Haematopoetic Stem Cells Flashcards
What are the two hallmarks of the haematopoietic system
- Blood has very high turnover and requires the production of billions of blood cells and immune cells daily
- In the adult, all blood lineages are derived from SCs residing in the bone marrow which must respond rapidly to fluctuations in demand.
What are the 3 characteristics of HSCs?
- Self renew to increase HSC numbers during fetal development, and maintain HSCs thoughout life → HSCs have some of the greatest capacity for self-renewal. Renew throughout the lifespan
- Produce haematopietic progenitors with extensive proliferation (in the fetal state, in the adult are more quiescent) and differentiation capacity that ultimately gives rise to all the different blood lineages
- Multipotent – regenerate multi-lineage hematopoeisis
Describe the assay to detect for the presence of myeloid progenitors
Colony Forming Cell (CFC) Assays for Myeloid Progenitors
Quantitive and qualitative assay:
• Perfomed in semi-solid media in the presence of cytokines required for proliferation and maturation of bloo cells
• Used to quantify progenitors through their ability to form colonies
• Allow the retrospective identification of biological potential – ie, multipotent vs restricted progenitors of myeloid lineage
• Cannot distinguish lymphoid progenitors present
• Cant distinguish stem cells from early progenitors, but can distinguish from late progenitors
• Cells cannot be recovered after the assay
- Colonies from a single cell formed after 1-2 weeks in GFs
- Cells slowy ‘drift’ from the centre of the colony
− CFU-GEMM → if you get granulocytes, erythroid, monocyte and megakaryocyte cells, it tells you the original cell was a multipotent progenitor −Often called CFU mixed, as doesn’t always have megakaryocytes
− CFU-GM → if you get only granulocytes and macrophages, it tells you the original cell was only bipotent
− BFU-E → only have erythroid cells, tells you the original cell was unipotent
Describe an in vitro assay for determining the presence of lymphoid progenitors
- Lymphoid cells require feeder cells to develop (use a stromal cell line)
− OP9 for B cells
− OP9-DL1 for T cells - Add exogenous cytokines such as IL-7 and Flt3 ligand. Culture for 1-2 weeks
- Then perform assays:
− Thymic organ reconstitution assay to test for T cells
− In vivo transplants (preferred, as have all the required cytokines)
− In vitro asssays
− Further expansion
Describe the CFU-S assay
Spleen Colony Forming Unit Assay (CFU-S)
• CFU-S cells are cells that, once injected into an irradiated recipient, home to the spleen and form macroscopic colonies that provide very short term (1-3 weeks) repopulation of the mouse
• These progenitors are actually more immature than CFCs but more mature than HSCs
Disadvantage:
• Requires mice
Describe the in vitro assays for HSC detection
Long-Term Culture Initiating Cells
1. Put test population into a dish with irradiated stromal feeder layer
2. Feeders produce cytokines needed for maintenance of HSCs as HSCs
3. Maintain them for a few weeks in culture
4. After a couple of weeks, even with the cytokines present, they will slowly begin to lineage commit
5. After 5 weeks, perform the CFC assay and analyse colonies.
• If all myeloid cells present, high chance that HSCs were initially present as other progenitors would have differentiated to the point that they would no longer be multipotent for the CFC assay
Cobblestone Area Forming Cells
- Again, put test population in dish with stromal feeder layer
- For unknown reasons, primitive cells migrate through the stromal layer and proliferative at time points defined by their primitiveness
- At sequential time points after initiation of the assay, individual wells are screened for the presence of absence of ‘cobblestone areas’
- Colonies that appear more later in time are derived form more primitive cell subsets
Advantages:
• Reflect a more primitive population than the CFC assay
• Correlate well with SC frequencies reported by the CFU-S
Describe the long-term reconstitution assay
• Reconstitutes all blood cell lineages when transplanted into an irradiated recipient
− Donor HPCs and mature cells present in the initial cell inoculum may be detectable in recipients up to 4 weeks post ransplant
− MPPs or short-term repopulating cells may produce transient haemopoesis for up to 4 months post transplant
− Long-term, multi-lineage donor-derived haematopoesisi should be detectable for > 4 months.
− However, originally people just used to see if the moust died at 4 months – if it did, there were no SCs in the inoculum
− This is letting the animals die uncessarily.
Describe the competetive repopulating assay and its limitations
- Measures the functional potential of the unknown source of HSCs against a set known number of HSCs
- Provides information about the capacity of the source of HSCs to repopulate compared with the competing bone marrow
- Cant distinguish between the number of HSCs and their quality
The quantitative limiting dilution assay is a variant, often called competitive repopulating unit assay:
• Based on the ability of HSCs to produce both myeloid and lymphoid progeny when transplanted into a conditioned recipient
• Series of dilutions of test cells are competed against a set number of regulator bone marrow cells
− Minimal number of HSCs allows for the optimal detection of HSCs at the single level, but doesn’t allow for the quality of the HSCs to be compared against a known standard
− If you use a standard, sufficient number of HSCs, the test cells are therefore measured relative to their ability tp effectively compete against the standard. If the inoculum has fewer or more HSCs than the standard, this will be reflected in the results of the assay.
