HSCs Flashcards

Question

how can HSCs be isolated using SLAM?

Answer 1

SLAM: Antibodies for CD48 and CD150 - CD150 positive and CD48 negative are the most long-term HSCs

Answer 2

Hoechst 33342 dye: - HSCs express ABC transporters which efflux the dye

Answer 3

yes: Can refine by using KSL with SP - Cells at tip of SP are the HSCs which are more quiescent

Answer 4

Colony assay - take bone marrow and put cells in semi-solid medium - Makes columns of cells – white spots - Microscope can see how morphology is different between colonies - CFU-E (erythrocytes) have haemoglobin – red so easily detected - Sparse growth = monocytes - Compact growth = granulocytes Can look at how mutations affect different lineage growth/proliferation in the colony - If colony isn’t forming, the mutated gene must be important

Answer 5

bone marrow transplants using congenic mice which differ in their CD45 subtype allele - host mouse is CD45.1 positive - donor mouse is CD45.2 positive - use antibodies to detect specific CD45 subtype alleles to see which cells were orginally in the animal, and which have been donated

Answer 6

Lethal irradiation of host, inject HSCs from healthy donor – see if it repopulates – if it does, they have self-renewal properties - Repeat in different host – repeat testing of self-renewal capacity - Won’t reject as they are congenic mouse

Answer 7

- Lethal irradiation of CD45.1 mouse - Inject donor CD45.2 BM cells via IV in the tail - Detect cells from donor and cells from host using antibodies for .1 and .2 - Competitor/reference – cells from mouse which express both .1 and .2

Answer 8

Competitor/reference mouse is a positive control which has HSCs expressing both CD45.1 and CD45.2 - Lethal irradiation of mice – deplete whole HSC population - Control checks that the blood was injected into correct vein – don’t want to miss the vein - Also keeps the animals alive

Answer 9

If CD45.2 cells only engraft, the mice will survive If they die, we don’t know why – cells may be non-functional, or too much irradiation

Answer 10

Cell autonomous: total K/O in all cells of mouse, not just in HSCs – mutant is everywhere - Transplant issues could be due to mutation in other cells, e.g. niche cells, not just in HSC - Take K/O BM, transplant into lethal irradiated but healthy mouse, everything else is WT in that mouse - if phenotype in healthy mouse is now the same as the mutant mouse, indicates the HSC mutation was cell autonomous - or, irradiate mouse containing mutation so HSCs are wiped out, see if mutant niche affects the phenotype

Answer 11

- intrafemural transplants - immunofluorescence - IVIS imaging - intravital microscopy

Answer 12

Intrafemural transplants: - lethally irradiate mouse - inject donor CD45.2 HSCs into the right knee - isolate bone marrow cells (CD45.1/CD45.2) and KSL cells (CD45.2) use flow cytometry to analyse

Answer 13

Injection into right knee: - If cells aren’t seen in the bone marrow of the right knee… There is a problem with BM retention If in right knee but not left knee… - Problem with migration/homing

Answer 14

If the CD45.2 cells of interest are not there, but the reference cells are there, then the gene of interest that is K/O must be important for homing to the bone marrow from the blood Or cells are not reaching bone marrow from IV injection Bone marrow produces cytokines for chemotaxis of cells – gene may impact cell reception of chemokines

Answer 15

Inject donor HSCs with IV, sacrifice mouse after 18 hours, take BM section, immunofluorescence study Stain HSCs with CD150 = red Mesenchymal cell: Nesting-GFP = green Lineage cocktail and CD48 = blue – excludes differentiated, mature cells Determine if red HSCs can migrate to endosteal niche

Answer 16

Anaesthetise mice - Inject HSCs cells with luciferin - glow - can see luciferase glow in thymus and spleen

Answer 17

Record movement of HSCs in vivo migrating into osteoblasts - Determine timing of movement - Compare to leukaemic cells for example

Answer 18

Conditional KO – induce mutation whenever wanted in specific tissue of interest, for example, c-myc: - Cre enzyme recognises palindromic sequence called floxP which has 2 loxP sites either side of gene of interest – Cre excises DNA in between loxP sites to create a KO - Cre only expressed in presence of interferon – induces KO under interferon conditions - Crossed MxCre mice with loxP-cMyc-loxP mouse in intron 1 after exon 3 - Cre deletes cMyc – K/O

Answer 19

When c-myc is K/O, bones of animal were paler = little haematopoises occuring - Mutant organs lack HSCs population – low HSC count over weeks post c-myc deletion KI67 is marker for quiescence: - Only proliferative cells will be positive for KI67, whereas HSCs will be negative - More quiescent cells when c-myc was K/O Lineage cocktail with KIT and SCA1: - Increased lineage negative cells with c-myc KO - KSL cells increased under c-myc KO

Answer 20

Interactions with N-cad, ICAM, integrins which maintain cells in quiescence, where they express low levels of cmyc - Cell divides via mitogenic signals: - Expansion – 2 daughter HSCs which have low c-myc levels - Homeostatic – 1 daughter cell has low c-myc, other is more committed with more c-myc expression - Depletion – both daughter cells are committed with high c-myc

Answer 21

LT HSCs have low c-myc:quiescent and self-renewing - Mitogenic signal leads to high c-myc expression in LT HSC, so it becomes ST HSC ST HSCs undergo expansion of c- myc for proliferation - maturing of cells means c-myc is switched off in c-myc K/O mice: - LT-HSCs continue to expand and cannot mature to MPPs as c-myc levels are too low - haematopoesis cannot progress, leading to cancer at level of too many LT-HSCs in high c-myc expression: - LT-HSCs continue to differentiate and are depleted - super expansion of progenitors which cannot switch off c-myc as it is overexpressed - cant produce mature cells - cancer at level of too many progenitors

Answer 22

Schwann cells surround HSC niche to help against myeloproliferative neoplasms (high number of mature cells which don’t function) - Mutation in JAK2 in 80% cases - humanised mouse with JAK2 mutant shows reduced mesenchymal stem cells and absence of sympathetic fibres and schwann cells JAK2 mutant depletes niche

Answer 23

beta3-adrenergic agonist can recover schwann cells - mature cell levels come down to baseline and are now functional - fibrosis is reduced when treated

Answer 24

Mutant HSCs produce IL-1b which destroy the sympathetic fibre glial cells Inflammation destroys mesenchymal stem cells – cytokines cause fibrosis and gliois B3-adrenergic agonist protects glial cells - More CXCL12 to recover normal haematopoiesis

Answer 25

use of bone marrow organoids in 3D derived from iPSCs - See interactions of components in the niche between leukaemic stem cells and normal HSCs