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CHAPTER 1
1 Haemopoiesis
This first chapter is concerned with the general the major haemopoietic organs and continue to pro-
aspects of blood cell formation (haemopoiesis). The duce blood cells until about 2 weeks after birth
processes that regulate haemopoiesis and the early (Table 1.1) (see Fig. 6.1b). The bone marrow is the
stages of formation of red cells (erythropoiesis), most important site from 6 to 7 months of fetal life.
granulocytes and monocytes (myelopoiesis) and During normal childhood and adult life the marrow
platelets (thrombopoiesis) are also discussed. is the only source of new blood cells. The develop-
ing cells are situated outside the bone marrow
sinuses and mature cells are released into the sinus
Site of haemopoiesis
spaces, the marrow microcirculation and so into the
In the first few weeks of gestation the yolk sac is the general circulation.
main site of haemopoiesis. However, definitive In infancy all the bone marrow is haemopoietic
haemopoiesis derives from a population of stem but during childhood there is progressive fatty
cells first observed on the dorsal aorta termed the replacement of marrow throughout the long bones
AGM (aorta-gonads-mesonephros) region. These so that in adult life haemopoietic marrow is con-
common precursor of endothelial and haemopoietic fined to the central skeleton and proximal ends of
cells (haemangloblasts) are believed to seed the the femurs and humeri (Table 1.1). Even in these
liver, spleen and bone marrow and from 6 weeks haemopoietic areas, approximately 50% of the
until 6–7 months of fetal life the liver and spleen are marrow consists of fat (Fig. 1.1). The remaining fatty
marrow is capable of reversion to haemopoiesis
Table 1.1 Sites of haemopoiesis. and in many diseases there is also expansion of
haemopoiesis down the long bones. Moreover, the
Fetus 0 –2 months (yolk sac) liver and spleen can resume their fetal haemopoietic
2 –7 months (liver, spleen) role (‘extramedullary haemopoiesis’).
5 – 9 months (bone marrow)
1
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2 CHAPTER 1
Pluripotent
stem cell
CFUGEMM
Mixed myeloid
progenitor cell
Lymphoid
stem cell
CFUbaso
BFUE CFUGMEo
Erythroid CFUEo
progenitors Eosinophil
CFUGM progenitor
CFUMeg Granulocyte
CFUE Megakary- monocyte Thymus
ocyte progenitor
progenitor
CFU-M CFU-G
B T NK
Red Platelets Mono- Neutro- Eosino- Baso- Lymphocytes NK cell
cells cytes phils phils phils
Fig. 1.2 Diagrammatic representation of the bone marrow pluripotent stem cell and the cell lines that arise from it.
Various progenitor cells can be identified by culture in semi-solid medium by the type of colony they form. Baso, basophil;
BFU, burst-forming unit; CFU, colony-forming unit; E, erythroid; Eo, eosinophil; GEMM, granulocyte, erythroid,
monocyte and megakaryocyte; GM, granulocyte, monocyte; Meg, megakaryocyte; NK, natural killer.
EHC01 8/10/06 4:46 PM Page 3
HAEMOPOIESIS 3
Self-renewal
Differentiation
and development
(a)
Mature
cells
Fig. 1.3 (a) Bone marrow cells are increasingly differentiated and lose the capacity for self-renewal as they mature.
(b) A single stem cell gives rise, after multiple cell divisions (shown by vertical lines), to >106 mature cells.
CFU (colony-forming unit)-GEMM (Fig. 1.2). The other cell line when the need arises. The development
bone marrow is also the primary site of origin of of the mature cells (red cells, granulocytes, mono-
lymphocytes (Chapter 8) which differentiate from cytes, megakaryocytes and lymphocytes) is consid-
a common lymphoid precursor. ered further in other sections of this book.
The stem cell has the capability for self-renewal
(Fig. 1.3) so that marrow cellularity remains constant
Bone marrow stroma
in a normal healthy steady state. There is consider-
able amplification in the system: one stem cell is The bone marrow forms a suitable environment
capable of producing about 106 mature blood cells for stem cell survival, growth and development. It is
after 20 cell divisions (Fig. 1.3). The precursor cells composed of stromal cells and a microvascular net-
are, however, capable of responding to haemopoietic work (Fig. 1.4). The stromal cells include adipocytes,
growth factors with increased production of one or fibroblasts, endothelial cells and macrophages and
Stem cell
Extracellular
matrix
Macrophage
Fat cell
Fig. 1.4 Haemopoiesis occurs in a
suitable microenvironment provided
by a stromal matrix on which stem
cells grow and divide. There are Endothelial cell Fibroblast
probably specific recognition and
adhesion sites (p. 11); extracellular Adhesion molecule Ligand
glycoproteins and other compounds
Growth factor Growth factor receptor
are involved in the binding.