• Number of mice negative for reconstitution measured
• Frequency of HSCs estimated using Poisson statistics → relies on the frequency of mice considered to have negative engraftment
• Success of this assay therefore relies on survival of the mice
But how do you detect the progeny of the stem cell – need to distinguish test cells from the standards
• different CD45 isoforms
• Can use GFP, or male/female cells (put male cells in female mouse, look for Y chromosome)
Important Factors to Consider in Limiting Dilution Assays:
Competing cells:
• Source of competing cells is a variability that can lead to different results between labs
Test cells, Unknown HSC Potential
• eg) LKS+ CD34+ cells are enriched with short term repopulating HSCs, but devoid of long term
• Best source of donor cells is whole bone marrow cells
Donor cell percentage used to determine number of negative mice
• Poisson statistics relies on frequency of mice considered to have negative engraftment
• Orignal experiment could reliably detect 5% donor cells – so
Describe the SRC assay
- Used for testing human HSCs retrospectively
- Same concept as CRU, but can inject human cells into a SCID mouse without rejection
- Identify human cell progeny based on human-specific markers
Describe the serial transplantation assay
• Most stringent
• LT-HSCs can self-renew for life (maybe longer, can be serially transplanted from mouse to mosue up to 7 times) but they do have a finite number of divisions in mice depending on strain. They are quiescent.
• ST-HSCs can be distinguished from LT-HSCs by CD34 expression, and they divide more frequently and thus have more limited self-renewal capacity
• MPPs are virtually indistinguishable from ST-HSCs except they are highly proliferative and give rise to lineage positive cells
➢ LT-HSCs will repopulate for more than 4 months, the rest wont
How can HSCs be isolated by flow?
- Test population labeled with fluorescent antibodies that mark specific cell populations
- Cells are run through the machine along with sheath fluid that passes the cells single file past a laser beam and fluorescent detector.
- Cells sorted based on their immunophenotype uing a FACS machine → flow with the addition of a charge to cells, allows the cells to be sorted into separate populations using charged plates.
- Lineage markers can all be in the same colour, as you are selecting against that
- Stem cell markers selected for using a series of gates.
- HSC → cKit + Sca-1 + Lin-
- CMP → cKit+ Sca-1 - CD34+ FcyR –
- GMP → cKit + Sca-1 - CD34+ FcyR hi
What is the frequency of LT repopulating cells generated from cell samples?
Unseparated bone marrow - 1 per 20,000
Sca-1 + cKit+ Lin- = 1 per 15
Thy-1 lo Sca-1 + cKit+ = 1 per5
Describe assays for cell cycle status
Stem cells have low Rhodamine 123 retention:
• Rh12 is a binding dye held in mitochondria
• More active cells have higher rention
Stem cells are in G0/G1:
• Hoechst 33342 DNA binding dye that can be used to distinguish between G0/G1 from cells in cycle
• Retained at low density in HSCs
→ SCs may therefore protect themselves through pumps and cell cycle inhibitors
Detection of Side Population Cells in the Bone Marrow
• SP cells exclude Hoechst 33342
• This is because they express high levels of the ABC transporter protein ABCG2
• Possible protection mechanism from toxins
P21 and quiescence:
• p21 encodes a cyclin dependent kinase inhibitor that prevents cell-cycle progression
• p21 expression is high in stem cells
• in p21 knockout animals, ability of cells to self-renew was impaired
• So if HSCs are cycling more frequently, they are losing self-renewal capcity → quiescence is an essential characteristic of HSCs
• HSCs get more proliferative as they differentiate
Describe the bone marrow microenvironment
• Contains haematopoetic and non-haematopoetic cells (stromal cells)
• Stromal cells = MSCs, fibroblasts, osteoblasts, fat cells, endothelial cells
• Trabecular region contains more LT-HSCs (sinusoidal and endosteal nice)
− Sinusoid – specialsied blood vessels allowing cells in and out of circulation
− Endosteum – interface between the bone and marrow
− Medullary cavity contains more ST-HSCs
What experiments suggested the presence of intrinsic and extrinsic factors?
Russel and Bernstein, 1960’s
• Mutant mouse (W) prone to anaemia and sensitive to radiation
• Had defects in HSC self renewal but could be cured by bone marrow transplant
− Normal bone marrow transplanted into W mouse → phenotype normal
− Bone marrow transplanted from W mouse into normal mouse → aneamia
• Demonstrates that W is a HSC instrinsic factor (expressed by HSCs)
Till and McCulloch, 1960’s
• Mutant mouse (S) had same phenotype but!
− Normal bone marrow transplanted into S mouse → anaemia
− Bone marrow transplanted from S mouse into normal mosue → normal
• Demonstrates that S is a HSC extrinsic factor (expressed by niche cells)
