Stem Cell Heirarchy Part One

Question 1

What type of cell is at the very top of the stem cell hierarchy?

Answer 1

Haemopoietic stem cell.

Question 2

Haematopoietic stem cell job description.

Answer 2

• Be versatile: give rise to the full range of differentiated cells.
• Be productive and adaptable: replace in excess of 10^11 blood cells per day normally, more in response to trauma or infection.
• Retain stemness: output required for several decades.

Question 3

How was clonal haematopoiesis discovered?

Answer 3

• 1961 In vivo colony formin assay showed about 1 in 1000 cells was colonogenic in mouse spleen.
• 1963 further work irradiated first cell and the proliferation of the lesions proved the circles of cells were definitely colonies that formed from the single transplanted cell.

Question 4

What was done to determine which cells could sustain haematopoiesis?

Answer 4

• Cells from colonies in the mouse spleen taken and transplanted to see what % of cells were still capable of forming new colonies and what % were already terminally differentiated in the originally colony.
• By this method of serial transplantation researchers were able to show that the stem cell can perpetuate clonal growth.
• Serial transplantation is still the gold standard in experiments to understand stemness.

Question 5

What is the most important characteristic of a stem cell?

Answer 5

They need to be able to retain stemness. They need to support haematopoiesis throughout life (or throughout serial transplantation in experimental systems).

Question 6

How do we identify a stem cell?

Answer 6

• depended very much on the invention of flow cytometry throughout the 1970s and 80s.
• Monoclonal antibodies became widely available in 80s and 90s.
• Early stem cell markers were first characterised in mice where it was found that these cells did not have any of the lineage markers, but did have the novel Thy1 and Sca-1 antigens.
• The use of flow cytometry to sort cells (e.g. by Thy1 and Sca-1, followed by transplantation allows assessment of the nature and frequency of the stem cell.

Question 7

What novel antigens were found on stem cells?

Answer 7

Early stem cell markers were first characterised in mice where it was found that these cells did not have any of the lineage markers, but did have the novel Thy1 and Sca-1 antigens.

Question 8

In clinical transplantation work what is the definition of a stem cell?

Answer 8

• In clinical transplantation work, any CD34+ haematopoietic cell is a stem cell.
• In research the definitions are much tighter.

Haematopoietic stem cells must be CD34+, CD38-,CD90+,CD45RA-.

Multipotent progenitor cells are CD34+, CD38-, CD90-, CD45RA-.

Lymphoid primed multipotent progenitor cells are CD34+, CD38-, CD90-, CD45RA+.

• In the past few years we are beginning to revise our ideas of how the hierarchy works. It seems now that the Megakaryocyte erythroid progenitor actually divides off from the Multi-potent progenitor before the choice between lymphoid progenitor and granulocyte monocyte progenitor differentiation.

Question 9

Have we identified and ‘stemness’ genes?

Answer 9

• Many candidate genes have been identified through gene array technology in sorted subpopulations.
• Candidate genes are validated by functional assays.
• If you look at some of the transcription factors involved in the regulation of haemopoiesis you will probably identify several that are also involved in leukaemia. It is also very much a cooperative effort between tf’s that specifies the next differentiation stage.

Question 10

Can stem cells be fully characterised phenotypically?

Answer 10

• Stem cells can be enriched but not fully characterised phenotypically. allowing their transcriptional program to be worked out.
  e. g. we can say that a CD34+ cell population is more likely to contain stem cells but cannot fully characterise them. Even within the CD34+ CD38- pops not all cells will be capable of recapitulating haematopoiesis following transplantation.

Question 11

What is the paradox of haematopoietic stem cells?

Answer 11

• They need to be able to give rise to differentiated progeny whilst themselves remaining undifferentiated.
• Need only 1 daughter cell to differentiate and the other to retain stemness = asymmetric division.

Question 12

What is meant by the asymmetric division of stem cells?

Answer 12

• A stem cell will normally divide asymmetrically so that one copy will retain stemness and the other copy will start the process of maturation and differentiation.

Question 13

How rare are haematopoietic stem cells?

Answer 13

• Very rare, about 1 in 300,000 BM MNC will engraft into a recipient mouse.

Question 14

Where in haematopoiesis hierarchy does the most cell division take place?

Answer 14

• The most cell division occurs somewhere at the progenitor cell level (Myeloid and Lymphid Progenitors) but not at the very top of the hierarchy.
• There is quiescence in the actual stem cells so this is not where the most proliferation is taking place.

Question 15

What is the function of stem cell quiescence?

Answer 15

• Quiescence prevents exhaustion.
• Quiescence protects the genome - if you were to have the cell at the top of a hierarch constantly dividing throughout life then you would be likely to end up with a very genomically damaged stem cell. In contrast, if you only have a very small amount of division at the top of the hierarch and then have the very fast proliferation somewhere further down the stem cells remain protected.

Question 16

How has quiescence in stem cells been estimated?

Answer 16

Quiescence has been estimated by 4 main methods:

1) . Gene marking studies to look at what cells are actually doing
2) . BrdU labelling to observe when cells are in S-phase.
3) . Telomere length measurement where the amount of telomere erosion can indicate how often a cell has divided.
4) . Subset transplantation can show us whether the subsets are dormant or proliferating.

From all of these experiments we have found that stem cells are usually quiescent. Only about 104 stem cells will occur in a 90 year lifespan = about 1 every 45 weeks.

Question 17

What are the 5 characteristics of stem cells?

Answer 17

1) . They support haematopoiesis throughout life.
2) . They give rise to differentiated progeny whilst themselves remaining undifferentiated.
3) . They can be enriched (but maybe not fully characterised) phenotypically.
4) . They are rare.
5) . They are usually quiescent.