EHC01 8/10/06 4:46 PM Page 4
4 CHAPTER 1
they secrete extracellular molecules such as collagen, interactions maintain stem cell viability and pro-
glycoproteins (fibronectin and thrombospondin) duction in the stroma including stem cell factor
and glycosaminoglycans (hyaluronic acid and (SCF) and Jagged proteins expressed on stroma and
chondroitin derivatives) to form an extracellular their respective receptors c-Kit and Notch expressed
matrix. In addition, stromal cells secrete several on stem cell.
growth factors necessary for stem cell survival.
Mesenchymal stem cells are thought to be critical
Stem cell plasticity
in stromal cell formation.
Stem cells are able to traffic around the body and There is some evidence that adult stem cells in
are found in peripheral blood in low numbers. different organs are pluripotent and can generate
In order to exit the bone marrow, cells must cross various types of tissue (Fig. 1.5). Studies in patients
the blood vessel endothelium and this process of and animals who have received haemopoietic stem
mobilization is enhanced by administration of cell transplants (Chapter 21) have suggested that
cytokines such as granulocyte colony-stimulating donor cells may contribute to tissues such as neu-
factor (G-CSF) or granulocyte–macrophage colony- rons, liver and muscle. The contribution of adult
stimulating factor (GM-CSF) (p. 97). The reverse donor bone marrow cells to non-haemopoietic tis-
process of stem cell homing appears to depend on sues is at most small. The persistence of pluripoten-
a chemokine gradient in which the stromal- tial stem cells in postnatal life, organ-specific stem
derived factor (SDF-1) is critical. Several critical cells and fusion of transplanted cells with host cells
Totipotent cell
Myeloid and
lymphoid cells
Liver, etc.
Epithelial
stem cell
Haemopoietic
stem cell
Fig. 1.5 (a) Cells in the early
embryo are able to generate all the
Neural tissues of the body and are known
tissues
Muscle, as totipotent. (b) Specialized adult
tendon, stem cells of the bone marrow,
cartilage, Neural nervous tissue, epithelial and
etc. Mesenchymal stem cell
stem cell other tissues give rise to
differentiated cells of the same
(b) Adult stem cells tissue and possibly to other
tissues (see text).
EHC01 8/10/06 4:46 PM Page 5
HAEMOPOIESIS 5
have all been proposed, however, to explain many of lineage whereas GATA-1 has an essential role in
the findings suggesting stem cell plasticity. erythropoietic and megakaryocytic differentiation.
Early cell
G-CSF
Proliferation
Monocyte
Differentiation
G-CSF
Neutrophil
Suppression
of apoptosis
G-CSF
Fig. 1.6 Growth factors may
stimulate proliferation of early
bone marrow cells, direct
differentiation to one or other cell G-CSF
Maturation
type, stimulate cell maturation,
suppress apoptosis or affect the
function of mature non-dividing
Late cell
cells, as illustrated here for
G-CSF Activation of
granulocyte colony-stimulating Functional phagocytosis,
factor (G-CSF) for an early activation killing, secretion
myeloid progenitor and a
neutrophil.
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6 CHAPTER 1
Table 1.2 General characteristics of myeloid and and thrombopoietin, made largely in the liver. An
lymphoid growth factors. important feature of growth factor action is that two
or more factors may synergize in stimulating a par-
Glycoproteins that act at very low concentrations ticular cell to proliferate or differentiate. Moreover,
Act hierarchically
the action of one growth factor on a cell may stimu-
Usually produced by many cell types
late production of another growth factor or growth
Usually affect more than one lineage
Usually active on stem/progenitor cells and on functional
factor receptor. SCF and Flt ligand (Flt-L) act locally
end cells on the pluripotential stem cells and on early myeloid
Usually show synergistic or additive interactions with and lymphoid progenitors (Fig. 1.7). Interleukin 3
other growth factors (IL-3) and GM-CSF are multipotential growth fac-
Often act on the neoplastic equivalent of a normal cell tors with overlapping activities. G-CSF and throm-
Multiple actions: proliferation, differentiation, bopoietin enhance the effects of SCF, Flt-L, IL-3 and
maturation, functional activation, prevention of GM-CSF on survival and differentiation of the early
apoptosis of progenitor cells haemopoietic cells.
