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HAEMATOPOIESIS
Dr.Michael Bennett
March 2007
HAEMATOPOIESIS
RED CELLS
GRANULOCYTES
MONOCYTES
PLATELETS
ERYTHROPOIESIS
MYELOPOIESIS
THROMBOPOIESIS
HAEMATOPOIESIS
1.
Examination of the blood
2.
Examination of the bone marrow
3.
Stem cells
4.
a.
b.
c.
Conditions for haematopoiesis
Bone marrow stroma
Adhesion molecules
Haemopoietic growth factors
5.
a.
b.
c.
Intracellular molecular mechanisms
The cell cycle
Apoptosis
Signal transduction
HAEMATOPOIESIS
1.
Examination of the blood
2.
Examination of the bone marrow
3.
Stem cells
4.
a.
b.
c.
Conditions for haematopoiesis
Bone marrow stroma
Adhesion molecules
Haemopoietic growth factors
5.
a.
b.
c.
Intracellular molecular mechanisms
The cell cycle
Apoptosis
Signal transduction
Normal blood count
Haemoglobin (Hb)
male
female
male
female
male
female
13.5 - 17.5 g/dl
11.5 - 15.5 g/dl
Red cells (RBC; erythrocytes)
4.5 - 6.5 x 1012/l
3.9 - 5.6 x 1012/l
Packed cell volume (PCV;haematocrit)
40 - 52%
36 - 48%
Mean corpuscular volume (MCV)
76 - 96 fl
Mean corpuscular haemoglobin(MCH)
27 - 34 pg
Mean corpuscular haemoglobin concentration (MCHC)
30 - 35 g/dl
Reticulocytes
0.5 - 2.0%
White cells (WBC; leucocytes)
total
4.0 - 11.0 x 109/l
neutrophils 2.5 - 7.5 x 109/l
lymphocytes 1.5 - 3.5 x 109/l
monocytes 0.2 - 0.8 x 109/l
eosinophils 0.04 - 0.44 x 109/l
basophils
0.01 - 0.1 x 109/l
Platelets (thrombocytes)
150 - 400 x 109/l
RED CELL
ABNORMALITIES
CAUSES
normal
macrocyte
Liver disease, alcoholism,
Oval in megaloblastosis
Target cell
Iron deficiency,liver disease,
haemoglobinopathies, post splenectomy
stomatocyte
Liver disease, alcoholism
Pencil cell
Iron deficiency
echinocyte
Liver disease, post splenectomy
acanthocyte
Liver disease, abetalipo-proteinaemia, renal
failure
RED CELL
ABNORMALITIES
CAUSES
microspherocyte
Hereditary spherocytosis, autoimmune
haemolytic anaemia, septicaemia
fragments
DIC, HUS, TTP,cardiac valves
elliptocyte
Hereditary elliptocytosis
tear drops
myelofibrosis
basket cell
Oxidant damage eg G6PD deficiency
Sickle cell
Sickle cell disease
microcyte
Iron deficiency, thalassemia
MEAN LIFE SPAN
Red cells
120 days
Platelets
8-10 days
Neutrophils 2-4 days
Monocytes months
Lymphocytes months
SITE OF HAEMOPOIESIS
YOLK SAC
FOETAL LIVER
and SPLEEN
BONE MARROW
up to 6 weeks
up to 6-7 months
from 6-7 months
BONE MARROW
Haematopoietic (Red Marrow)
all bones at birth
adults – axial skeleton and proximal
long bones
Fatty marrow (Yellow Marrow)
begins to replace red marrow
from 5-7 years
HAEMATOPOIESIS
1.
Examination of the blood
2.
Examination of the bone marrow
3.
Stem cells
4.
a.
b.
c.
Conditions for haematopoiesis
Bone marrow stroma
Adhesion molecules
Haemopoietic growth factors
5.
a.
b.
c.
