Haematopoietic stem cells

• What is haematopoiesis?

  The production of all haematopoietic (blood) cells in the body - including white blood cells, red blood cells, and platelets
• What are the origins of blood cells?

  Haematopoietic stem cells

  • Rare population of cells
  • Able to self-renew and give rise to differentiated cells of all haematopoietic lineages
• What is the process of producing red blood cells like?

  Progression between different cell types is sequential and continuous - there is an additive effect where all cells are dividing/deifferentiating
• Where does haematopoiesis occur?

  Occurs predominantly in the red bone marrow (medullary)

  
  

  Also occurs during embryonic and foetal development in liver and the spleen - known as physiologic extramedullary haematopoiesis

• What is reconstitution (of a haematopoietic system)?

  The ability of HSCs to repopulate the HSC population when infused into an irradiated animal with a non-functional haematopoietic system

  • Long-term (LT) HSCs - reconstitute in the long term, replenish the HSC pool (most primitive type of HSCs)
  • Short-term (ST) HSCs - gives rise to short-lived progenitor cells that subsequently differentiate to blood cell lineages, replenish the blood cell pool
  • A single HSC is capable of reconstituting the whole haematopoietic system
• What is the current model of haematopoiesis?

  • Continuum of differentiation (rather than defined individual subpopulations of cells) into branches until mature cells of the blood are reached
  • Subtly different characteristics and cell surface markers
• What are haematopoietic stem cells (HSCs)?

  • Multipotent primitive cells that can develop into all types of blood cells, including myeloid-lineage and lymphoid-lineage cells
  • Most widely studied and best characterised of all adult stem cells
  • Fairly quiescent and divide every so often - daughter cells proliferate and expand to maintain a pool of cells
• Where are HSCs found?

  Several organs such as...

  • Peripheral blood
  • Bone marrow
  • Umbilical cord blood
• What are examples of markers of HSCs?

  • CD34 - expressed on HSCs and progenitors from early stages of foetal development to adult bone marrow
  • Thy1 (CD90) - glycophosphatidylinositol (GPI) anchored cell surface protein shown to enrich for functional HSCs
  • CD49f (Integrin subunit alpha 6)
• Regulating haematopoiesis in in vitro studies

  • Differentiation pathways already identified - from HSCs down to the various cells of the blood
  • GFs and small molecules which induce expansion and differentiation down the different lineages already identified - thus potential to generate specific cells of the haematopoietic system
  • Therapeutic use - use combination of markers to identify and sort cells, and induce differentiation to produce RBCs rather than isolating from patients
• Regulating haematopoiesis in in vivo studies

  • More complex and controlled than in vitro - need to account for additional influence on signalling by the niche
  • Niche implements all kinds of gradients of GFs and signalling molecules as well as contact between neighbouring cells
  • To induce differentiation down lineages, select cells expressing the right markers for the mature phenotypes of these particular cells
• Why are HSCs the best characterised and most widely used stem cells?

  • Tissue accessibility and ease of sampling - relatively non-invasive
  • Tissue properties - soft with high cellularity, easy to fragment and disaggregate into individual cells
  • Relative ease of identification - mature cells and their immediate precursors are morphologically distinct
  • Robust and reliable in vivo reconstitution assays
  • Straightforward delivery of 'test' cells/cell - tail-vein infusion or direct injection
  • Availability of congenic/mutant/engineered mouse strains
• What is the stem cell niche?

  Microenvironment where stem cells reside and receive signals for self-renewal and differentiation

  
  

  Cells always surrounded by neighbouring cells

  • Direct cell-cell interactions
  • Interaction with surrounding ECM
  • Exposure to different gradients of soluble markers (GFs, small molecules, chemokines, cytokines, etc.)
  • Mechnical forces acting on the cell
  • Oxygen gradient
  • Combination of factors influencing gene expression and cell activity
• How does the stem cell niche influence cell fate processes?

  Cell fate processes like replication, differentiation, migration, and apoptosis are influenced by the microenvironment and combination of signalling within it

  
  

  All cells recieve subtly different signalling factors - identity of cells differs subtly too
• What is the HSC niche?

  >80% of HSCs exist in a perivascular niche, where they are mostly found near sinusoids (leaky vessels which radiate out from the central vein, facilitating efficient exchange of blood cells and molecules)

  • Non-diving HSCs are enrinched in central marrow
  • Dividing HSCs are enriched in endosteal region

  
• What characteristics do endosteal HSCs show over central marrow HSCs?

  Greater potential for in vivo..

  • Homing
  • Lodgement
  • Reconstitution

  Transplanted HSCs will home to the endosteal region when injected into irradiated animals
• How is HSC fate regulated?

  Intrinsic and extrinsic cellular factors

  • Exposure to different signalling environments and molecules influences quiescent or active states
  • Quiescent HSCs can become activated - then either return to quiescence or be influenced to divide asymmetrically or symmetrically
• What are the main signalling cells and factors of the HSC niche?