These factors maintain a pool of haemopoietic
stem and progenitor cells on which later acting
Table 1.3 Haemopoietic growth factors.
factors erythropoietin, G-CSF, M-CSF, IL-5 and
Act on stromal cells thrombopoietin act to increase production of one or
IL-1 other cell lineage in response to the body’s need.
TNF Granulocyte and monocyte formation, for example,
can be stimulated by infection or inflammation
Act on pluripotential stem cells
through release of IL-1 and tumour necrosis factor
SCF
Flt-L
(TNF) which then stimulate stromal cells to produce
growth factors in an interacting network (Fig. 7.4).
Act on multipotential progenitor cells In contrast, cytokines such as transforming growth
IL-3
factor-β (TGF-β) and γ-interferon (IFN-γ) can exert
GM-CSF
a negative effect on haemopoiesis and may have a
IL-6
role in the development of aplastic anaemia (p. 244).
G-CSF
Thrombopoietin
HAEMOPOIESIS 7
SCF
PSC
IL-3 IL-3
TPO
CFUGEMM
GM-CSF GM-CSF
BFUEMeg
CFUGMEo
BFUE
IL-5
CFUE CFUM CFUG
Fig. 1.7 A diagram of the role of growth factors in normal haemopoiesis. Multiple growth factors act on the earlier
marrow stem and progenitor cells. EPO, erythropoietin; PSC, pluripotential stem cell; SCF, stem cell factor; TPO,
thrombopoietin. For other abbreviations see Fig. 1.2.
molecules, resulting in their aggregation. Receptor signal for the different processes (e.g. proliferation
aggregation induces activation of the JAKs which or suppression of apoptosis) mediated by growth
now phosphorylate members of the signal trans- factors.
ducer and activator of transcription (STAT) family A second smaller group of growth factors, includ-
of transcription factors. This results in their dimer- ing SCF, Flt-3L and macrophage colony-stimulating
ization and translocation from the cell cytoplasm factor (M-CSF) (Table 1.3), bind to receptors that
across the nuclear membrane to the cell nucleus. have an extracellular immunoglobulin-like domain
Within the nucleus STAT dimers activate transcrip- linked via a transmembrane bridge to a cytoplasmic
tion of specific genes. A model for control of gene tyrosine kinase domain. Growth factor binding
expression by a transcription factor is shown in results in dimerization of these receptors and con-
Fig. 1.9. The clinical importance of this pathway is sequent activation of the tyrosine kinase domain.
revealed by the finding of an activating mutation of Phosphorylation of tyrosine residues in the receptor
the JAK2 gene as the cause of polycythaemia rubra itself generates binding sites for signalling proteins
vera (p. 230). which initiate complex cascades of biochemical
JAK can also activate the MAPK pathway which events resulting in changes in gene expression, cell
is regulated by Ras and controls proliferation. PI3 proliferation and prevention of apoptosis.
kinases phophorylate inositol lipids which have a
wide range of downstream effects including activa-
The cell cycle
tion of AKT (protein kinase) B leading to block of
apoptosis and other actions (Fig. 1.8, 19.2). Different The cell division cycle, generally known simply
domains of the intracellular receptor protein may as the cell cycle, is a complex process that lies at
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8 CHAPTER 1
Growth
factor
Plasma membrane
PI3Kinase
JAK
AKT
JAK
Blocked
STATs RAS
apoptosis
RAF
Nucleus
Active STAT dimers MAP kinase Fig. 1.8 Control of haemopoiesis
by growth factors. The factors act
MYC, FOS
on cells expressing the
corresponding receptors. Binding
M Gene
of a growth factor to its receptor
expression
activates the JAK/STAT, MAPK
Activation of
gene expression and hosphatidyl-inositol3-kinase
(PI3K) pathways (Fig. 19.2) which
leads to transcriptional activation
G2 G1
of specific genes. E2F is a
transcription factor needed for
cell transition from G1 to S phase.
