lecture 22: Hematopoietic Stem Cells Flashcards
1
Q
How many publications in this field over the last 40 years?
A
- 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
Q
How well understood are HSCs?
A
- 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
Q
What is the classical view of renewal and regeneration in adult tissues?
A
- continuously renewing
- bone marrow
- gastrointestinal tract
- skin
- conditionally renewing
- lung
- kidney
- non-renewing
- brain
- skeletal muscle
- coinciding with a decreasing rate of regeneration
4
Q
Why do we have continuously regenerating tissues such as in the bone marrow?
A
- 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 x109cells/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
Q
What is the classical stem cell hierarchy?
A
- 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
- progenitor 1
- SC2
- P3
- M5
- M6
- P3
6
Q
What is transdifferentiation?
A
- M1 → M2
- M1 →→ M3
7
Q
What is transdetermination?
A
- SC1 → P3 → M5/M6
- P2 → M2
8
Q
What is dedifferentiation?
A
- M1 → P2
- M3 → SC1
9
Q
What is the haematopoietic stem cell hierarchy?
A
- stem cells
- committed progenitors
- maturing cells
- matured cells
- two major arms: lymphoid arm, myeloid arm
10
Q
What is developmental haematopoiesis?
A
- 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
- primitive
11
Q
What is key transcriptional regulation of haematopoiesis?
A
- 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
Q
How can we deconstruct the adult haematopoietic stem cell hierarchy?
A
- biomarkers/phenotype ↔ in vivo assay/transplantation assays ↔ in vitro surrogate assay/stem cell assay ↔ biomarkers/phenotype
13
Q
What were pioneering experiments that aiding understanding of HSCs?
A
- 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
Q
What is limiting dilution stem cell transplantation?
A
- 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
Q
What are surrogate stem cell assays?
A
- 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
Q
What is the isolation, enrichment, and characterisation of HSCs?
A
- 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
Q
What is immunoprofiling of HSC?
A
- 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
Q
How did we find out about transcriptional regulation of haematopoeisis?
A
- animal knock out models
19
Q
How do we know what we know?
A
- 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
Q
What is the controversy about HSC niche?
A
- 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
Q
What is the history of transplantation?
A
- 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
