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Molecular mechanisms in immune cell differentiation and function > Haematopoietic Stem Cell Differentiation > Flashcards

Haematopoietic Stem Cell Differentiation Flashcards

1
Q

What is haematopoiesis?

A

Process of blood production.

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2
Q

Where does haematopoiesis take place?

A

Firstly, in the yolk sac, then in the fetal liver, aorta-gonad-mesonephros, placenta and bone marrow.

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3
Q

Which haematopoietic stem cells have self-renewal properties?

A

Long-term haematopoietic stem cells (LT-HSC), short-term haematopoietic stem cells (ST-HSC) and multipotent progenitors (MPP).

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4
Q

What can be found in bone marrow niche?

A

Haematopoietic stem cells and stromal cells. Stromal cells include endothelial cells, perivascular cells, sympathetic nerve fibres, megakaryocytes, macrophages, T cells, adipocytes, and osteoblasts.

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5
Q

What are the 2 types of bone marrow niches?

A
  • Endosteal niche (close to the bone)
  • Central niche/perivascular niche (further away from the bone).
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6
Q

In which niche are the majority of HSCs?

A

Central/perivascular niche.

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7
Q

What characterizes the endosteal niche?

A

Higher Calcium and lower oxygen environment. The endosteal niche is used for long-term HSC storage in a quiescent state.

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8
Q

What characterizes the central niche?

A

Lower Calcium and higher oxygen environment. The central niche is used for short-term HSC storage in a process of proliferation and differentiation.

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9
Q

When does haematopoiesis start in humans?

A

5 weeks of gestation.

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10
Q

What progenitors do we have in haematopoiesis?

A
  • Common myeloid progenitors
  • Common lymphoid progenitors
  • Megakaryocyte/erythrocyte progenitor
  • Granulocyte/monocyte progenitors.
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11
Q

What are the characteristics of progenitors?

A

They are highly proliferative and more committed than stem cells. However, they don’t have self-renewal properties.

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12
Q

What are the characteristics of mature cells?

A

They are the most committed. They will perform a function, they do not proliferate, and they do not have self-renewal properties.

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13
Q

Is haematopoiesis happening continuously in order?

A

No, there are intermediates, and they can shift from one side to the other.

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14
Q

Do progenitors in the fetal liver derive from HSCs?

A

No, there is a study which states that the progenitors in the fetal liver come from a separate branch of haematopoietic stem cells.

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15
Q

Do HSCs need to last a lifetime?

A

Yes, LT, ST and MPPs need to last a lifetime. It has been shown in a study where mice were irradiated and then given an injection with bone marrow cells, and they saw that the animal did not die but recovered the full blood. This means that there was a stem cell population which repopulated the whole bone marrow.

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16
Q

What is the lineage capacity for stromal stem cells in bone marrow?

A

They can differentiate into:
- osteoblasts,
- chondrocytes
- adipocytes

17
Q

How does nonmyelinating Schawnn cell control HSC?

A

It secretes TGF-beta, which is a signal for HSC to maintain quiescence.

18
Q

Does bone marrow niche interact with HSCs?

A

Yes, there are many ways in which this interaction occurs, for example, by specific receptors or cytokines. The interaction can predict the fate of HSCs.

19
Q

What are potential fates of HSCs?

A
  • Apoptosis
  • Quiescence
  • Division in 3 different ways:
  • Expansion (two identical HSCs identical to parental HSC)
  • Depletion (two more proliferative HSCs than the parental HSC)
  • Homeostatic self-renewal (one identical to parental and one more proliferative).
20
Q

How can bone marrow niche support HSCs?

A

They can signal for:
- self-renewal
- quiescence
- proliferation
- engraftment
- homing

21
Q

What is the most common disease arising from the dysregulation of haematopoiesis?

A

Leukaemia (acute myeloid leukaemia, acute lymphocytic leukaemia).

22
Q

How can we isolate HSC using a sorter? Which markers can we use?

A
  • KSL (Kit, Sca-1, Lineage) - LT-HSC, ST-HSC, MPP
  • SLAM (CD150, CD48)
  • Side population (SP): Hoechst 33342
23
Q

How does KSL work?

A

Combine the bone marrow with a cocktail of antibodies, which will contain the following:
- lineage-negative antibodies (Ter119, Mac1, B220, CD5, CD8a and Gr1),
- c-kit antibody
- Sca-1 antibody
As they will all be fluorescent green, we first need to set a profile where green fluorescence is on the Y axis (lineage), and the X axis will be the forward scatter (the cell size). We treat cells only with the lineage cocktail. On the bottom are lineage-negative cells, which don’t express any markers from the lineage cocktail.
Now from only lineage negative cells we need to treat them with anti-c-kit and anti-Sca-1, c-Kit on Y axis, Sca-1 on. X-axis and we need double-positive cells, and there is our cell population (Kit+Sca+Lin-). These are our LT-HSC, ST-HSC and MPPs.

24
Q

How can we isolate each subset after isolating a population of mixed LT-HSC, ST-HSC, and MPPs by KSL?

A

We can use markers like Flt-3 and CD34.
- MPPs will be double positive,
- ST-HSC will be positive for CD34 and negative for Flt-3
- LT-HSC will be positive for Flt-3 and negative for CD34

25
Q

How does SLAM work?

A

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

26
Q

How does the side population work?

A

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.

27
Q

What properties of HSCs can we study?

A
  • proliferation and differentiation capacity
  • Stem cell properties: repopulation capacity
  • Homing and niche retention
28
Q

How can we assess the proliferation and differentiation capacity?

A

By colony assay

29
Q

How does colony assay work?

A
  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
30
Q

How can we assess the repopulation capacity?

A

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.

31
Q

How can we assess homing?

A

By intrafemural transplants KSL

32
Q

How do intrafemural transplants KSL work?

A

Molecular mechanisms in immune cell differentiation and function (2 decks)

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