E2F is inhibited by the tumour
E2F suppressor gene Rb
S
(retinoblastoma) which can be
Rb indirectly activated by p53. The
synthesis and degradation of
different cyclins (not shown)
p53 stimulates the cell to pass through
the different phases of the cell
DNA damage cycle. The growth factors may
also suppress apoptosis by
activating protein kinase B.
HAEMOPOIESIS 9
the heart of haemopoiesis. Dysregulation of cell division is accomplished, and cytokinesis in which
proliferation is also the key to the development of cell fission occurs.
malignant disease. The duration of the cell cycle Interphase is divided into three main stages: a
is variable between different tissues but the basic G1 phase in which the cell begins to commit to
principles remain constant. The cycle is divided in replication, an S phase during which DNA content
to the mitotic phase (M phase), during which the cell doubles (Fig. 1.10b) and the chromosomes replicate
physically divides, and interphase during which the and the G2 phase in which the cell organelles are
chromosomes are duplicated and cell growth occurs copied and cytoplasmic volume is increased. If cells
prior to division (Fig. 1.10). The M phase is further rest prior to division they enter a G0 state where they
partitioned into classical mitosis in which nuclear can remain for long periods of time. The number
of cells at each stage of the cell cycle can be assessed
by exposing cells to a chemical or radiolabel that
gets incorporated into newly generated DNA or by
M phase
flow cytometry.
The cell cycle is controlled by two checkpoints
which act as brakes to coordinate the division
process at the end of the G1 and G2 phases. Two
M G0 major classes of molecules control these check-
Cdk2 G2 G1 points, cyclin dependent protein kinases (Cdk) which
Cdk2
phosophorylate downstream protein targets and
Cyclin
Cyclin cyclins which bind to Cdks and regulate their activ-
B
S E
ity. An example of the importance of these systems
Cyclin is demonstrated by mantle cell lymphoma which
Cdk2 A
results from the constitutive activation of cyclin D1
Interphase as a result of a chromosomal translocation (p. 212).
(a)
Apoptosis
4c Apoptosis is a regulated process of physiological
DNA content
10 CHAPTER 1
pathway is via the release of cytochrome c from example is the translocation of the BCL-2 gene to
mitochondria. Cytochrome c binds to Apaf-1 which the immunoglobulin heavy chain locus in the
then activates caspases. DNA damage induced by t(14; 18) translocation in follicle centre lymphoma.
irradiation or chemotherapy may act through this Overexpression of the BCL-2 protein makes the
pathway. The protein p53 has an important role in malignant B cells less susceptible to apoptosis.
sensing DNA damage. It activates apoptosis by Apoptosis is the normal fate for most B cells
raising the cell level of BAX which then increases undergoing selection in the lymphoid germinal
cytochrome c release (Fig. 1.11). P53 also shuts centres.
down the cell cycle to stop the damaged cell from Several translocations leading to the generation of
dividing (Fig. 1.8). The cellular level of p53 is rigidly fusion proteins such as t(9; 22), t(1; 14) and t(15; 17)
controlled by a second protein MDM2. Following also result in inhibition of apoptosis (Chapter 10). In
death, apoptotic cells display molecules that lead to addition, genes encoding proteins that are involved
their ingestion by macrophages. in mediating apoptosis following DNA damage, such
As well as molecules that mediate apoptosis as p53 and ATM, are also frequently mutated and
there are several intracellular proteins that protect therefore inactivated in haemopoietic malignancies.
cells from apoptosis. The best characterized exam-
ple is BCL-2. BCL-2 is the prototype of a family of
Transcription factors
related proteins, some of which are anti-apoptotic
and some, like BAX, pro-apoptotic. The intracellular Transcription factors regulate gene expression by
ratio of BAX and BCL-2 determines the relative controlling the transcription of specific genes or
susceptibility of cells to apoptosis and may act gene families. Typically, they contain at least two
through regulation of cytochrome c release from domains: a DNA-binding domain such as a leucine
mitochondria. zipper or helix-loop-helix motif which binds to a
Many of the genetic changes associated with specific DNA sequence, and an activation domain
malignant disease lead to a reduced rate of apopto- which contributes to assembly of the transcription
sis and hence prolonged cell survival. The clearest complex at a gene promoter.
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HAEMOPOIESIS 11