Haematopoietic Stem Cells

What are the most studied adult stem cells?

HSCs

How many nucleated cells are in the human bone marrow?

1×10^12

How many active HSCs are within the adult bone marrow?

50,000-200,000 (1/10million nucleated cells)

How often do HSCs divide?

Once every 40 weeks?

What is the key characteristic of HSCs?

They produce progenitors that provide every cell in the blood system.

What is haematopoeisis?

The production of haematopoietic blood cells.

What is the rate of blood cell production?

4-5×10^11 cells a day (HSCs divide rarely but proliferation of daughter cells).

What do HSCs give rise to?

Myeloid & lymphoid progenitors.

Where does haematopoiesis occur in prenatal stages?

Yolk sac and liver.

Where does haematopoiesis occur in postnatal stages?

Vertebrae and pelvis and sternum.

Where does haematopoiesis occur around birth?

Bone marrow, femur and tibia.

What is the process where fat replaces haematopoietic marrow in long bones?

Marrow involution.

How has our understanding of the haematopoietic differentiation hierarchy evolved from 2000-now?

2000: stepwise hierarchy with distinct myeloid and lymphoid branches, 2005-16: heterogeneity in the HSC pool with multipotent progenitors, now: continuous differentiation model with overlapping fates.

What is the current view of haematopoietic differentiation?

A continuum model with lineage-based HSCs, fate decision occur early and more gradually with early transcriptional priming toward specific lineages (less hierarchical and more dynamic).

What is the history of HSC research?

Radiation damage from atomic bomb testing let to bone marrow transplant research that let to the discovery of HSCs.

What are 6 characteristics of HSCs that make them well studied?

Tissue accessibility and ease of sampling (bone marrow aspirate and peripheral blood), soft tissue properties with high cellularity and easy to disaggregate), morphologically distinct making them easy to identify when stained, robust in vivo reconstitution assays, straightforward delivery of cells (tail-vein infusion of direct BM injection), can do lineage tracing in vivo to track fate and developmental potential.

What is the basic test of reconstitution?

Give mouse a sublethal/lethal dose of ionising radiation to wipe out existing system. Intravenous injection of HSCs causes recovery as cells home to BM and produce new blood cells. Otherwise mouse dies.

What are long-term HSCs?

Self-renew over lifespan with the ability to differentiation into all types of blood cells (maintains whole HSC system). They divide infrequently to maintain quiescence and preserve regenerative capacity.

What are short-term HSCs?

Limited self-renewal capacity and more involved in progenitor cells. Contribute to haematopoiesis for weeks/months and provide an immediate response to the body’s need for new blood cells (infection etc).

What is the main marker for HSCs?

CD34+

What are the cell-surface markers for LT-HSCs?

Lin-CD34+CD38-CD45RA-CD49f+CD90+ ±Rholo

What are the cell-surface markers for ST-HSCs?

Lin-C34+CD38-CD45RA-CD49f-CD90+

What is rho (rhodamine 123)?

Membrane dye for mitochondria (indicator of mitochondrial membrane potential).

Why should cells be enriched?

For therapeutic use or further study.

What regulates HSC differentiation and lineage commitment?

Cytokines, growth factors, signalling from the niche.

Give an example of cytokines/growth factors important for differentiation.

IL-7 for common lymphoid progenitor cells, and IL-3/GM-CSF/M-CSF for common myeloid progenitor cells.

Explain the stem cell niche.

All forces/interactions acting on the cells influence cell fate. Different parts of the body and within tissues have different signalling.

Give 3 examples of different interactions within the niche.

Physical signals from ECM components, soluble signals from cytokines/chemokines, cell-cell interactions through cadherins.

How can you identify each lineage of HSC?

Marker combinations using flow cytometry.

What are the 4 cell fate processes?

Quiescence, proliferation, maintenance, retention.

Explain quiescence.

Dormancy to prevent exhaustion and maintain long-term regenerative capacity.

Explain proliferation.

Division to produce stem cells and differentiated cells that is important for injury/infection.

Explain maintenance.

Preservation of the HSC pool involving mechanisms that balance quiescence/proliferation, DDR, and protect cells from stress/ageing.

Explain retention.

Mechanisms that keep HSCs in their niche important for correct function and stability.

Why can change the balance of fate determination in the niche?

Changes to signalling.

Where is the HSC niche?

In close proximity (within 10um) to sinusoids in red bone marrow.

What is yellow bone marrow?

Adipocytes and MSCs (increases in long bones as we age).

Why have transgenic mice been helpful in HSC studies?

Helped us to determine the HSC niche through similarities between haematopoietic systems.

What are the drawbacks of using transgenic mice in HSC studies?

Very different anatomy and long bones of mice contribute to haematopoiesis greater than those of humans.

Why has the identification of the HSC niche been difficult?

The rarity of HSCs in the high cellular content of bone marrow.

What techniques enabled the identification of the HSC niche?

Marker identification and imaging techniques (such as optical clearing of bones to overcome opacity, reduce light scattering and enable deeper imaging with microscopy).

What identifies HSCs from other cellular cells in the bone marrow in imaging studies?

Co-expression of c-Kit and ACAT-GFP, and round morphology.

