5 blood Flashcards
(23 cards)
What are the lifespans of different blood cells?
RBC in circulation = 120 days
some lymphoid cells = 20 years or more
Neutrophils = only in circulation or tissues for a few days
Platelets = 9-12 days
Where is the main site of blood formation in adults?
Bone marrow
What are haematopoietic stem cells?
Have the unique ability to develop into all types of blood cells, including red blood cells, white blood cells, and platelets.
Can self-renew and give rise to another 2 stem cells or progenitors which divide to become differentiated mature cells
How are progenitors more proliferative than stem cells?
Their number vastly exceeds number of stem cells.
Their self-renewing capacity is more limited, and they are committed to differentiate.
In some context, referred to as transit amplifying cells
What is a precursor?
A cell that precedes another cell in a lineage.
Can be a stem cell or a progenitor
The term doesn’t make statement about a cell’s self-renewal capacity
What is the gold standard assay for haematopoietic stem cell activity?
Long-term repopulation assay.
Intravenously inject bone marrow cells into tail of irradiatied mouse - after 16 weeks, see the blood cells that are in circulation are now proliferated from the donor.
Problem is done in vivo, maintaining mouse for 16 weeks is expensive
How does the CFU-S assay improve on the LTR assay?
12 days after mice injected with BM cells, macroscopic colonies develop in spleen.
These were derived from proliferative cells
DIssociated spleen colony and injected cells into secondary mouse, new spleen colonies formed –> some cells retain CFU-S activity and had undergone self-renewal divisions to give rise to different cells in the new colonies.
Fewer colonies formed in 2nd mouse –> oligopotent progenitors with limited self-renewal capacity –> erythromyeloid progenitors, not HSCs.
Cheaper option, mice only need to be kept for 12 days rather than the 16 weeks to count for long-term survival
Describe the in vitro colony assay developed by bradley and metcalfe (1996)
In original form of assay:
- feeder cells suspected on solid bottom layer of agar (produce things like cytokines and
other molecules which encourage the survival, proliferation and differentiation of the haematopoietic progenitors included in the cell suspension support development of cells in the top layer)
- BM cells placed in semisolid layer above - progenitors recognise signals and form colonies in semisolid medium
These days, use methylcellulose and specifically produced cytokine cocktails instead of agar and feeder cells.
CFU-C assay (colony forming unit culture assay)
Describe FAC (FLuorescence activated cell) sorting
Prospectively sort HSC and progenitor cells.
- Presence of proteins on surface of blood stem and progenitor cells detected with help of antibodies - If antibodies are fluorescently labelled, they can be used for fluorescence-activated cell sorting
Antibodies are bound to the cell surface
Cells are run individually through a laser beam, the emitted fluorescence of each is detected
The sorter deflects the cells downstream into separate tubes
Progenitor cells that are double positive for the c-Kit and Sca-1 antigens are highly enriched for haematopoietic stem cells.
How are FAC-sorted cells tested in long-term repopulation assay?
C-kit, Sca-1 double positive cells are gated, sorted and transplanted into irradiated mice
16 weeks later, peripheral blood analysed.
Some of these cells have repopulation the blood system of the recipient in long term.
These days, most long-term repopulation assays are competitive repopulation assays where we always inject two different type of cells
Donor (one of interest) and competitor cells (makes sure mouse survives if donor cells fail)
carry different alleles of CD45 surface marker – donor all CD45.1, competitor are .1/2
At different points after transplantation, peripheral blood is isolated and analysed by flow cytometry to determine the contribution of donor and competitor cells to different white blood cell types.
CD45 is only expressed on surface of WBC so contribution to RBC reconstitution cannot be determined
How did experiments show HSC first form in the embryo (mammals)?
Dissociated mouse and human embryos of different stages and tested in LTR assay in irradiated mice.
Mouse - LTR HSCs can first be identified from E11 (not time point where you first find blood cells in embryo - already in YS and into circulation at E8.25)
Humans - LTR HSCs first appear E32
Describe the in vitro evidence for the existence of the haemangioblast
Mesodermal cells (Bry:GFP+ and Flk1+) that are present in ESC-derived embryoid bodies and in posterior primitive streak of E7.5 mouse embryos give rise to mixed blast colony made up of blood progenitors and endothelial progenitors.
Blast colony (BL-C) forming cells in haemangioblast (mix of 2 types of progenitors) - separate cells and put under specific conditions –> some of cells turn to either endothelial cells or haematopoietic cells
Describe primitive haematopoiesis in the mouse YS
Progenitors (CFU-EryP) first detectable at E7 in IVCA and enter circulation as erythroblasts at E8.25. Mature in circulation and persist until just after bith.
In mouse Gata1 KO, primitive RBC progenitors form but fail to mature and mice are pale and anemic before they die between E10.5-E11.
Scl/Tal1 acts upstream of Gata1 and is another TF expressed in developing primitive RBC of YS. Mutant mice embryos normal until E8.5 then retarded and pale with no primitive RBC progenitors in YS mesoderm at E9.
KO mice died at E9.5
How do primitive RBC differ from mature RBC?
Much bigger
Express embryonic globins but later switch to adult globins.
Nucleated
Describe the pro-definitive wave of haematopoiesis.
CFU-EryP go into circ at E8.25. From now on, new erythroid progenitors appear than generate RBCs that look like adult ones.
Progenitors present at E9.25 form large burst colonies (BFU-Es) - thought that these mature into CFU-E progenitors which then differenitate futher - BFU-E and CFU-E are pro-definitive eryP than enter circulation and seed foetal liver where they mature first into erythroblasts and eventually into enucleated pro-definitive RBCs.
