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