lecture 22: Hematopoietic Stem Cells Flashcards Preview

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Flashcards in lecture 22: Hematopoietic Stem Cells Deck (21):
1

How many publications in this field over the last 40 years?

  • Haematopoietic/Hematopoietic Stem Cells = 82970
  • Bone Marrow Transplantation = 76542
  • HSC Transplantation = 43112 
  • i.e. quite a lot of information
  • very had to discern where to start when you are dealing with such a big topic
  • 2012 breakdown of publications by stem cell type → about 1/4 are HSCs, burgeoning MST, also iPS and hESC

2

How well understood are HSCs?

  • most extensively studied and characterised stem cell population 
  • robust strategies for elucidating the HSC compartment has paved the way for the characterisation of other stem cell populations 
  • HSC are only stem cell population routinely utilised in the clinic
  • hierarchical organisation
  • isolation
  • enrichment 
  • differentiation 
  • niche
  • in vitro functional assays
  • in vivo assays 
  • transplantation
  • therapeutics 

3

What is the classical view of renewal and regeneration in adult tissues?

  • continuously renewing 
    • bone marrow 
    • gastrointestinal tract 
    • skin
  • conditionally renewing
    • lung
    • kidney
  • non-renewing
    • brain
    • skeletal muscle
  • coinciding with a decreasing rate of regeneration 

4

Why do we have continuously regenerating tissues such as in the bone marrow?

  • cellular requirement for turnover
  • cells that have a short half-life / life-span 
  • need variants in the cells that are being produced, constantly, in order for body to effectively be able to search for evading pathogens/respond to particular stresses etc 
  • bone marrow
    • granulocytes: 120 x10cells/day, 5400 kg per 70 years, 1 days turnover 
    • erythrocytes: 200, 460, 120 
    • lymphocytes: 20, 275 
    • platelets: 150, 40, 7-14 
  • gut mucosa: 56, 6850 
  • skin: 0.7, 86, 7
  • this level of turnover requires a very organised system

5

What is the classical stem cell hierarchy?

  • intrinsic properties of stem cells:
    • renewal 
    • high proliferative potential
    • clonal repopulation 
    • multilineage differentiation 
  • decreasing probability of quiescence 
  • diminishing regenerative capacity 
  • dimishing proliferative potential 
  • progressive lineage restriction 
  • stem cell 1
    • progenitor 1
      • mature cell 1 (M1)
      • M2
    • P2
      • M3 
      • M4 
  • SC2 
    • P3
      • M5
      • M6

6

What is transdifferentiation?

  • M1 → M2 
  • M1 →→ M3 

7

What is transdetermination?

  • SC1 → P3 → M5/M6 
  • P2 → M2 

8

What is dedifferentiation?

  • M1 → P2 
  • M3 → SC1 

9

What is the haematopoietic stem cell hierarchy?

  • stem cells 
  • committed progenitors 
  • maturing cells
  • matured cells  
  • two major arms: lymphoid arm, myeloid arm

10

What is developmental haematopoiesis?

  • mouse 
    • E6.5: mesoderm formation
    • 7.5: yolk sac blood islands 
    • circulation beginnging ~ 9
    • 10.5: Aorta Gonad Mesonephros  
    • 14.5: foetal liver stage 
    • 18.5 – adulthood: bone marrow
  • human:
    • 17: yolk sac
    • 23: 1st hepatic colonization 
    • 27: arterial clusters 
    • 30: 2nd hepatic colonization 
    • 10.5 weeks: bone marrow colonization 
  • important to understand because regeneration recapitulates ontogeny 
  • multiple sites of haematopoiesis during development 
  • occurs through two distinct waves
    • primitive 
      • dedicated primarily to production of erythroid progenitors 
      • allow circulation to start occuring
    • definitive 
      • controlled haematopoiesis
      • point at which it is maintained for life 

11

What is key transcriptional regulation of haematopoiesis?

  • ETV2:
    • ETS family member regulating early haematopoietic development 
    • ETV2-/- mouse embryos fail to develop yolk sac blood islands, primitive haematopoiesis is absent and vasculogenesis abrogated 
  • SCL:
    • basic helix-loop-helix transcription factor crucial to haematopoeitic development
    • SCL -/- mouse embryos lack yolk sac haemat. and die at E9.5 
  • RUNX1: 
    • transcription factor expressed in haemat. cells and endothelial tissues in contact with nascent blood cells 
    • RUNX1-/- mouse embryos fail to undergo definitive haemat. and die from severe haemhorrage at E12.5 

12

How can we deconstruct the adult haematopoietic stem cell hierarchy?