What is c-Kit?

Receptor tyrosine kinase highly expressed on HSCs critical for survival, proliferation and maintenance.

What is Acat-GFP?

Acetyl-coA acetyltransferase involved in lipid metabolism that labels the unique metabolic profile of HSCs when fused to GFP.

What is the sinusoid?

Large continuous blood vessels that span the bone marrow.

Where are non-dividing HSCs enriched?

Central marrow (20%).

Where are dividing HSCs enriched?

Endosteal region (80%).

Where do implanted HSCs home to/reconstitution happen?

Endosteal region.

What intrinsic factors determines the fate of HSCs (cell-cycle entry or quiescence)?

Transcription factors, cell cycle regulators, epigenetic landscape.

What external factors trigger cell-cycle entry opposed to quiescence?

ROA, hypoxia, SCF (kit factor)

What does symmetric division achieve?

Expansion (HSC + HSC) or differentiation (HPC + HPC)

What does asymmetric division achieve?

Maintenance (HSC+ HPC)

What promotes maintainence in the HSC niche?

Leptin receptor (Lepr+) perivascular stromal cells, endothelial cells and NG2+ pericytes secrete maintainence factors.

What factors secreted by cells in the niche promote maintenance?

CXCL12 and SCF

What enables HSC mobilisation (BM —> blood)?

Circadian release of noradrenaline that downregulates CXCL12, and secretion of granulocyte-colony stimulating factor.

What factors promote quiescence in the HSC niche?

TGFbeta and CXCL4

What factors promote proliferation in the niche?

Notch ligands (jagged-1) and thrombopoietin.

What factors promote retention in the niche?

Pleiotrophin, CXCL12

What percentage of HSCs are perivascular?

80%

When tracking HSCs in live mice using microscopy, are LT-HSC or MPP closer to the endosteum?

LT-HSC

Where are MPPs located?

50/50 split between transitional zone vessels and sinusoids (migrated in relation to the parent HSCs).

Do HSCs exist in a hypoxic environment?

No, the oxygen concentration in LT-HSCs and MPPs is basically the same as the extracellular space around the blood vessels (normal concentration you find in tissue).

Are LT-HSCs or MPPs more motile (higher displacement)?

MPPs

How can you stimulate LT-HSCs to move?

Cyclophosphamide and granulocyte colony stimulating factor but depends on the environment they are in.

What type of of bone are LT-HSCs most motile in?

Mixed-type

What type of bone are MPPs most motile in?

Mixed and resorption type.

How are different bone types created?

From the different extents of bone remodelling.

What is resorption type bone?

Bone tissue is broken down and new bone is deposited.

What is deposition type bone?

The bone tissue is not broken, new bone is deposited on top of the existing bone.

What is associated with ageing?

Reduced DDR (GIN), ROS accumulation, epigenetic drift (change in DNA methylation patterns).

Do young or old HSCs have a good balance of myeloid and lymphoid progenitors?

Young.

What happens in older HSCs?

Clonal expansion and greater proliferation in certain cells resulting in an imbalanced proportion of myeloid and lymphoid progenitor cells. Reduced lymphoid cells results in weaker immune system and increased myeloid cells increases risk of malignancies.

What is the role of osteopontin?

Negative regulator of proliferation.

What is the role of adipocytes?

Downregulates proliferation.

Role of adipocytes in ageing.

More fat in older tissue, adds more of a brake to proliferation

Why does age increase the risk of malignancies?

Bone loss decreases the level of osteopontin which increases proliferation.

How does age affect the niche?

Reduced CXCL12/SCF, reduced Jagged (less notch signalling), reduced adrenergic nerve fibres (less mobilisation into blood)

What is the phenomenon caused by ageing?

Clonal haematopoiesis of indeterminate potential (CHIP).

How many protein coding mutations exist in the elderly HSC pool?

350,000-1.4million

Does CHIP affect other things than blood cancer?

Yes, coronary heart disease (1.8 increased hazard ratio)l

What is the only established stem cell therapy?

HSC (bone marrow) transplant.

What is myeloablative conditioning?

Total body irradiation and/or chemotherapeutics at high doses which don’t recover the haematopoietic system.

What can HSC transplantation treat?

Mostly leukaemia like AML, CML and ALL, but also immune disorders (SCID) and blood disorders (sickle cell anaemia).

Explain how autologous HSC transplants for blood cancers work.

Pretreatment with G-CSF to mobilise HSCs to peripheral blood and G-PBMCs are collected from blood or bone marrow. HSCs are selected based on their markers and frozen. Patient gets irradiated to kill remaining cancer cells and any remains HSCs. Frozen HSCs are infused back, home to the bone marrow and produce new blood cells.

What is the limitation of autologous HSC transplantation?

High risk of infection and GvHD.

What is graft vs host disease?

Transplanted donor cells recognise the recipients tissues/cells as foreign and attack them, mostly affecting the skin, liver and GI tract.

How to avoid GvHD?

Enrich for CD34+ cells from G-PBMC as it reduces the number of immune cells in transplant graft.

How can HSC transplants treat other conditions?

Defective HSCs can be corrected with gene editing and reconstitute the haematopoietic system without the defect.