Haematopoietic Stem Cell Differentiation Flashcards

Question

How does SLAM work?

Answer 1

You also need to use the lineage negative cocktail, c-Kit, and Sca-1. Then, you can use CD150 and CD48. - LT-HSC are CD150 positive, CD48 negative

Answer 2

By using Hoechst 33342. HSCs express ABCD2 transporters when the cell gets something cytotoxic (dye), which they pump out. This will give you a side population outside of the main population, which will be HSCs.

Answer 3

- proliferation and differentiation capacity - Stem cell properties: repopulation capacity - Homing and niche retention

Answer 4

By colony assay

Answer 5

1. Take the bone marrow 2. Put a limited amount of cells into a semi-solid medium 3. You will end up with different colonies of cells 4. Look under the microscope to assess the morphology of each colony to tell which cells you have in which colony

Answer 6

Bone marrow transplant We can use congenic mice, which differ only in CD45.1 and CD45.2 1. Lethaly irradiate mouse with CD45.1 2. Inject bone marrow cells from CD45.2 mouse 3. Use antibodies against CD45.1 and CD45.2 using flow cytometry. This way, we can see cells from each mouse. There will be a population which will be double positive (competitor/reference). We use it to know if we successfully injected the cells into the vein. Another use of competitor population is if we inject two different cell types, we can see which one is more proliferative.

Answer 7

By intrafemural transplants KSL

Answer 8

- Lethal irradiation - inject WT KSL (CD45.2) and total BM (CD45.1/CD45.2) into the knee of the mouse - Check with flow cytometry

Answer 9

Because with intravenous you are not sure if you managed to inject the cells into the vein. When it comes to homing bone marrow produces chemokines to attract cells to the bone marrow and if you don't get the cells there then you dont know if its because of gene X that you are looking at is important for coding of receptors or if you missed the vein. This does not happen with intrafemural injection.

Answer 10

There is a problem with retention.

Answer 11

There is a problem with homing.

Answer 12

- You can inject cells into the vein, then sacrifice the mouse, take a piece of bone marrow and check it by immunofluorescence. You can stain your HSCs for CD150, and to exclude other cells, you can use another colour for the CD48 lineage cocktail and Nestin for mesenchymal stem cells (stromal cells). This way, you can see if cells can migrate into the bone marrow niche. - You can also check it in vivo by anaesthetising the mouse, injecting luminescent cells and checking with luciferin the cells that glow, and you can check if these cells are migrating to the bone marrow.

Answer 13

It recognises the LoxP sequence, and if there are two LoxP sequences, cre will bind to them, excise the DNA between them, and it created a KO.

Answer 14

When Wilson et al. did the KO of c-myc, they saw a decrease in the number of cells in BM, spleen and thymus, and bones were very pale as if no hematopoiesis was taking place. - There is a low expression of c-myc in LT-HSCs, when they receive mitogenic signals, c-myc expression increases so that cells can start maturing (ST-HSC), and once they mature, c-myc expression is turned off. - When there is c-myc deficiency, then cells stay as LT-HSC with low c-myc expression, so there is no expansion and there is accumulation of LT-HSC (leukaemia) - When there is overexpression of c-myc then cells expand but c-myc is not switched off so they cant progress to become mature cells also there is no LT-HSCs as they progress to ST-HSCs (leukaemia)

Answer 15

Promotes maturation and proliferation.

Answer 16

It is a group of disorders (cancers) where there is too many blood cells produced but they do not function properly. They are characterised by the mutation in JAK2.

Answer 17

They KI a mutation in JAK2 and mimicked the myeloproliferative neoplasm disorder in mice. They found that this mutation was affecting the bone marrow niche (less stromal cells, Schwann cells and almost absent sympathetic fibres). Then they found that treatment with beta3-adrenergic agonist blocks MPN and rescues Schwann cells. This shows that sympathetic nerve fibres are important in haematopoiesis.

Answer 18

Mutant haematopoietic stem cells are producing a lot of IL-1beta and it is destroying glial cells. Also the inflammatory response causes destruction of stromal cells and the cytokine storm leads to fibrosis and osteoporosis.

Answer 19

It protects glial cells, increases the production of CXCL12, and restores the normal function of haematopoiesis.

Answer 20

Irradiate the mouse, and if it dies, then there is an issue with self-renewal, and if it didn't then there is no issue with self-renewal.