Intracellular molecular mechanisms
The cell cycle
Apoptosis
Signal transduction
BONE MARROW
Haematopoietic (Red Marrow)
all bones at birth
adults – axial skeleton and proximal
long bones
Fatty marrow (Yellow Marrow)
begins to replace red marrow
from 5-7 years
MARROW TREPHINE
MARROW ASPIRATE
CELLS IN THE BONE MARROW
1. MYELOID PRECURSORS for neutrophils,
eosinophils, basophils and monocytes
2. ERYTHROID PRECURSORS
3. MEGAKARYOCYTES
4. LYMPHOCYTES
5. PLASMA CELLS
6. RETICULOENDOTHELIAL CELLS
7. FAT CELLS
8. OSTEOBLASTS
9. OSTEOCLASTS
Bone Marrow Differential %
Blasts
Promyelocytes
Neutrophil Myelocytes
Eosinophil Myelocytes
Metamyelocytes
Neutrophils
Eosinophils
Basophils
Erythroblasts
Lymphocytes
Plasma Cells
Monocytes
Macrophages
Myeloid : Erythroid ratio
95% range
0.0 - 3.0
3.2 - 12.4
3.7 - 10
0 - 2.8
2.5 - 5.9
21.9 -49.2
0.3 -4.2
0 - 0.4
13.0 - 40.1
6.0 - 20.0
0 - 1.2
0 - 2.6
0 - 1.8
1.1 - 5.2
mean
1.4
7.8
7.6
1.3
4.1
34.8
2.2
0.1
25.3
13.1
0.6
1.3
0.4
2.5
ERYTHROBLAST
NORMOBLASTIC ERYTHROPOIESIS
proerythroblast
basophilic
erythroblast
polychromatic
erythroblast
pyknotic
erythroblast
MYELOPOIESIS
myeloblast
promyelocyte
metamyelocyte
myelocyte
primary
secondary indentation
immature granulation granulation of nucleus
nucleolus
no nucleolus
no granulation
polymorph
stab
EOSINOPHIL
EOSINOPHIL
MYELOCYTE
BASOPHIL
MYELOCYTE
BASOPHIL
Myelopoiesis
Cirulating
neutrophils
Stem
cells
Marginating
neutrophils
Progenitor
cells
Myeloblasts
promyelocytes
myelocytes
Mitotic pool
Metamyelocytes
Bands
Segmented neutrophils
Post-mitotic pool
bone marrow
6-10 days
blood
6-10 hrs
MEGAKARYOCYTES
PLATELETS
MONOBLASTS
PROMONOCYTES
MONOCYTES
monocyte
migration
sites
LYMPHOBLASTS
LYMPHOCYTES
PERIPHERAL BLOOD LYMPHOCYTES
A. T CELLS (65-80%) CD2 POS
CD4 helper - majority in PB
CD8 suppressor/cytotoxic - majority in BM
B. B CELLS (5-15%) CD 19, 20, 22 POS
endogenous Ig molecules on surface membrane
C. Natural Killer Cells
kill target cells without MHC restriction
LYMPHOCYTE PRODUCTION
Although marrow is a major site of “virgin” lymphocyte
production, most circulating cells are produced in lymphoid
tissue such as lymph nodes, spleen, thymus and lymphoid
tissue of the GI and respiratory tracts.
Lymphoblasts are difficult to differentiate from other
blast cells.
HAEMATOPOIESIS
1.
Examination of the blood
2.
Examination of the bone marrow
3.
Stem cells
4.
a.
b.
c.
Conditions for haematopoiesis
Bone marrow stroma
Adhesion molecules
Haemopoietic growth factors
5.
a.
b.
c.
Intracellular molecular mechanisms
The cell cycle
Apoptosis
Signal transduction
PLURIPOTENT STEM CELLS
LINEAGE COMMITTED
PROGENITOR CELLS
RECOGNIZABLE
MARROW PRECURSORS
PROGENITOR
CELLS
endothelial cells
HSC
hemangioblast
Hematopoietic stem cell
CLP
CMP
MEP
MKP
Plts
EP
RBC
GMP
GP
grans
MonoP
monos
ERYTHROID BURST
MARROW CULTURE : GE AND EOSINOPHIL COLONY
HAEMOPOIETIC STEM CELL
CD38- CD34+
1 in 20x106
Nucleated cells
in marrow
Capacity for
Renewal
Differentiation
After 20 divisions
106 mature cells
are formed
Found also in
peripheral blood
STEM CELL PLASTICITY
Stem cells may be totipotent and generate all
tissues of the body
HAEMATOPOIESIS
1.
Examination of the blood
2.
Examination of the bone marrow
3.
Stem cells
4.
a.
b.
c.
Conditions for haematopoiesis
Bone marrow stroma
Adhesion molecules
Haemopoietic growth factors
5.
a.
b.
c.
Intracellular molecular mechanisms
The cell cycle
Apoptosis
Signal transduction
STROMAL CELLS
1. MACROPHAGES
2. FAT CELLS
3. ENDOTHELIAL CELLS
4. FIBROBLASTS
EXTRACELLULAR MATRIX
1.
2.
3.
4.
5.
FIBRONECTIN
HAEMONECTIN
LAMININ
COLLAGEN
PROTEOGLYCANS eg heparan, chondroitin
ADHESION MOLECULES
Glycoprotein molecules that mediate
attachment of haemopoietic cells,
leucocytes and platelets to the
extracellular matrix, endothelium and
each other.