CFU-EryP generate erythroblasts that enter circulation where they mature
Scl/Tal1 mutant embryonic stem cells generate no blood cells at all.
acts upstream of Gata1 and Pu.1 during haematopoiesis
What is the timing of the 3 waves of haematopoiesis in mouse embryos?
- E7-8 Primitive H that comes from the haemangioblast generates PRBC in YS
- E8-10 Pro-definitive H generates that comes from veinous endothelial cells and arterial endothelial cells –> turn haemogenic and give rise to blood progenitors –> give rise to definitive wave of blood cells
If from haemogenic endothelial cells in arteries –> give rise to pre-HSCs (seed foetal liver) - E11 Definitive H starts with formation fo the first LTR HSCs (only wave derived from stem cells)
Describe definitive haematopoiesis
E11, several sites in mouse and circulation makes it difficult to determine where HSCs first emerge.
Aorta gonads mesonephros generates HSCs autonomously which are localised to the ventral aspect of the dorsal aorta.
Endothelial cells undergo endothelial to haematopoietic transition (EHT).
HSCs form in dorsal aorta, umbilical artery (blood out of placenta) and the vitelline artery (blood into the YS).
Pre-HSCs that require culture express endothelial marker VE-cadherin and blood cell marker C-kit. As these turn into LTR-HSCs, they switch off endothelial markers but maintain c-kit.
C-kit cells also found in vessels of YS sac once PRBC are in circulation. Timing suggests they’re pro-definitive progenitors airsing from haemogenic endothelium. Unlike HSCs they form in arteries and veins
Describe the dissociation/reaggregation assay to define surface phenotype of pre-HSCs
Modified verision of AGM explant culture that involved dissociation and reaggregation of AGM cells to identify cells that can turn into LTR-HSCs.
target surface marker negative AGM cells of non-transgenic embryo isolated and aggregated with positive cells from GFP-positive embryo - aggregate allowed to develop for 4 days and cells tested for LTR-HSC activity in irradiated mice.
If the GFP-expressing test cells give rise to LTR-HSCs, GFP+ cells will be found in the peripheral blood of the recipient
Describe how YS cells contribute to adult haematopoiesis in mammals.
LTR assay fails to detected LTR-HSC activity in YS cells but doesn’t rule out possibility that YS-derived cells contribute to adult haematopoiesis.
At embryonic stages, HSCs and progenitors would normally seed foetal liver at day 12 and not the bone marrow.
Interestingly E9.5, YS cells can contribute if transplanted into foetal liver of newborn - can’t be detected as LTR stem cells at E9.5 simply because they cannot reach the bone marrow.
Results show that E9.5 YS cells contain newborn-repopulating cells - constitute HSC precursors that can seed the foetal liver and the placenta where they are educated to become HSCs that seen the bone marrow
What are the two sites that accumulate large numbers of HSCs?
Placenta and foetal liver.
Pre-HSCs form in the AGM region and in the YS, they enter circulation and seed the foetal liver where they mature into transplantable LTR HSCs (explains huge number of LTR-HSCs on E12)
Describe how Runx1/AML1 signalling is involved in haematopoiesis.
Required for endothelial to haematopoietic transition at the onset of pro-definitive and definitive haematopoiesis.
Hetero mutant carriers are viable but homo mutant die during embryogenesis (most before E11.5). Form primitive RBCs but foetal liver remains pale –> defect in pro-definitive erythropoiesis and mutants display massive haemorrhages.
Runx1 double KO have fairly normal pRBCs but no enucleated RBC in peripheral blood and no stained haematopoietic progenitors in sections of foetal liver –> suggests failure of foetal liver haematopoiesis and that Runx1 is required for pro-definitive wave.
YS and AGM explants of Runx1 +/+, +/- and -/- embryos were cultured ex vivo (mice).
Their survival was examined 6 months post transplantation. Runx1 -/- cells did not support long-term reconstitution –> Runx1 is needed to make LTR HSCs.
Dorsal aorta of homo neg Runx1 embryos lacks intra-aortic c-Kit + cells.
HECs fail to undergo endothelial-to-haematopoietic transition in the absence of Runx1.
C-Kit + cell clusters detected in YS of WT embryos at the time when pro-definitive blood cells form. These clusters are missing in Runx1-/- embryos.
This suggests Runx1 is needed for the formation of pro-definitive blood progenitors in YS – immunohistochemistry shows c-Kit and Runx1 co-expressed in spindle shaped ECs of the YS that give rise to c-kit-positive round blood cells. Pro-definitive progenitors arise from haemogenic endothelial cells in a Runx1-dependent manner.
Describe how Notch1 is involved in haematopoiesis.
Mutant embryos die after E9.5 due to defect in angiogenic vessel remodelling but develop normal pRBCs and almost normal numbers of early CFU-Cs in YS.
Do not form any YS pre-HSCs with newborn repopulation capacity.
Formation of adult-repopulating HSC formation in AGM region cannot be assessed because they would die 2 days before that would develop.
Lacz transgenic Notch1++ or Notch1—ESC injected into WT non-transgenic blastocysts to generate chimeric embryos > YS, foetal liver and BM tissues prepared at different time points and tested in colony essay to see whether lacZ+ cells form the colonies.
Early on, WT and Notch mutant cells contribute equally to CFU-Cs generated during pro-definitive wave of haematopoiesis.
Later when CFU-Cs are derived from definitive HSCs only WT cells can contribute to CFU-C formation.
–> Notch1 not needed for primitive and pro-definitive haematopoiesis
–> Notch1 essential for angiogenic remodelling, arterial specification and definitive haematopoiesis
Why does arterial specification appear to be needed for HSC formation?
Haematopoietic stem cells form only in arteries, including dorsal aorta, vitelline artery and the umbilical artery
