Flashcards in SC9 Deck (42):
what is haematopoiesis
when HSCs give rise to all other blood lineages, including myeloid and lymphoid progeny
describe Haematopoesis in development
occurs in wave
1st wave: occurs in yolk sac outside of the embryo (cannot engraft) and then enter the embryo via the vasculature
2nd wave: occurs in the liver (more proliferative than adult SCs) highly enriched cells
3rd wave: occurs as soon as bone cavity is formed, SC colonise and become productive
what are the first difintive HSCs and where are they found
- definitive: found early as possible in development and can be transplanted into irradiated mouse.
** intraembryonics HSCs: develop in the aorta gonad mesenephros (AGM) region.
on the floor of the dorsal aorta which is surrounded by mesenchymal cells.
In this region some of the endothelial cell develop into HSCs moving in circulation.
Describe the experiment showing new HSC emergence in zebra fish embryo
used non invasive high resolution imaging of live zebra fish embryos to show that HSCs emerge directly from the endothelium of the aortic floor.
* in this model the developing SCs pulls adjacent cells together to maintain the vessel and then the newly formed HSCs move away.
how does zebra fish embryonic HSC differ from mammal
in mammals new HSC go to the blood stream, in fish HSCs just move away.
why are zebra fish useful in study of development
as they are transparent so development can be seen in vivo.
what happens in adult haematopoesis
HSCs give rise to all cell types in haemtopoetic system.
what is the current understanding of haemotopoiesis lineages
original understanding was that the system was strictly heirachal, with lineages being related in certain ways.
however now believe there are more routes than original predicted.
for example the common myeloid progenitoy may not exist in reality and may actually be 19 sub populations.
the exact route from a stem to a differentiated cell can vary.
give an example of a HSC regulation factor
HoxB4: homeobox gene: positive regulator of self-renewal and expansion.
ES and iPSC have high levels
give two examples of HSC TFs
1. Gfi1: negative regulator of HSC proliferation. KO leads to HSC that proliferate more.
2. c-myc: promotes differentiation and proliferation
give three examples of HSC cell cycle inhibitors and what are they?
proteins involved in maintaining SC quiescence and preserving from aging.
p21, p27 and p16
what does p21 do in HSCs
inhibits HSC cycle
p21 KO mice rapidly lose HSC due to their fast proliferation and exhaustion.
what does p27 do in HSCs
does not affect HSCs directly
but p27 KO have an expanded progenitor pool
what does p16 do in HSCs
KO leads to reduction of HSC in young mice but rescues HSCs in old mice
what are two examples of chromatin and histone modifiers of HSCs
Bmi-1 and MII
what does Bmi-1 do in HSCs
polcomb gene neccessary for maintenance of Lt-HSC. KO loses HSC rapidly
what does MII do in HSCs
neccessary for HSC development.
MII deletion in adult mice leads to no overt phenoptype, however MII deficient BM fails to reconstitute recipient mice.
give an example of mircoRNA processing in HSCs
essential for HSC persistance in vivo.
In particular, Mir-125a controls HPC apoptosis by down regulating pro-apoptotic genes such as Bak1.
give an example of a tumour suppressor in HSCs
PTEN: indispensable in HSC maintenacne (but not leukemia maintainence).
How is the haemotopoietic system robust?
lots of redundant systems.
KOs often have no phenotypes until stress is applied.
Treatment for therapy targets therefore need to be important for Leukemia cells than healthy HSCs. CD44 KOs can engraft into normal HSCs byt not leukemia
How can HSC fitness be tested
Competative BM transplantation
describe a competitive BM transplantation
1. BM mixed 1:1 from competitor mouse with CD45.1 or with a donor mouse (either a WT control or altered (KO)) CD45.2. injected into CD45.1 mouse.
2. Host is irradiated so injected BM repopulates system
3. flow cytometry can identify how many cells are CD45.1/2 using Abs.
4. can test how altered KO SC are doing compared to control at repopulating tissue.
5. control mice have 50:50 reconstitution.
why would the BM from another mouse not be rejected
as HSC niche is an immuno priveledged site.
Describe serial transplant and why it is useful
alter cells in BM and move into a recipient, remove and repeat then looking for results for tertiary recipient.
-- allows for subtler phenotypes to be shown.
give an example of a subtler phenotype only shown via serial transplants
DK11 - wnt signalling inhibitor by competing for receptors.
initially mice have no phenotype but after serial transplantation/stress the recipients dont survive therefore inhibition of self-renewal occurs
Why are wnt signalling pws difficult to study
due to a high activity in a no. of systems/ dose dependencay
Give an example of a KO that has no apparent phenotype and 4 reasons why this is unexpected
beta (alpha/gamma) catenin KO
unexpected due to related activity of:
1. tumour suppressor APC
2. GSK3 beta
4. wnt pws
how does tumour suppressor APC involve beta catenin and how is it important for HSCs
regulates the function of HSCs through a beta catenin paw and is indispensable for HC survival.
how does GSK3 beta involve beta catenin and how is it important for HSCs
key enzyme in wnt signalling, it phosphorylates beta catenin leading to its degradation.
GSK3beta inhibitors increase HSC ability to repopulate transplanted recipients by expanding progenitors and reatining HSCs.
how does Dkk1 involve beta catenin and how is it important for HSCs
dk11 inhibits wnt/beta catenin pw via reduction in beta catenin and increase in OCT4 expression.
Expression of Dkk1 by osteoblasts impairs HSC function.
why is wnt signalling important for HSCs
wnt stimulates self-renewal of atleast a sub-population of HSCs.
wnt signalling levels need to be within optimum levels.
how do osteoblasts support HSC generation
by secreting GFs
what affect does killing off osteoblasts with suicide genes and ganciclovir have on BM
BM empties out, however repopulation is still possible.
what is PTH and what does it do
a hormone important in bone remodelling treatment expands the osteoblast compartment and HSC pool via Notch signalling.
what is BMP cytokine and what does it do
BMP cytokine = bome morphogenic protein.
signalling regulates osteoblast no. and HSC no.
what does colagen1.3 TK do
ablates osteoblast population and severely alters BM hematopoesis, reversible so not all HSCs must be ablated.
what does in vivo imaging of HSC injection into irradiated and non irradiated mice show
in non irradiated: HSCs dont engraft and are further away from osteoblasts
in irradiated: HSCs engraft and are closer to osteoblasts and vessels
What are the two hypothesised niches hor HSCs
*osteoblasts and blood vessels both seem to be important.
what is the initial working model for the HSC niche
quiescent SCs are found close to osteoblasts and move towards the central marrow while differentiating.
what is the current working model for for HSC niche
specific types of vessels are found in both HSCs and mesenchymal cells and regulate hematopoesis.
* LT-HSC (long term): preferentially localise near osteoblasts.
* endosteum vessels are surrounded by Nestin+ mesencymal cells (SC marker) and very closely asssociated with HSCs
what are mesenchymal cells
in vivo: give rise to bone/cartilage and fat
in vitro: give rise to all plus astrocytes/fibroblasts and myocytes.
* still difficult to identify - bad marker (selection is via good adherance to plastic)