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)

Properties of the adult hematopoietic stem cell

Adult = 4 weeks old (mouse); 2 – 4 years (human)

Quiescent: Majority (75-80% in G0 at any one time)

Multipotent: Can differentiate into any hematopoietic lineage (clonal)

Able to self-renew: Long-term vs. short-term

Only cell capable of long-term hematopoietic reconstitution (Makes bone marrow transplant possible)

After transplant, all hematopoiesis is derived from long-term HSCs > 16 weeks in the mouse

Reside in the bone marrow; can be “mobilized” to enter the periphery

Definitive Test of HSC Function

Repopulation Assays

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

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

- Sca-1 (generated from antibody library raised against marrow progenitors)

- c-kit (mutated W/Wv mice, which have severe anemia)

- Lineage-negative, c-kit(HI), Sca-1(HI) (LSK) cells contain 100% of hematopoietic stem cells under normal conditions

c-ki

t

Sca-1

Prospective Isolation of HSCs

Strategy has been refined… 1)  To isolate distinct populations

2)  To determine other HSC markers

3)  To isolate HSCs to near-purity

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-

Prospective Isolation of HSCs

In Vivo Lifespan of Purified Hematopoietic Populations

Multipotent Progenitors (MPP)

Lymphoid Progenitors Myeloid Progenitors

Mitotic Long Term (LT)-HSC

Short Term (ST)-HSC

Quiescent Long Term (LT)-HSC

(> 4 months)

(≈ 2 – 3 months)

(≈ 6 – 8 weeks)

(≈ 6 – 8 weeks) (≈ 1 – 2 weeks)

Where do HSCs live? The HSC Niche

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

Interactions between HSC and niche critical for homing and retention

Mobilization protocols seek to inhibit these interactions (e.g. SDF-1α:CXCR4)

Reya and Clevers, 2005

Quiescent HSC

“Activated” HSC

Self-renewal Differentiation

Basic Model of HSC Fate

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

- What is it for? Current models/concepts: 1) Dormant HSCs can be activated and then resume dormancy 2) Protects against endogenous/genotoxic stress, replication errors

e.g. Old story: HSCs more radioresistant than progenitors

NHEJ (low-fidelity) vs. HR (high-fidelity) repair

New story: Radioresistance a feature of HSCs and quiescent HSCs may acquire more mutations than proliferating HSCs

3) Conserves long-term HSC function (replicative senescence)

(Mohrin, et al Cell Stem Cell, 2010)

Bone marrow has limited capacity for serial transplant (≈ 4 rounds)

HSC Quiescence

Strong link between quiescence and function (“exhaustion”)

Loss of function Cdkn1a, Gfi1, Mll, Pten, Fbxw7, Pbx1

Increased cell-cycle entry

Reduced HSC Function

Loss of function Mef

Decreased cell-cycle entry

Increased HSC Function

HSC Quiescence

1)  Positive Regulation (Examples) - Stem cell factor (ligand for c-kit) - Thrombopoietin - SDF-1α (necessary for HSC homing and retention) - Hypoxic profile (high Hif-1α, low O2 tension)

Extrinsic Factors

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

Strong link between quiescence and function (“exhaustion”)

HSC Quiescence: Role for Metabolism

Hypoxic profile (high Hif-1α, low O2 tension) - observed regardless of local oxygenation Metabolic profile of HSCs 1)  have fewer mitochondria than progenitors

2)  utilize anaerobic glycolysis for generating ATP (Simsek, et al., 2010)

3)  anaerobic glycolysis maintains HSC quiescence

loss of Pdk2/4 results in decreased quiescence and stem cell function (Takubo, et al, 2013)

Glucose Pyruvate

Lactate

Acetyl-CoA

(Anaerobic glycolysis)

(Aerobic glycolysis) Pyruvate dehydrogenase

Pdk

Turnover Rate of HSCs

- Summation of data (Chesier, 1999; Kiel; 2007; Nygren, 2008; Wilson, 2008; Foudi, 2009; Takizawa, 2011) - On average, HSCs divide once per 17.8 – 39 days - Presence of two distinct populations

1) Divides once per 9 – 36 days 2) Divides once per 56 – 145 days

Turnover Rate of HSCs

Takizawa, 2011

Clonal succession

Dynamic repetition

High-turnover population responsible for daily hematopoiesis and low-turnover population acts as dormant reserve

HSCs “take turns” proliferating and contributing to hematopoiesis before resuming relative dormancy

HSC Self-Renewal and Differentiation

How does an HSC decide to self-renew or differentiate?

- Self-Renewal: a process by which a mitotic HSC generates daughter cell(s) that retain the parent stem cell phenotype

Processes active or passive? Instructive or stochastic?

How do HSCs decide their lineage fate?

Zon, 2008

- Self-renewal is an intrinsic process that is influenced by environment

- Switch between self-renewal and differentiation regulated by competition between transcription factors

How does an HSC decide to self-renew or differentiate?

Pre-Determined Fates of HSCs

Challen, et al., Cell Stem Cell

As age increases, the percentage of myeloid cells of the total bone marrow also increases

What is the predicted relative distribution of HSC types as mice age?

Environment age partly regulates lineage bias

Ergen A V et al. Blood 2012;119:2500-2509

©2012 by American Society of Hematology

Rantes is a regulator of age-related lineage bias

Ergen A V et al. Blood 2012;119:2500-2509

©2012 by American Society of Hematology

Kim , et al Cell Stem Cell, Volume 14, Issue 4, 2014, 473 - 485

Dynamics of HSPC Repopulation Using Nonhuman Primate Model

Time

Key points: 1)  Initial repopulation is dominated by short-term HSCs/progenitors 2)  Long-term hematopoiesis maintained by mix of lineage-biased and balanced clones 3)  Different clones “emerge” at different times (clonal succession and diversity)

Hematopoiesis: Local versus systemic bacterial infection.

Takizawa H et al. Blood 2012;119:2991-3002

©2012 by American Society of Hematology

HSC Activation: Response to Inflammation

(King and Goodell, 2011)

MPP

Lymphoid Progenitors

Myeloid Progenitors

Mitotic LT-HSC

Quiescent LT-HSC

PAMPs: Direct effects on HSPCs

HSPCs exhibit high levels of TLR2 and 4

Myeloid Effector Cells

Dendritic Cells (reduced B cells)

LPS

Increased Proliferation

Increased Differentiation

PAMPs: Indirect effects on HSPCs

Mitotic LT-HSC

Quiescent LT-HSC HSPCs do not express TLR3 Dendritic Cells Macrophages

pIpC (dsRNA) Type I IFN

Increased Proliferation

LSK, CD150+ CD48- 0 hrs + pIpC; 48 hrs + pIpC; 96 hrs

Ki-6

7

Hoescht

(Zhao, et al, 2014)

Danger Signals are Converted into Cytokine Signals by HSPCs

MPP

Increased Cytokines

Mitotic LT-HSC

Quiescent LT-HSC

Myeloid Effector Cells

PAMPs (e.g. LPS)

Danger Signals are Converted into Cytokine Signals by HSPCs

PAMPs (e.g. LPS)