Myeloid cell differentiation Flashcards
How do HSCs choose their lineage?
The chromatin is regulating which genes are turned on and off and which transcription factors are to be expressed which is giving the identity to the cell.
If cells go into a particular direction, can they still change their fate?
In healthy, in vivo setting if a cell commits towards certain lineage then it usually does not change their lineage anymore. However, in the lab it can be manipulated by turning on different transcription factors.
What guides the cell fate decisions?
- Intrinsic factors (transcription factors which regulate genes)
- Extrinsic factors (molecules, cytokines and cells around them which give signals)
What do colony-stimulating-factors (CSF) do?
They can make progenitor cells to differentiate into a particular colony, e.g. macrophage-colony-stimulating- factor will make them to differentiate into macrophages.
What is a semi-solid medium?
It is a very thick medium and you can vortex your cells there and each cell will be in one place and it will not diffuse, then these cells if are progenitors will differentiate into certain cells and for colonies which can be observed under a microscope.
What are examples of extrinsic factors?
- IL-3 is required for proliferation, it is required in differentiation into many different lineages and is not lineage specific (megakaryocyte, erythrocyte, granulocyte, monocyte etc.)
- TPO - specific for megakaryocytes
- EPO - specific for erythrocytes
- M-CSF and GM-CSF - specific for monocyte/macrophage
- GM-CSF - specific for granulocytes
What signalling pathway is typical for type I cytokines (IL-3, IL-5, IL-6, GM-CSF, G-CSF)
JAK/STAT signalling pathway
How does JAK/STAT pathway work?
- When cytokine engages with its receptor, that leads to the phosphorylation of tyrosines by JAK kinase. This gives a signal to recruit other proteins, here STAT which gets activated by phosphorylation. Once activates it travels into the nucleus, finds specific DNA motifs and promotes expression of target genes. Then STAT becomes inactivated and travels back to the cytosol.
- Genes which are expressed as a result of JAK/STAT pathway are essential for cell survival, cell proliferation and functional activation
What are transcription factors?
They are proteins found in the nucleus which bind to specific sequences in the DNA and induce changes in the transcriptional program. They can have activator or repressor function.
What is the importance of chromatin with transcription factors?
As transcription factors recognise a short sequence, which we will find a lot within the genome, the chromatin, which is tightly wrapped around nucleosomes it does not give access to transcription factors to bind.
Do transcription factors work alone or in complexes?
They are usually in complexes to get the enhancer-promoter to be open and be active.
What happens after transcription factor binds?
It recruits co-activators and chromatin remodelling complexes, which allows other transcription factors to bind as well
What are examples of intrinsic factors?
- PU-1 (granulocyte, monocyte)
- GATA-1 (megakaryocyte, erythrocyte)
- C/EBP - neutrophil/eosinophil
-Gfi-1 - neutrophil, known for its inhibitory function
How is the balance kept?
- You need a balance as if you will have a lot erythrocytes, you will have good transport of oxygen but if you dont have myeloid cells then you will have problem when you get an infection. So the balance need to be kept and then for example if you lose red blood cells or require more oxygen for example mountain climbing then there will be more EPO produced etc.
- If you need CMP to differentiate into an erythroid cell then you need signals for erythroid genes and transcription factors but at the same time you need to reduce the signals for the myeloid genes and other way around.
How do we get a myeloid lineage?
Myeloid transcription factors will give positive feedback to themselves and other myeloid transcription factors and will inhibit the expression of erythroid transcription factors. E.g. PU-1 will bind to GATA-1 and repress GATA-1 bound genes.
Where is PU-1 expressed?
It is expressed in both myeloid and lymphoid cells, however, it is quickly turned off in the lymphoid lineage, only B cells retain the expression but at lower levels than myeloid cells. It is required for granulocyte/monocyte lineage and is turned off in megakaryocyte/erythrocyte lineage
What happens to PU-1 KO mice?
They die soon after birth, they have a lack of myeloid cells, B and T cells. Even PU-1 expression is turned off in T cells to allow them to mature, they still need PU-1 to get to that stage (Ka Sin Mak et al. 2011).
How can we track the PU-1 expression in different progenitor cells?
- KI GFP into PU-1 gene
- Flow cytometry for Lineage-negative cells
- Separate GFP positive and GFP negative cells from each other
- By flow cytometry, you can look at different cell populations and their GFP expression (Back et al. 2005)
How does PU-1 act negatively on erythropoiesis?
- it’s KO causes premature differentiation of erythrocytes which leads to their apoptosis.
- it’s overexpression leads to anaemia due to the block in erythrocyte differentiation.
How do we know that PU-1 is an important regulator of myeloid differentiation?
Because when Iwasaki et al. 2005 make conditional KO mouse they saw decrease in. HSCs, loss of repopulation ability and absence of CMPs and GMPs. When excised from CMPs and GMPs cells did not mature properly, hence PU-1 is important in all stages of myeloid differentiation (Iwasaki et al. 2005)
How do GMPs become macrophages or neutrophils?
- GMP has expression of PU-1 and C-EBPalpha
- To become neutrophil the expression of C/EBPalpha increases and PU-1 expression stays the same. This recruits Gfi-1
- To become macrophage the expression of PU-1 increases and C/EBPalpha expression stays the same. This recruits Egr-1.2 and Nab-2
- Gfi-1 and Egr-1.2/Nab-2 inhibit each other
How can cytokines modulate cell fate decisions?
They do that by regulating lineage transcription factor.
What Dahl et al. 2003 did is they had PUER cells and they gave tamoxifen which binds to PU-1 ER complex and leads to PU-1 transport into the nucleus.
- If cells got low Pu-1 concentration they became neutrophils
- If cells got high PU-1 concentration they became macrophages
- If cells got high PU-1 concentration and IL-3 it supported their differentiation into macrophages
- But if cells got high PU-1 concentration and then G-CSF they differentiated into neutrophils
What is GATA-1
A transcription factor, regulator of erythroid/megakaryocyte lineage. GATA-1 can bind on its own but usually, it binds as a complex which gives more specificity.
What does master regulator mean?
- It is a transcription factor which is essential for specific lineage differentiation
- It represses other potential lineage programs
- They can reprogram certain specific cell lineage when introduced into cells
