones taken
two pluripotent states in the early embryo
E2.5-4.5 = ESC
express
-Klfs, Rex1, Esrrb
-Oct4, Nanog, Sox2 (pluripotency markers?)
-are LIF and BMP dependent
-FGF induces differentiation
E5.5-7.5 = EpiSC
express
-Fgf5, T-bra, Foxa2
-Oct4, Nanog, Sox2 (P markers)
-FGF/activin dependent
-LIF does nothing
using pluripotent stem cells to model embryonic development in vitro
can differentiate them in a 2d flat structure
differentiating pluripotent stem cells in 2d culture to make neuromesodermal progenitors (NMP)
remove the self renewal factors
add in Wnt and FGF
causes differentiation from ES cell to cell types in primitive streak region - NMPs
these NMPs can male cell types of clinical interest
cell types generates by NMPs
NMPs used to generate cells that are neural crest derived cell type that can ONLY be generated by NMPs
NMPs active during axis elongation
if can generate derivative of NMP then it will be trunk like derivative and not anterior like
the derived neural crest like cells have markers for trunk like markers and neural crest markers
so by following the logic of the embryo -can make in vitro cell types of potential clinical interest
eg can generate neural crest cells that end up above the adrenal gland
defects can come from these cells
so generating them can be of clinical use possibly
clinical application 1 of embryonic pluripotent cells
reversal of diabetes with embryonic pluripotent cell derived insulin producing cells
generating pancreatic beta cells
treating parkinsons by generating dopaminergic neurons
clinical applicaiton 2 of embryonic pluripotent cells
testing pharmaceuticals
can test new pharmaceutical products on specialised ES-derived cells
