SBM HEMATOLOGY October !"#$%&!

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HEMATOPOIESIS
20!"

Christopher Lowrey, MD
(c.lowrey@dartmouth.edu)

Learning objectives:
1. Be able to recognize normal human peripheral blood cells.
2. Understand how hematopoietic stem cells give rise to mature blood cells.
3. Be able to explain how sites of hematopoiesis change during development.
4. Understand how hypoxia stimulates production of red blood cells.
5. Understand the role of hematopoietic growth factors in regulating blood cell production.
6. Understand the rationale for hematopoietic stem cell transplantation.

Reading: Up-to-Date: “Overview of hematopoiesis and stem cell function.”

N o te: The most important concepts for you to know are underlined in the text of these notes.

I. Types of cells in the blood.

The cellular portion of blood is composed of seven distinct types of cells (Figure 1):

Neutrophil (immature)
Platelet
Red Blood Cell

Eosinophil

Neutrophil (mature)

Basophil

Lymphocyte
Monocyte

Figure 1. Normal Human Blood Cells

a. Red cells – carry oxygen
b. Platelets – participate in clot formation, a critical component of hemostasis.
c. Lymphocytes (T and B cells) – respond to viral and other types of infection.
d. Monocytes, Neutrophils, Eosinophils, Basophils – respond to bacterial, fungal, and parasitic
infections.

Hints for identifying the different types of WBC’s: lymphocytes generally have small round
nuclei, minimal cytoplasm and no granules, monocytes have more cytoplasm and an indented nucleus,
basophils have intense blue granules, eosinophils have intense red granules that are stained by the dye
“eosin”, mature neutrophils have 3 “lobes” and pale granules, immature granulocytes are known as “bands”
with a nucleus that has not yet separated into 3 lobes.

Clinical Correlation – The Complete Blood Count (CBC)

WBC – total number of all white blood cells per cubic mm (i.e., microliter).
Platelet Count - total number of platelets per cubic mm (i.e., microliter).
Hemoglobin & Hematocrit – these parameters are typically used instead of the “red blood cell count”
to quantify the number of red cells. See figure 2.

Revised September 2009 September 25, 2009

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A B

Hematocrit =
Volume Plasma/Volume of Red Cells
Hematocrit
Hemoglobin = WBC Platelets
Spectrophotometric measurement Hemoglobin
hemoglobin absorbance

Figure 2. A) Calculation of hemoglobin and hematocrit values. B) Typical clinical

short-hand often used to record the CBC.

II. The Concept of the “Hematopoietic Stem Cell.”

All the cells of the blood arise from a common hematopoietic stem cell (HSC) (Figure 3). HSCs were first
identified in the 1950’s and were the first (and for many years the only) example of a cell that could give
rise to many other types of more mature cells. They were first identified by showing that the hematopoietic
system of lethally-irradiated mice could be regenerated by injecting bone marrow from untreated donor
mice. A single HSC can regenerate the blood system of a mouse – including giving rise to more HSCs.

HEMATOPOIETIC GROWTH FACTORS

IL-2
NK lymphocytes
LYMPHOID IL-2
STEM CELL T lymphocytes
(IL-6)
B lymphocytes
MATURE CELLS

(TPO)/IL-11
Stem Cell Plts
Factor(SCF)
STEM GM-CSF
CELL Monos

CFU-GEMM G-CSF/GM-CSF
MYELOID PMNs
PRECURSOR EPO RBCs

CFUs MATURE CELLS

Figure 3. All blood cells arise from a common precursor -

the hematopoietic stem cell (HSC).

Approximately 1 in 10,000 bone marrow cells are HSCs in adult humans. They are typically identified by
the presence of a cell surface protein known as CD34. The function of this protein is not known. It is also
present on early myeloid cells and on some leukemia cells. It is estimated that humans have approximately
50,000 stem cells. At any given time most of these cells are in the G0 phase of the cell cycle with only a
small proportion contributing to hematopoiesis at any one time. The quiescent stem cells are thought to be
resistant to toxic insults such as radiation or chemicals because they are able to repair their DNA before
entering the cell cycle at some later time point.

