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Hematopoiesis
- Daily turnover of blood cells (70 kg human) 1,000,000,000,000 total cells 200,000,000,000 red blood cells 70,000,000,000 neutrophils
- Process of generation of mature blood cells
Hematopoiesis and the Microenvironment
(Figure by Winslow & Kibiuk; Stem Cells: Scientific Progress and Future Research Directions, 2001)
Definition of the adult hematopoietic stem cell
Adult = 4 weeks old (mouse); 2 – 4 years (human)
Multipotent: Can differentiate into any hematopoietic lineage
Able to self-renew: Long-term vs. short-term
Only cell capable of long-term hematopoietic reconstitution (Makes bone marrow transplant possible)
Reside in the bone marrow; can be “mobilized” to enter the periphery
Quiescent: Majority (75-80% in G0 at any one time)
Rossi, et al., Cell Stem Cell, 2012
Tests of HSC Function
Lineage depletion (negative selection)
- Based on findings that cells that gave rise to B-cell colonies in vitro were B220-negative (B220 = B-cell specific antigen)
Immunophenotyping of HSCs
Prospective isolation
- Use of antigens expressed on the surface of putative HSCs
- Detection using multiparameter flow cytometry
Whole Bone Marrow
Sid
e S
catte
r
Lineage Markers
5.0%
Lin- Cells
c-ki
t
Sca-1
0.18%
CD
150
CD48
0.03%
CD
34
Forward Scatter
Lin-, Sca-1+, c-kit+ CD150+, CD48-
0.01%
CD34-
Immunophenotyping of HSCs
Testing HSC Function through BMT
Test Cells +
Competitor/ Radioprotective
Cells
Test and Donor (preferably competitor too) must be distinguishable e.g. CD45 markers, GFP
Differences in engraftment must be attributable to test variable
Lethal Irradiation
Analyze blood every 4 weeks Analyze marrow at 16 weeks
Evidence of HSC requires multi-lineage engraftment at > 16 weeks
At earlier time points, myeloid cells (short life-span) surrogate of HSCs
In Vivo Lifespan of Purified Hematopoietic Populations
Multipotent Progenitors (MPP)
Lymphoid Progenitors Myeloid Progenitors
Long Term (LT)-HSC
Short Term (ST)-HSC
(> 4 months)
(≈ 2 – 3 months)
(≈ 6 – 8 weeks)
(≈ 6 – 8 weeks) (≈ 1 – 2 weeks)
Testing HSC Self-Renewal through Serial BMT
Bone Marrow Cells
Primary Recipient
HSC
HSC MP
Secondary Recipient
Tertiary Recipient
Repopulating Potential Decreases After Each Transplant
Serial Transplant Mimics HSC Aging
CD34-
Ki-6
7
Hoescht Dye
Ki-6
7
CD34+
Lin-, Sca-1+, c-kit+ CD150+, CD48-
22.6% 5.12%
72.2%
22.1% 0.42%
78.5%
HSC Quiescence
LT-HSC
ST-HSC
Proliferation of Hematopoietic Stem and Progenitors
Multipotent Progenitor
Short Term HSC
Long Term HSC
Committed Progenitor
HSC Quiescence
1) Positive Regulation (Examples) - Stem cell factor (ligand for c-kit) - Thrombopoietin - SDF-1α (necessary for HSC homing and retention) - Wnt Ligands (e.g. Wnt5a) - Hypoxic profile (high Hif-1α, low O2 tension)
2) Negative Regulation (Examples) - Bone marrow injury (molecular data unclear) - Mobilization of stem cells to the periphery - Reactive oxygen species (increased oxidative stress) - Bacterial infection via interferon alpha/gamma Link between quiescence and function (“exhaustion”) is frequently observed but is not absolute; context is critical
- Cell Cycle Regulators (Cdkn1a) - Transcription Factors (e.g., Gfi1, Mll, Pten, Fbxw7, Pbx1)
- Pdk2/4 (maintains quiescence through anaerobic glycolysis)
HSC Quiescence
4) Dormant HSCs can be activated and then resume dormancy
3) Conserves long-term HSC function (replicative senescence)
1) Quiescent: Majority (75-80% in G0 at any one time)
2) Adult quiescent HSCs are superior to cycling HSCs in repopulating hematopoiesis
5) Stress vs. homeostasis is likely a critical variable
4) Dormant HSCs can be activated and then resume dormancy (Wilson, et al., Cell, 2008)
Label Retaining Assay – Cell Division Inversely Associated with Label Intensity
5-Fluorouracil: Induces HSC Proliferation
BrdU: Thymidine Analogue Incorporates Into DNA
BrdU+ Cells at day 0 post-chase
BrdU+ Cells at 70 days post-chase
Does Activation During Stress Differ than Homeostasis? (Qiu, et al., Stem Cell Rep, 2014)
Label Retaining Assay – TetON-GFP
Does Activation During Stress Differ than Homeostasis? (Qiu, et al., Stem Cell Rep, 2014)
The HSC Microenvironment
Schofield R, Blood Cells, 1978
- Hypothesized the presence of a stem cell niche in the marrow
1. Defined anatomical site
2. Allows for maintenance of stem cell
3. Prevents differentiation
4. Niche space is limited
5. Occupation of niche by differentiated cell causes reversion to stem cell phenotype
Specialized microenvironment that supports HSC function
Anatomy of the HSC Niche
HSCs preferentially reside in the trabecular bone area
Evidence suggests HSCs reside at or near the endosteal surface (Distinct perivascular niche?)
Endosteum is comprised of multiple cell lineages (osteoblast, vascular, MSCs)
Niche regulates HSC function by: 1) Cell-cell contact 2) Release of soluble factors
Reya and Clevers, 2005
Reciprocal Transplantation Assay
W (White, severe anemia) Sl (Steel, severe anemia)
Donor
Host
Wild-type mice
No effect on anemia
Rescued anemia
Rescued anemia
No effect on anemia
W = c-kit receptor Sl = stem cell factor
HSC Fate: Stochastic or Instructive?
As age increases, the percentage of myeloid cells of the total bone marrow also increases
Blue = α, myeloid biased Magenta = β, balanced Green/Yellow = γ + δ, lymphoid biased
Pre-Determination of HSC Fate
Hundreds of mice transplanted with single purified HSC (Dykstra, et al, Cell Stem Cell, 2007)
Differentiation programs can be stable, but there is significant conversion between programs
Pre-Determination of HSC Fate
Secondary Transplants Blue = α, engrafted, durable self-renewal Magenta = β, engrafted, durable self-renewal Green/Yellow = γ + δ, did not engraft, no self-renewal
Challen, et al., Cell Stem Cell, 2010
Pre-Determination of HSC Fate
Side-Population: Dye Efflux Property of HSCs
Sanjuan-Pla, et al., Nature, 2013
Hierarchy of Pre-Determined HSCs
vWF-eGFP Transgenic Mice
Hierarchy of Pre-Determined HSCs
vWF-GFP+ HSCs can repopulate GFP+/GFP- HSCs but GFP- HSCs can not repopulate GFP+ HSCs Platelet/Myeloid biased HSCs are more apical than lymphoid biased HSCs
Hematopoietic Hierarchy: An Evolving Model
Megakaryocyte/ Myeloid
Progenitors
Lymphoid Progenitors
Mitotic HSC (Platelet/Myeloid
Biased)
Mitotic HSC (Lymphoid
Biased)
Quiescent HSC
Mediate Reconstitution During BMT
Mechanisms Controlling Dormancy/Self-Renewal
Critical for Leukemia
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