ADHESION MOLECULES
These molecules on leucocytes are termed
receptors and they bind to ligands on
surfaces of target cells
EXPRESSION OF
ADHESION MOLECULES
MAY BE MODIFIED QUANTITATIVELY OR
FUNCTIONALLY BY INTRA OR
EXTRACELLULAR FACTORS
eg upregulation by IL1,TNF, INF-γ, viral
infection
IMPORTANCE OF
ADHESION MOLECULES
1.INFLAMMATORY AND IMMUNE
RESPONSES
2.PLATELET AND LEUCOCYTE VESSEL WALL
INTERACTIONS
3.ON TUMOUR CELLS – MODE OF SPREAD
AND TISSUE LOCALISATION
ADHESION MOLECULES
1. Immunoglobulin superfamily – Antigen receptors, T-cell
receptors, growth factor receptors
2. Selectins – attach leucocytes and platelets to vessels
walls
3. Integrins – mediate cell adhesion during migration and
signals for growth and development
4. Cadherins
5. Syndecams
6. ADAMs
HAEMOPOIETIC GROWTH FACTORS
1. ERYTHROPOIETIN
2. G-CSF
3. GM-CSF
4. M-CSF
5. SCF
6. THROMBOPOIETIN
7. INTERLEUKINS
8. TNF
9. Flt LIGAND
10. INF   
HAEMOPOIETIC GROWTH FACTORS
general properties
1. Glycoproteins
2. Act locally where produced or systemically
3. Act at low concentrations
4. Specific receptors on target cells
5. Produced by many cell types
6. Synergistic effects
7. Action of one factor may stimulate production of
another growth factor or receptor
8. May effect more than one lineage
9. Multiple actions - self renewal of stem cells,
differentiation of lineage-committed progenitor cells,
maturation, functional activation, prevention of apoptosis
GROWTH FACTORS IN NORMAL HAEMATOPOIESIS
SCF
GM-CSF
GM-CSF
IL-5
ERYTHROPOIETIN
Source : Kidney and liver
34 kd glycoprotein
Lineage specific
Level regulated by a simple feedback mechanism in
response to oxygen tension
Uses: 1.
2.
3.
4.
5.
Anaemia of chronic renal failure
AIDS HIV infection
Cancer patients receiving chemotherapy
MDS
Autologous blood transfusion
Peritubular
Interstitial
cells of
outer cortex
Peritubular
Interstitial
cells of
outer cortex
G-CSF
SOURCE : stromal cells
endothelial cells
monocytes and macrophages
ACTIVITY : Induces proliferation and maturation
of neutrophil progenitors
Mobilises myeloid stem cells into PB
Activates neutrophil function and prolongs
their life span
CLINICAL
APPLICATIONS
Causes bone pain
1. Post chemotherapy
2. After BMT
3. collection of stem cells from PB
4. Kostman’s syndrome
THROMBOPOIETIN
Originally cloned as the ligand
for the c-mpl receptor
THROMBOPOIETIN
ACTIVITY
lineage dominant factor megakaryocyte
progenitor cell proliferation, maturation
and platelet production. Affects also
erythropoiesis and pluripotent stem cell
proliferation
LEVELS OF
THROMBOPOIETIN
(TPO) INVERSELY
RELATED TO
PLATELET COUNTS
THROMBOPOIETIN
APPICATIONS
not yet established a single dose may increase
platelets from day 4, peaking even after day 8
half life 20-30 hours
HAEMATOPOIESIS
1.
Examination of the blood
2.
Examination of the bone marrow
3.
Stem cells
4.
a.
b.
c.
Conditions for haematopoiesis
Bone marrow stroma
Adhesion molecules
Haemopoietic growth factors
5.
a.
b.
c.
Intracellular molecular mechanisms
The cell cycle
Apoptosis
Signal transduction
The Cell Cycle
During development from stem to fully differentiated, cells in
the body alternately divide (mitosis) and "appear" to be resting
(interphase). This sequence of activities exhibited by cells is
called the cell cycle.
Interphase, which appears to the eye to be a resting stage
between cell divisions, is actually a period of diverse activities.
Those interphase activities are indispensible in making the next
mitosis possible.
Interphase
Lasts at least 12 to 24 hours in
mammalian tissue. During this period,
the cell is constantly synthesizing RNA,
producing protein and growing in size. It
can be divided into 4 steps: Gap 0 (G0),
Gap 1 (G1), S (synthesis) phase, Gap 2
(G2).
Gap 0 (G0)
There are times when a cell will leave
the cycle and quit dividing. This may be a
temporary resting period or more
permanent. An example of the latter is a
cell that has reached an end stage of
development and will no longer divide.