Translational Science Correlation – Hematopoietic Stem Cell “Plasticity”

Recent evidence, mostly from mouse experiments, has suggested the possibility that HSCs can also
generate non-hematopoietic cells. For example, injecting HSCs into mice that have had experimentally
induced heart attacks may lead to regeneration of cardiac myocytes that are derived from the HSCs. Similar
results have been provided for regeneration of chemically damaged liver cells. Despite the fact that the
concept of HSC “plasticity” remains controversial, several early human trials of autologous (from the same
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person) HSC transplant for patients after myocardial infarction

have been performed – so far there is no conclusive evidence of
HSC-mediated cardiac repair or of improved survival.

Clinical Correlation – Bone Marrow Transplantation

Figure 4. Dr. E. Donnell Thomas - winner
of the 1990 Nobel Prize for Physiology and
Medicine for his work on Bone Marrow
Transplantation.
bone marrow transplantation.
Based on the ability to reconstitute the hematopoietic system of
mice and other animals following lethal irradiation, researchers in
the 1960’s began treating patients with life-threatening diseases of
blood stem cells (mostly leukemia and other blood cancers) with
stem cell transplants. The idea was that lethal doses of irradiation
and/or chemotherapy drugs could be used to eliminate the
abnormal stem cells but that this would also eliminate normal
stem cells – which could be replaced by stem cells from a normal
donor. Approximately 8,000 of these allogeneic (from another
person) transplants were performed in 2006. These transplants
offer the only hope for a cure for many patients. Dr. E. Donnell
Thomas (Figure of the University of Washington received the
1990 Nobel Prize in Physiology & Medicine for his work on

III. The location of Hematopoiesis Changes During Human Development.

In the first few weeks of gestation, the yolk sac is the main site
for hematopoiesis. Early in embryonic development, the first
HSCs arise in the wall of the developing aorta and then migrate to
the fetal liver and spleen. From 6 weeks until 6-7 months of fetal
life, the liver and spleen are the main organs for production and
continue to be sites until about 2 weeks after birth. The bone
marrow is the most important site from 6-7 months of fetal life
and soon after birth is the only source of new cells. Developing
cells are situated outside the bone marrow sinuses and mature cells
are released into the sinus spaces, the marrow microcirculation and
the general circulation.

In infancy, all the bone marrow is hematopoietic but during

childhood, the marrow is progressively replaced by fat in the long
bones. In the adult, hematopoietic marrow is confined to the axial
skeleton and proximal ends of the femurs and humeri. Even in the
hematopoietic areas, the marrow consists of 50% fat. In times of
need, the marrow in these sites replaced by fat may produce
hematopoietic cells. The liver and spleen may also become a
production site (called extramedullary hematopoiesis).

Figure 5. The location of hematopoiesis changes during normal human development.

Bone marrow
biopsies are typically
performed at the posterior
Iliac crest.

Fig 6: Infant vs. adult sites of hematopoiesis.

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Bone Hematopoietic Cells

A Fat
B
Myeloid precursors Erythroid precursors

2
1
2 3
3

Megakaryocytes
(platelet precursors)

Figure 7. Normal adult bone marrow morphology. A) Biopsy core from the posterior iliac
crest of a person without hematologic disease. B) High-power view of hematopoietic cells
From a normal bone marrow aspirate. Numbers refer to progressively more mature cells.
Erythroid nuclei are round and become progressively condensed prior to enucleation. C)
Lower power view than in B, showing megakaryocytes.

IV. Control of Hematopoiesis – The Critical Role of Hematopoietic Growth Factors

The hematopoietic system is far and away the most prolific of the body’s organs with approximately 106 red
cells and 104-5 white cells being produced every second. This equates to roughly 1016 blood cells in a
lifetime. The hematopoietic system is also highly regulated as too few or too many of each cell type can
lead to serious medical complications. The body must be able to rapidly increase production of red cells
following hemorrhage or red cell destruction. Similarly, WBC production can be rapidly accelerated during
infection.

The production of each type of blood cell is regulated by a specific combinations of hematopoietic growth
factors – also known as cytokines or interleukins (i.e. IL-3, interleukine-3) . These are shown in Figure 8
and their properties are listed in Table 1. The growth factors responsible for hematopoietic development are
glycoprotein hormones. They may be produced by local cells in the marrow (like t-cells, macrophages,
endothelial cells and fibroblasts) or come through the circulation from distant sites (e.g. erythropoietin from
the kidney). The biological effects of these factors are mediated by specific cell surface receptors, which send
signals to the nucleus to respond in certain ways.

The most important factors for you to know about are Stem Cell Factor (aka, c-kit ligand), IL-3, IL-2, GM-
CSF, G-CSF and erythropoietin. See table 1 for detailed information about these factors.

