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6 HAEMATOPOIESIS Dr.Michael Bennett March 2007

7 HAEMATOPOIESIS RED CELLS ERYTHROPOIESIS GRANULOCYTES MYELOPOIESIS
MONOCYTES PLATELETS THROMBOPOIESIS

8 HAEMATOPOIESIS Examination of the blood Examination of the bone marrow
Stem cells Conditions for haematopoiesis Bone marrow stroma Adhesion molecules Haemopoietic growth factors Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

9 HAEMATOPOIESIS Examination of the blood Examination of the bone marrow
Stem cells Conditions for haematopoiesis Bone marrow stroma Adhesion molecules Haemopoietic growth factors Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

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14 CAUSES RED CELL normal Liver disease, alcoholism,
ABNORMALITIES normal Liver disease, alcoholism, Oval in megaloblastosis macrocyte Iron deficiency,liver disease, haemoglobinopathies, post splenectomy Target cell Liver disease, alcoholism stomatocyte Iron deficiency Pencil cell Liver disease, post splenectomy echinocyte Liver disease, abetalipo-proteinaemia, renal failure acanthocyte

15 CAUSES RED CELL ABNORMALITIES Hereditary spherocytosis, autoimmune haemolytic anaemia, septicaemia microspherocyte DIC, HUS, TTP,cardiac valves fragments Hereditary elliptocytosis elliptocyte myelofibrosis tear drops Oxidant damage eg G6PD deficiency basket cell Sickle cell disease Sickle cell Iron deficiency, thalassemia microcyte

16 MEAN LIFE SPAN Red cells days Platelets days Neutrophils 2-4 days Monocytes months Lymphocytes months

17 SITE OF HAEMOPOIESIS YOLK SAC up to 6 weeks
FOETAL LIVER up to 6-7 months and SPLEEN BONE MARROW from 6-7 months

18 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

19 HAEMATOPOIESIS Examination of the blood Examination of the bone marrow
Stem cells Conditions for haematopoiesis Bone marrow stroma Adhesion molecules Haemopoietic growth factors Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

20 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

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23 MARROW ASPIRATE MARROW TREPHINE

24 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

25 Bone Marrow Differential %

26 ERYTHROBLAST

27 NORMOBLASTIC ERYTHROPOIESIS
polychromatic erythroblast proerythroblast pyknotic erythroblast basophilic erythroblast

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30 MYELOPOIESIS promyelocyte metamyelocyte polymorph myelocyte myeloblast
stab indentation of nucleus primary granulation secondary granulation no nucleolus immature nucleolus no granulation

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37 EOSINOPHIL MYELOCYTE EOSINOPHIL

38 BASOPHIL MYELOCYTE BASOPHIL

39 Segmented neutrophils
Myelopoiesis Cirulating neutrophils Marginating neutrophils Stem cells Progenitor cells Metamyelocytes Bands Segmented neutrophils Myeloblasts promyelocytes myelocytes Mitotic pool Post-mitotic pool bone marrow blood 6-10 days 6-10 hrs

40 MEGAKARYOCYTES PLATELETS

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45 MONOBLASTS PROMONOCYTES MONOCYTES

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48 monocyte migration sites

49 LYMPHOBLASTS LYMPHOCYTES

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51 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

52 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.

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55 HAEMATOPOIESIS Examination of the blood Examination of the bone marrow
Stem cells Conditions for haematopoiesis Bone marrow stroma Adhesion molecules Haemopoietic growth factors Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

56 PLURIPOTENT STEM CELLS
LINEAGE COMMITTED PROGENITOR CELLS RECOGNIZABLE MARROW PRECURSORS

57 PROGENITOR CELLS endothelial cells HSC Hematopoietic stem cell
hemangioblast CLP CMP MEP GMP GP MonoP MKP EP grans monos Plts RBC

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59 ERYTHROID BURST

60 MARROW CULTURE : GE AND EOSINOPHIL COLONY

61 CD38- CD34+ HAEMOPOIETIC STEM CELL 1 in 20x106 Nucleated cells
in marrow Capacity for Renewal Differentiation After 20 divisions 106 mature cells are formed Found also in peripheral blood

62 Stem cells may be totipotent and generate all tissues of the body
STEM CELL PLASTICITY Stem cells may be totipotent and generate all tissues of the body

63 HAEMATOPOIESIS Examination of the blood Examination of the bone marrow
Stem cells Conditions for haematopoiesis Bone marrow stroma Adhesion molecules Haemopoietic growth factors Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

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65 STROMAL CELLS MACROPHAGES FAT CELLS ENDOTHELIAL CELLS FIBROBLASTS

66 EXTRACELLULAR MATRIX FIBRONECTIN HAEMONECTIN LAMININ COLLAGEN
PROTEOGLYCANS eg heparan, chondroitin

67 ADHESION MOLECULES Glycoprotein molecules that mediate attachment of haemopoietic cells, leucocytes and platelets to the extracellular matrix, endothelium and each other.