  Cells

  • Lepr+ perivascular stromal cells - release CXCL12 and SCF
  • Endothelial cells
  • NG2+ pericytes - moderate level of CXCL12
  • Schwann cells - release TGFb

  Main regulators of quiescence

  • CXCL12 (or SDF1) - quiescence + retention
  • Stem cell factor (SCF, or KIT ligand) - self-renewal + maintenance of cells
  • TGFb

  Regulator of activeness

  • Circadian release of noradrenaline is responsible for HSC mobilisation (BM to blood) via. downregulation of CXCL12
  • Notch signalling - promotes osteogenesis, increases SCF levels
• What factors negatively regulate the HSC niche?

  
  

  • Osteopontin (OPN) is secreted by bone cells and stops HSC proliferation - this affects HSC pool size
  • Adipocytes - makes the HSC niche more fatty with age
  • Reduced Jagged 1 = less Notch signalling - reduced proliferation
  • Reduced numbers of adrenergic nerve fibres
• What are the results of HSC ageing?

  The process of aging in HSCs is driven by both cell-intrinsic and extrinsic factors, which lead to a reduction in blood cell production and impairment of immune system function

  • Reduced DNA damage repair – accumulation of mutations in DNA
  • Accumulation of ROS – damage DNA resulting in mutations
  • Shift in polarity of cytoskeletal proteins & epigenetic markers
  • Epigenetic drift – differences in methylation patterns of DNA which affect overall proliferation of HSCs
• What is the difference between young and old HSC pools?

  • Young - have both LT- and ST-HSCs, as well as MPPs, CMPs, and CLPs
  • Old - have clonal expansion of selective HSCs, as certain cells within population proliferate more regularly than others and form clones
  • Dysregulation in balance between CMP and CLP results in more myeloid cells - causing immunosenence and increased propensity for myeloid malignancy
• What is clonal haematopoiesis of indeterminate potential (CHIP)?

  • Haematopoiesis of a specific clone of individual HSCs that are overrepresented in the pool of blood cells
  • Greatest contribution from mutations in genes involved in epigenetic regulation - large percentage of blood cells derived from one single clone
  • Prevalent in the elderly
  • Does not necessarily cause malignancy but may predispose
• What are the therapeutic applications of HSCs?

  • HSC transplantation - currently the only established stem cell therapy
  • Reprogramming the immune system - to be resistant to infection, scaling up RBCs or other cells of the blood etc.
• Describe HSC transplantation

  • Autologous (patient-derived) or allogeneic (donor-derived)
  • Donors ideally are fully HLA-matched siblings, or unrelated but still matched
  • Risks include graft versus host disease (GvHD) - where graft's immune cells recognise the host as foreign and attack the recipient's body cells - can use magenetic activated sorting to enrich for CD34+ HSCs
  • May require immune system ablation to ensure better homing
  • Use granulocyte stimulating factor to assist in HSC mobilisation into the peripheral blood
• What is aplastic anaemia?

  Condition that occurs when the bone marrow cannot produce enough new blood cells

  Commonly seen in post-war soldiers which had been exposed to radiation that destroyed their haematopoietic system
• Where does haematopoiesis occur?

  Occurs predominantly in the red bone marrow (medullary)

  • In children and mice, occurs in long bones, e.g. femur and tibia
  • In adults, occurs mostly in the spine (vertebrae) and hips, ribs, skull and breastbone (sternum)

  Also occurs during human embryonic and foetal development in liver and the spleen - known as physiologic extramedullary haematopoiesis

  
• Where are HSCs located?

  In adult humans

  • Red bone marrow within the spongy bone (towards the ends of the bone)

  In mice

  • Bone marrow within the long part of the bone - in adult humans, the equivalent space is yellow marrow

  Identifying the exact niche in situ has been hampered by the rarity of HSCs among the very cellular human bone marrow - made more difficult to study as taking HSCs out of their niche can change the signalling

  
• What are the types of bone marrow blood vessel?

  Series of vessels organised in a specific order

  • Arteries are longitudinally aligned along diaphysis (length) of long bones - infiltrate into BM via. branching into smaller arterioles
  • Arterioles become smaller in diameter and progress into endosteal region - connecting to smaller diameter transition zone (TZ) capillaries near the bone surface
  • TZ vessels connect to the large diameter sinusoids that feed blood into the central sinous through which it leaves the bone marrow in venous circulation

  Laminin (ECM protein) distrubted all throughout

  
• What is the difference between asymmetric and symmetric division?

  Asymmetric division

  • (a) 1 stem cell and 1 differentiated daughter cell
  • (b) 2 distinctly differentiated daughters

  Symmetric division

  • (c) 2 identical stem cells (expanding population)
  • (d) 2 identical differentiated daughter cells (differentiating)

  
• What is the difference between young and old HSC pools?

  Young

  • LT-HSCs and ST-HSCs
  • ST-HSCs give rise to multipotent progenitors (MPPs) - enter either CMP or CLP lineage
  • Balanced innate and adaptive immunity

  Old

  • Clonal expansion of selective HSCs - certain cells within population proliferate more regularly than others and form clones
  • Aged HSCs with altered differentiation potential
  • Dysregulation in balance between CMP and CLP - results in more CMP than CLP
  • More myeloid = immunosenence (reduced function of immune system) and increased propensity for myeloid malignancy