Clinical Correlation – Growth Factors

Several of the hematopoietic growth factors are now produced commercially for use in specific clinical
situations. For example, kidney failure is often accompanied by inadequate erythropoietin production
leading to anemia. Erythropoietin injections are now routinely used for patients with chromic renal failure.
We’ll discuss the therapeutic use of several of these cytokines in more detail in the Pharmacology portion
of SBM.
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IL-2,4,6,7,10,13,14,15 B lymphocytes
LYMPHOID IL-2,4,6,7,9,12,15
STEM CELL T lymphocytes
IL-2,12,15
NK lymphocytes
IL-3 MATURE CELLS

Stem Cell Thrombopoietin

Plts
Factor(SCF),
STEM IL-3 GM-CSF
CELL Monos

MYELOID G-CSF/GM-CSF
PMNs
PRECURSOR
CELL Erythropoietin
RBCs

MATURE CELLS

Figure 8. Production of hematopoietic cells is under the regulation of lineage-specific

growth factors.

Figure 9. The stages of erythropoiesis.

Kinetics of Hematopoiesis
1. Basal rates of hourly bone marrow production are thought to be 1010 erythrocytes and 108 leucocytes per
hour. Production can be increased about 8 times baseline upon demand.
2. Each RBC lives 120 days. There are 20-30 times more RBCs in the peripheral blood than in the
marrow. The stimulus to production is O2 levels in the kidney which leads to EPO production.
3. The ratio of WBCs to RBCs in the marrow is 3 or 4:1. It takes about 10 days to produce mature
WBCs - the stimulus is inflammation, invasion of microbes. Each poly lives 6-7 hours. neutrophils,
bands and metamyelocytes don't divide.
4. Platelets are stimulated by thrombopoietin (stimulus=bleeding). They take 10 days to produce.
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The production of erythropoietin by the kidney in response to its oxygen (O2) supplies. Erythropoietin stimulates
erythropoiesis and so increases O2 delivery.

Figure 10 shows production and effects of erythropoietin

From Hoffbrand & Pettit, Essential Haematology. 1993. Figure 2.4, page 15.

A B
Epo level
Normal Epo
10000
response to anemia
1000
100 Impaired Epo
Response to anemia
10

0 5 10 15 Hgb

Figure 11. Reticulocyte response to anemia. A) Blood smear showing mixture of mature red cells and reticulocytes.
B) The normal response to anemia involves and exponential rise in erythropoietin levels. In patients with
kidney disease the response can be impaired.

Clinical Correlation – The reticulocyte count and erythropoietin levels

When evaluating a patient with anemia, one of the first questions to ask is whether the problem is with
erythropoiesis (decreased red production) or whether it is due to some other effect such as bleeding or hemolysis.
The normal response of the body to anemia is increase production of erythropoietin in the kidney. This in turn
leads to increased production of red cells – and increased numbers of reticulocytes in the blood. These cells still
have substantial amounts of messenger RNA which can be detected by staining techniques (Figure 11A). If the
number of reticulocytes is increased in response to anemia, then the problem is not on in red cell production. If the
reticulocyte count is low in a patient with anemia, then one might ask whether the Epo level is appropriately
increased. As shown in Figure 11B, erythropoietin levels normally increase exponentially in response to anemia.
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Hematopoiesis

V. Case Study
A 19-year-old former high school football player presents with extreme fatigue, dyspnea on exertion, fevers,
diffuse bruising and nose bleeding. CBC shows WBC of 0.2 (normal 4-10) and only 20% neutrophils, hemoglobin
of 6 (normal 13-16) and platelets of 4 (normal 145-450) =
Bone Marrow Biopsy pancytopenia. He is given red cell and platelet transfusions and broad-
spectrum antibiotics are administered. A bone marrow biopsy is
Patient performed and, as shown in the figure, is markedly hypocellular. A
diagnosis of aplastic anemia is made. The etiology of this disease is
autoimmune destruction of the HSCs First-line treatment is treatment
with very strong immunosuppression (anti-thymocyte globulin and
cyclosporine – don’t need to know these). While 75% of patients
respond to this therapy with recovery of counts, he does not. A
decision is made to pursue allogeneic HSC transplantation. While
this is being arranged he receives G-CSF, erythropoietin and weekly
platelet transfusions (there is no clinically-available cytokine for
Normal platelets) to help support his blood counts. A donor is identified and
his transplant successfully reconstitutes his hematopoietic function.