Hematopoiesis. - Process of generation of mature blood cells. - Daily turnover of blood cells (70 kg human)

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1 Hematopoiesis - Process of generation of mature blood cells - 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

2 Hematopoiesis and the Microenvironment (Figure by Winslow & Kibiuk; Stem Cells: Scientific Progress and Future Research Directions, 2001)

3 Definition of the adult hematopoietic stem cell Adult = 4 weeks old (mouse); 2 4 years (human) Reside in the bone marrow; can be mobilized to enter the periphery 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) Quiescent: Majority (75-80% in G0 at any one time)

4 Rossi, et al., Cell Stem Cell, 2012 Tests of HSC Function

5 Immunophenotyping of HSCs 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) Prospective isolation - Use of antigens expressed on the surface of putative HSCs - Detection using multiparameter flow cytometry

6 Immunophenotyping of HSCs Whole Bone Marrow Lin- Cells Side Scatter 5.0% c-kit 0.18% Lineage Markers Sca-1 CD34 Lin-, Sca-1+, c-kit+ CD150+, CD48- CD % CD % Forward Scatter CD48

7 Testing HSC Function through BMT Lethal Irradiation Test Cells + Competitor/ Radioprotective Cells 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 Test and Donor (preferably competitor too) must be distinguishable e.g. CD45 markers, GFP Differences in engraftment must be attributable to test variable

8 In Vivo Lifespan of Purified Hematopoietic Populations Long Term (LT)-HSC (> 4 months) Short Term (ST)-HSC ( 2 3 months) Multipotent Progenitors (MPP) ( 6 8 weeks) Lymphoid Progenitors Myeloid Progenitors ( 6 8 weeks) ( 1 2 weeks)

9 Testing HSC Self-Renewal through Serial BMT Primary Recipient Secondary Recipient Tertiary Recipient Bone Marrow Cells HSC HSC MP Repopulating Potential Decreases After Each Transplant Serial Transplant Mimics HSC Aging

10 HSC Quiescence Lin-, Sca-1+, c-kit+ CD150+, CD48- Ki-67 ST-HSC 22.6% 5.12% CD % CD34- LT-HSC 22.1% 0.42% Ki % Hoescht Dye

11 Proliferation of Hematopoietic Stem and Progenitors Long Term HSC Short Term HSC Multipotent Progenitor Committed Progenitor

12 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 O 2 tension) - Pdk2/4 (maintains quiescence through anaerobic glycolysis) - Cell Cycle Regulators (Cdkn1a) - Transcription Factors (e.g., Gfi1, Mll, Pten, Fbxw7, Pbx1) 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

13 HSC Quiescence 1) Quiescent: Majority (75-80% in G0 at any one time) 2) Adult quiescent HSCs are superior to cycling HSCs in repopulating hematopoiesis 3) Conserves long-term HSC function (replicative senescence) 4) Dormant HSCs can be activated and then resume dormancy 5) Stress vs. homeostasis is likely a critical variable

14 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

15 Does Activation During Stress Differ than Homeostasis? (Qiu, et al., Stem Cell Rep, 2014) Label Retaining Assay TetON-GFP

16 Does Activation During Stress Differ than Homeostasis? (Qiu, et al., Stem Cell Rep, 2014)

17 The HSC Microenvironment Specialized microenvironment that supports HSC function Schofield R, Blood Cells, 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

18 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

19 Reciprocal Transplantation Assay W (White, W = c-kit severe receptor anemia) Sl Sl (Steel, = stem severe cell factor anemia) Wild-type mice Donor Host No effect on anemia Rescued anemia Rescued anemia No effect on anemia

20 HSC Fate: Stochastic or Instructive? As age increases, the percentage of myeloid cells of the total bone marrow also increases

21 Pre-Determination of HSC Fate Hundreds of mice transplanted with single purified HSC (Dykstra, et al, Cell Stem Cell, 2007) Blue = α, myeloid biased Magenta = β, balanced Green/Yellow = γ + δ, lymphoid biased

22 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 Differentiation programs can be stable, but there is significant conversion between programs

23 Pre-Determination of HSC Fate Side-Population: Dye Efflux Property of HSCs Challen, et al., Cell Stem Cell, 2010

24 Hierarchy of Pre-Determined HSCs vwf-egfp Transgenic Mice Sanjuan-Pla, et al., Nature, 2013

25 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

26 Hematopoietic Hierarchy: An Evolving Model Quiescent HSC Mechanisms Controlling Dormancy/Self-Renewal Critical for Leukemia Mitotic HSC (Platelet/Myeloid Biased) Mitotic HSC (Lymphoid Biased) Mediate Reconstitution During BMT Megakaryocyte/ Myeloid Progenitors Lymphoid Progenitors