SC9

Question 1

what is haematopoiesis

Answer 1

when HSCs give rise to all other blood lineages, including myeloid and lymphoid progeny

Question 2

describe Haematopoesis in development

Answer 2

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

Question 3

what are the first difintive HSCs and where are they found

Answer 3

• 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.

Question 4

Describe the experiment showing new HSC emergence in zebra fish embryo

Answer 4

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.

Question 5

how does zebra fish embryonic HSC differ from mammal

Answer 5

in mammals new HSC go to the blood stream, in fish HSCs just move away.

Question 6

why are zebra fish useful in study of development

Answer 6

as they are transparent so development can be seen in vivo.

Question 7

what happens in adult haematopoesis

Answer 7

HSCs give rise to all cell types in haemtopoetic system.

Question 8

what is the current understanding of haemotopoiesis lineages

Answer 8

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.

Question 9

give an example of a HSC regulation factor

Answer 9

HoxB4: homeobox gene: positive regulator of self-renewal and expansion.
ES and iPSC have high levels

Question 10

give two examples of HSC TFs

Answer 10

1. Gfi1: negative regulator of HSC proliferation. KO leads to HSC that proliferate more.
2. c-myc: promotes differentiation and proliferation

Question 11

give three examples of HSC cell cycle inhibitors and what are they?

Answer 11

proteins involved in maintaining SC quiescence and preserving from aging.
p21, p27 and p16

Question 12

what does p21 do in HSCs

Answer 12

inhibits HSC cycle
p21 KO mice rapidly lose HSC due to their fast proliferation and exhaustion.
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Question 13

what does p27 do in HSCs

Answer 13

does not affect HSCs directly

but p27 KO have an expanded progenitor pool

Question 14

what does p16 do in HSCs

Answer 14

KO leads to reduction of HSC in young mice but rescues HSCs in old mice

Question 15

what are two examples of chromatin and histone modifiers of HSCs

Answer 15

Bmi-1 and MII

Question 16

what does Bmi-1 do in HSCs

Answer 16

polcomb gene neccessary for maintenance of Lt-HSC. KO loses HSC rapidly

Question 17

what does MII do in HSCs

Answer 17

tritorax gene
neccessary for HSC development.
MII deletion in adult mice leads to no overt phenoptype, however MII deficient BM fails to reconstitute recipient mice.

Question 18

give an example of mircoRNA processing in HSCs

Answer 18

DICEE
essential for HSC persistance in vivo.
In particular, Mir-125a controls HPC apoptosis by down regulating pro-apoptotic genes such as Bak1.

Question 19

give an example of a tumour suppressor in HSCs

Answer 19

PTEN: indispensable in HSC maintenacne (but not leukemia maintainence).

Question 20

How is the haemotopoietic system robust?

Answer 20

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

Question 21

How can HSC fitness be tested

Answer 21

Competative BM transplantation

Question 22

describe a competitive BM transplantation

Answer 22

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.

Question 23

why would the BM from another mouse not be rejected

Answer 23

as HSC niche is an immuno priveledged site.

Question 24

Describe serial transplant and why it is useful

Answer 24

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.

Question 25

give an example of a subtler phenotype only shown via serial transplants

Answer 25

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

Question 26

Why are wnt signalling pws difficult to study

Answer 26

due to a high activity in a no. of systems/ dose dependencay

Question 27

Give an example of a KO that has no apparent phenotype and 4 reasons why this is unexpected

Answer 27

``` beta (alpha/gamma) catenin KO unexpected due to related activity of: 1. tumour suppressor APC 2. GSK3 beta 3. Dkk1 4. wnt pws ```

Question 28

how does tumour suppressor APC involve beta catenin and how is it important for HSCs

Answer 28

regulates the function of HSCs through a beta catenin paw and is indispensable for HC survival.

Question 29

how does GSK3 beta involve beta catenin and how is it important for HSCs

Answer 29

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.

Question 30

how does Dkk1 involve beta catenin and how is it important for HSCs

Answer 30

dk11 inhibits wnt/beta catenin pw via reduction in beta catenin and increase in OCT4 expression. Expression of Dkk1 by osteoblasts impairs HSC function.

Question 31

why is wnt signalling important for HSCs

Answer 31

wnt stimulates self-renewal of atleast a sub-population of HSCs. wnt signalling levels need to be within optimum levels.

Question 32

how do osteoblasts support HSC generation

Answer 32

by secreting GFs

Question 33

what affect does killing off osteoblasts with suicide genes and ganciclovir have on BM

Answer 33

BM empties out, however repopulation is still possible.

Question 34

what is PTH and what does it do

Answer 34

a hormone important in bone remodelling treatment expands the osteoblast compartment and HSC pool via Notch signalling.

Question 35

what is BMP cytokine and what does it do

Answer 35

BMP cytokine = bome morphogenic protein. | signalling regulates osteoblast no. and HSC no.

Question 36

what does colagen1.3 TK do

Answer 36

ablates osteoblast population and severely alters BM hematopoesis, reversible so not all HSCs must be ablated.

Question 37

what does in vivo imaging of HSC injection into irradiated and non irradiated mice show

Answer 37

in non irradiated: HSCs dont engraft and are further away from osteoblasts in irradiated: HSCs engraft and are closer to osteoblasts and vessels

Question 38

What are the two hypothesised niches hor HSCs

Answer 38

endosteal niche vascualture niche *osteoblasts and blood vessels both seem to be important.

Question 39

what is the initial working model for the HSC niche

Answer 39

quiescent SCs are found close to osteoblasts and move towards the central marrow while differentiating.

Question 40

what is the current working model for for HSC niche

Answer 40

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

Question 41

what are mesenchymal cells

Answer 41

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)

Question 42

what are the therapeutic applications for mesenchymal BM cells

Answer 42

1. have good anti-inflammatory function and improve BM engraftent. 2. Also improve results of several therapies with good immunomodulatory effect. 3. however have very poor homing to BM and reside only transiently in lung and spleen and dont engraft there.