  • biomarkers/phenotype ↔ in vivo assay/transplantation assays ↔ in vitro surrogate assay/stem cell assay ↔ biomarkers/phenotype

13

What were pioneering experiments that aiding understanding of HSCs? 

  • transplantation experiments
    • Lorenz - 1951
    • irradiated mice - transplanted normal bone marrow
  • colony forming assays 
    • till and McCulloch - 1961 
    • in vivo colony forming assay (CFU-S)
    • irradiated mice 
    • IV injection of cells 
    • colonies of mature haemat. cells in spleen 
  • in vitro surrogate assays 
    • bradley and metcalf (1966)
    • bone marrow suspensions 
    • cultured cells in bito in semi-solid state 
      • media and agar of methylcellulose 
    • measured colony number, size, morphology 
    • added cytokines (GM-CSF, G-SCF, M-CSF, IL-3 etc) to assess cytokine directed differentiation 
    • feeder cells
    • bone marrow cells
    • cytokines 

14

What is limiting dilution stem cell transplantation?

  • used two different alleles of CD45 (leucocyte common antigen - Ly5.1 and Ly5.2) in mice to discriminate between host and donor 
  • transplanted different number of donor cells
  • look for circulating blood cells from donor in recipient irradiated mice to determine incidence of stem cells 

15

What are surrogate stem cell assays?

  • immunophenotyping 
    • measured expression of Sca-1 CD34; c-kit; Thy-1, Mac-1; lineage markers 
    • takes hours 
  • clonogenic assays 
    • measures cytokine-stimulated colony growth in semi solid agar or methylcellulose
    • takes 7-14 days 
  • liquid culture assay 
    • measures long term culture initiating cells (LTCIC) 
    • measures cobbestone area forming assay (CAFC) 
    • takes weeks
  • transplantation 
    • measures long term reconstitution potential
    • takes months 

16

What is the isolation, enrichment, and characterisation of HSCs?

  • bone marrow
  • low density cells 
  • negative lineage selection
    • put in markers for ones we want to get rid of 
    • FACS or magnetic beads 

  • flow cytometry analysis and sorting 
  • uses Nycoprep 1.077g/cc3
  • further subsetting
    • antibodies/fluorochromes/membrane markers 
  • gene expression profiling 
  • cell cycle analysis 
  • clonal analysis 
  • transplantation 
  • density gradient centrifugation → know density of mononuclear cells 

17

What is immunoprofiling of HSC?

  • can classify different progenitor cells through the hierarchy based on expression of cell surface markers 
  • has to be confirmed by using in vitro and in vivo essays
  • some surface markers are shared between mice and humans and some are not
  • also some specific markers for certain subpopulations etc 
  • similarity but also discrepancies between animal models  

18

How did we find out about transcriptional regulation of haematopoeisis?

  • animal knock out models

19

How do we know what we know?

  • technological advances 
  • flow cytometric analysis 
  • in vitro clonogenic assays 
    • proliferative capacity 
    • differentiative capacity 
    • cytokine driven for differentiation 
  • in vivo assays 
    • clonogenic transplantation 
    • repopulation transplantation 
    • knockout mice 
    • lineage tracing 

20

What is the controversy about HSC niche?

  • one or two niche?
  • Long term in endosteum
  • activated in perivascular niche 
  • endosteal cells had:
    • greater proliferative potential
    • greater affinity for endosteum
    • greater transplant potential  

21

What is the history of transplantation?

  • up to 1968 a bit of a disaster 
  • rescue of irradiated mice by spleen protection and transplantation (Jacobson - 1949( 
  • resceu of irradiated mice by bone marrow transplantation (Lorenz- 1951) 
  • first use of allogenic bone marrow transplant in leukaemic patients (thomas - 1957) 
  • histocompatibility determines outcome of transplantation in dogs with matched littermates (Epstein and Storb 1968) 
  • HLA matched siblings bone marrow donors (Thomas 0 1971) 
  • immune suppression by drugs and T-cell depletion (GVHD), patient-donor compatibility, mobilisation, understanding of haematopoiesis