Gap 1 (G1)
Cells increase in size in Gap 1, produce
RNA and synthesize protein. An
important cell cycle control
mechanism activated during this
period (G1 Checkpoint) ensures that
everything is ready for DNA
synthesis.
S Phase
To produce two similar daughter
cells, the complete DNA
instructions in the cell must be
duplicated. DNA replication occurs
during this S (synthesis) phase.
Gap 2 (G2)
During the gap between DNA synthesis
and mitosis, the cell will continue to
grow and produce new proteins. At the
end of this gap is another control
checkpoint (G2 Checkpoint) to
determine if the cell can now proceed
to enter M (mitosis) and divide.
Mitosis or M Phase
Orderly division into two similar
daughter cells.
Mitosis is much shorter than interphase,
lasting one to two hours. As in both G1
and G2, there is a Checkpoint in the
middle of mitosis (Metaphase
Checkpoint) that ensures the cell is
ready to complete cell division.
G1 & G2 CHECKPOINTS
CONTROLLED BY
1. Cyclin dependent protein kinases (Cdk)
that phosphorylate downstream
protein targets
2. Cyclins which regulate Cdks
THE CELL CYCLE
APOPTOSIS
Programmed cell death
APOPTOSIS
CASPASES
Cysteinyl Aspartate-Specific Proteases
Many caspase isoforms promote apoptosis
activated by two main pathways:
1.the death receptor pathway
2. the mitochondrial pathway
DEATH RECEPTOR PATHWAY
EXTRINSIC
Fas (CD-95) and TNF receptor
are membrane proteins exposed at the surface of cells
Binding of FasL and TNF
results in the formation of a death inducing signalling
complex (DISC) which contains Fas-associated death
domain protein
MITOCHONDRIAL PATHWAY
INTRINSIC
Mitochondria release cytochrome C
cytochrome C binds to Apaf-1
aggregate to form apoptosomes
Activate caspase-9
BAX
Promotes apoptosis
causes holes in the mitochondrial
membrane and cytochrome C to leak
out
p53 induces the transcription of BAX
after DNA damage
CD95L
Cell death
CD95
Active
Caspase
Procaspase
Death
Domain
Bcl-2
Anti-apoptotic
stabilise mitochrondrial membrane
Increased expression of the Bcl-2 gene
may lead to neoplasia eg in follicular
lymphoma t(14;18)
IMPORTANCE OF
APOPTOSIS
Normal modeling and embryonal development
Removing calls with DNA damage, viral
infection, cancer cells
Cellular immune function
LACK OF APOPTOSIS IN CANCER
Bcl-2 over expression in follicular lymphoma
p53 mutation or lack leads to a poor prognosis in
leukemia
SIGNAL TRANSDUCTION
CELL SIGNALING
cells respond to external stimuli
and respond by :
1. Changes in metabolism
2. Changes in electrical charge across
plasma membrane
3. Changes in gene expression
SIGNAL (Ligand)
RECEPTOR
SIGNAL TRANSDUCTION
NUCLEUS
GENE EXPRESSION
3 major pathways
in signal transduction
1. JAK/STAT
2. MAP kinase (mitogen activated protein)
3. PI3 kinase (phosphatidylinositol 3)
All involve phosphorylation of tyrosine
residues
JAK kinase
=
Just Another Kinase
JANUS god of gates and pathways
Type I cytokine receptors
eg for Epo, G-CSF, GM-CSF,TPO, IL
lack a cytoplasmic tyrosine kinase
domain
Use the JAK-STAT pathway to
initiate signalling
Signal
Transducers
and
Activators
of Transcription
STRUCTURE OF JAK2
Four-point-one
Ezrin
Radixin
Moesin domain
Epo R
• exists in a homodimeric state in the absence of
ligand with the cytoplasmic domains 73 Å apart
• JAK bound to receptor by FERM domain to box 1
in a conformation that JH2 inhibits JH1
• upon ligand binding – conformational shift
bringing the cytoplasmic domains 39 Å apart
• The tethered JAK2 also undergoes a
conformational change which results in a loss of
apposition of JH2 to JH1 allowing its kinase
activity.
The juxtaposition of the 2 tethered JAK2
kinases also allows cross activation and
initiation of signal transduction by
phosphorylation of :
1. tyrosine residues within the receptor
2. Molecules that promote cell survival and
proliferation eg STATs ,PI3K, Mapks.
3. Molecules that limit cell signaling eg
SHP1, SHIP1 phosphatases
JAK2 (V617F)
Somatic
Does not occur in normal population
Polycythemia Vera 90-95%
Essential Thrombocythemia 50-70%
Idiopathic Myelofibrosis 40-50%
Rare in other hematological conditions
THE END