68 ADHESION MOLECULES These molecules on leucocytes are termed receptors and they bind to ligands on surfaces of target cells

69 EXPRESSION OF ADHESION MOLECULES
MAY BE MODIFIED QUANTITATIVELY OR FUNCTIONALLY BY INTRA OR EXTRACELLULAR FACTORS eg upregulation by IL1,TNF, INF-γ, viral infection

70 IMPORTANCE OF ADHESION MOLECULES
INFLAMMATORY AND IMMUNE RESPONSES PLATELET AND LEUCOCYTE VESSEL WALL INTERACTIONS ON TUMOUR CELLS – MODE OF SPREAD AND TISSUE LOCALISATION

71 ADHESION MOLECULES Immunoglobulin superfamily – Antigen receptors, T-cell receptors, growth factor receptors Selectins – attach leucocytes and platelets to vessels walls Integrins – mediate cell adhesion during migration and signals for growth and development Cadherins Syndecams ADAMs

72 HAEMOPOIETIC GROWTH FACTORS
ERYTHROPOIETIN 6. THROMBOPOIETIN G-CSF INTERLEUKINS GM-CSF TNF M-CSF Flt LIGAND SCF INF   

73 HAEMOPOIETIC GROWTH FACTORS
general properties Glycoproteins Act locally where produced or systemically Act at low concentrations Specific receptors on target cells Produced by many cell types Synergistic effects 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

74 GROWTH FACTORS IN NORMAL HAEMATOPOIESIS
SCF GM-CSF GM-CSF IL-5

75 ERYTHROPOIETIN Source : Kidney and liver 34 kd glycoprotein
Lineage specific Level regulated by a simple feedback mechanism in response to oxygen tension Uses: 1. Anaemia of chronic renal failure 2. AIDS HIV infection 3. Cancer patients receiving chemotherapy 4. MDS 5. Autologous blood transfusion

76 Peritubular Interstitial cells of outer cortex Peritubular Interstitial cells of outer cortex

77 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 Post chemotherapy APPLICATIONS After BMT 3. collection of stem cells from PB 4. Kostman’s syndrome Causes bone pain

78 Originally cloned as the ligand
THROMBOPOIETIN Originally cloned as the ligand for the c-mpl receptor

79 THROMBOPOIETIN ACTIVITY lineage dominant factor megakaryocyte
progenitor cell proliferation, maturation and platelet production. Affects also erythropoiesis and pluripotent stem cell proliferation

80 LEVELS OF THROMBOPOIETIN (TPO) INVERSELY RELATED TO PLATELET COUNTS

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82 THROMBOPOIETIN APPICATIONS
not yet established a single dose may increase platelets from day 4, peaking even after day 8 half life hours

83 HAEMATOPOIESIS Examination of the blood Examination of the bone marrow
Stem cells Conditions for haematopoiesis Bone marrow stroma Adhesion molecules Haemopoietic growth factors Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

84 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.

85 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).

86 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.

87 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.

88 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.

89 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.

90 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.

91 G1 & G2 CHECKPOINTS CONTROLLED BY
Cyclin dependent protein kinases (Cdk) that phosphorylate downstream protein targets 2. Cyclins which regulate Cdks

92 THE CELL CYCLE

93 APOPTOSIS Programmed cell death

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95 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

96 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

97 MITOCHONDRIAL PATHWAY INTRINSIC
Mitochondria release cytochrome C cytochrome C binds to Apaf-1 aggregate to form apoptosomes Activate caspase-9

98 p53 induces the transcription of BAX
Promotes apoptosis causes holes in the mitochondrial membrane and cytochrome C to leak out p53 induces the transcription of BAX after DNA damage

99 Cell death CD95L CD95 Active Caspase Death Domain Procaspase

100 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)

101 IMPORTANCE OF APOPTOSIS
Normal modeling and embryonal development Removing calls with DNA damage, viral infection, cancer cells Cellular immune function

102 LACK OF APOPTOSIS IN CANCER
Bcl-2 over expression in follicular lymphoma p53 mutation or lack leads to a poor prognosis in leukemia

103 SIGNAL TRANSDUCTION

104 cells respond to external stimuli
CELL SIGNALING cells respond to external stimuli and respond by : Changes in metabolism Changes in electrical charge across plasma membrane Changes in gene expression

105 SIGNAL (Ligand) RECEPTOR SIGNAL TRANSDUCTION NUCLEUS GENE EXPRESSION

106 3 major pathways in signal transduction
JAK/STAT MAP kinase (mitogen activated protein) PI3 kinase (phosphatidylinositol 3) All involve phosphorylation of tyrosine residues

107 JAK kinase = Just Another Kinase

108 JANUS god of gates and pathways

109 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

110 Signal Transducers and Activators of Transcription

111 STRUCTURE OF JAK2 Four-point-one Ezrin Radixin Moesin domain

112 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.

113 The juxtaposition of the 2 tethered JAK2 kinases also allows cross activation and initiation of signal transduction by phosphorylation of : tyrosine residues within the receptor Molecules that promote cell survival and proliferation eg STATs ,PI3K, Mapks. Molecules that limit cell signaling eg SHP1, SHIP1 phosphatases

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115 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

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117 THE END


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