Prasit Chanarat Clinical microscopy AMS. CMU.

HEMOPOIESIS

Objectives 1. Describe the ontogeny of hemopoiesis

2. Discuss the theories of hemopoiesis 3. Discuss the totipotential stem cells, committed stem cells, progenitor sells, precursor cells 4. Discuss a regulation of hemopoiesis and hemopoietic proliferation 5. Describe composition and function of blood

Whole Blood

• Fluid - Plasma • Cell - WBC (Leukocytes) - Platelets (Thrombocytes) - RBC (Erythrocytes)• Clotted blood ----- Serum• Anticoagulated blood ----- Plasma• Fibrinogen ----(thrombin)-> Fibrin• Antocoagulants : EDTA, Heparin• EDTA : removes Ca+2

• Heparin : neutralizes thrombin

Plasma

• Protein : Albumin, Globulin (alpha-1, alpha-2, Beta, gamma), fibrinogen, clotting factors (Coagulation factors)• Amino acids, peptides, glucose, lipids, lactic acid, enzymes, vitamins, hormones, BUN, uric acid, creatinine, electrolytes (Na+, K+, Ca+2, Mg+2, Ci-, HCO

3-, SO4

-2, etc.)

Hemopoiesis

Hemapoiesis (Hematopoiesis) Hemo = Hemato = Blood Poiesis = Production

Hemopoiesis system Bone marrow, spleen, liver, thymus, lymphnode

Hematopoiesis Production and

Development of Blood Cells Pluripotent Stem Cell - Continuous Self-

Replication- Gives Rise to All Blood Cell Lines

Growth Factors (Cytokines) - Promote Differentiation Into Mature Blood Elements

Hemopoiesis Phases

1. Embryonic or prenatal phase - mesoblastic peroid - hepatic period - myeloid period

2. Post-natal phase - medullary hemopoiesis - extramedullary hemopoiesis

Hemopoiesis

PHSC CSC Diff. Compartment Circulation

<------ Proliferation ---------------------> Differentiation --------------------> Maturation -----------------------> Release ------------------------->

PHSC = Pluripotent hemopoietic stem cellsCSC = Committed stem cells

Theories of Hemopoiesis

I. Monophyletic theory Totipotential stem cells II Polyphyletic theory Dualists Trialists Polyphyletists

Stem Cells and theirTherapeutic Potential

What are “stem cells”?

Stem cells

Embryonic Adult

The defining properties of a stem cell

- It is not terminally differentiated.

- It can divide without limit.

- When it divides, the daughter cell has 2 “choices”:

• Remain a stem cell.

• Terminally differentiate.

Varying Potentials of Stem Cells:

– Totipotent cells. Fertilized oocyte (zygote) & progeny of the first two cell divisions. Cells able to form the whole organism.

– Pluripotent cells. Obtained from the inner cell mass of the blastocyst, able to differentiate into almost all cells of the three germ layers – but not into an embryo.

– Multipotential cells. Found in most tissues, these cells can produce a limited range of differentiated cell lineages appropriate to their location (e.g., Hematopoietic stem cells from the bone marrow).

– Unipotential cells. Cells capable of generating only one cell type (epidermal stem cells, adult liver stem cells).

Leukopoiesis

Objective:

1. Classification of WBC

2 . Leukopoiesis

2 1. Granulopoiesis

2 2. Monopoiesis

2 3. Lymphopoiesis

2 4. P lasmopoiesis

3. Factors affecting leukopoiesis

Classification of WBC

1. Shape of the nucleus

11. mononuclear : Lymphocyte, Monocyte

1 2. Polymorphonuclear : PMN, Eos, Baso

2. Granules

2 1. Granulocytes : PMN, Eos, Baso

22. Agranulocyte : Lymphocyte, Monocyte

3. Site of origin

Myeloid cells / Lymphoid cells

4. Function / Phagocyte :

- Macrophage Monocyte

- Microphage PMN

Leukopoiesis Study

1. Morphology

2 . Biochemical markers

3 . Immunological markers

4 . Gene markers

Stage of Granulopoiesis

Stem cell

Myeloblast

Promyelocyte

Basophilic Neutrophilic Eosinophilic

Myelocyte Myelocyte Myelocyte

Metamyelocyte Metamyelocyte Metamyelocyte

Band Band Band

Basophil Neutrophil (PMN) Eosinophil

Regulation of Neutrophil Production

Bone Marrow Blood stream Tissue

Proliferatrion Circulating Move freely

compartment compartment

Storage Marginating Move

compartment compartment restricted

(( ((( (( ((((((((((

( (((((( (((((((

Neutropenia : PMN decrease

Neutrophilia : PMN increase

- -Cytokine : Multi CSF (IL 3)

-GM CSF

-G CSF

-(6

Granulopoietin (Granulocytic

growth factor)

Eosinophilia : Eos. increase

-Cytokine : IL 3

-5IL

( ( -( ((

- ECF A (Eosinophilic chemotactic factor of anaphylaxis)

((((( ((((((((: .

-Cytokine : GM CSF

-4IL

-9IL

Monopoiesis

Stem cell

Monoblast

Promonocyte

Monocyte

Monocytosis : Monocyte increase

-Cytokine : IL 3

-GM CSF

-M CSF

Tissue macrophage

Osteoclast : Bone

Macrophage : Lymphoid tissue

( (((((( (((( ( ((((( Microglia cell : Brain

Serosal macrophage : Pleural cavity

Alveolar macrophage : Lung

Lymphopoiesis

Within thymus : is not antigen dependent

• Within lymphnode / spleen : mediated by

antigen stimulation

- molecular nature of antigen

- antigen dose

- circulating antibody levels

- handling ofantigen by RE cells

- - proliferation of thymus derived cells

- thymic humoral factors

Central Lymphoid Peripheral Lymphoid

(((( (( (-((( (( ( ( (

Stem Th Ts

cel l s

- Bursal B lymph Plasma cell

equivalent

( ( (

Stage of T-cell differentiation

- Thymic cortex Stage I (Early thymocyte)

- Stage II (Common thymocyte(

- Thymic medulla Stage III (Late thymocyte)

- Peripheral blood Mature cell

- -Activated T cell

-(See picture of Immunophenotype of Ontogeny T cell)

Biochemical marker

Thymocyte

-Stage I II III T cell

-TdT ++ ++ +

(( ( ( ( ( ( ( ( ( ( PNP + + ++ ++

dCK +++ + + +

- 5 ’ + + / + + + +

Tdt = Terminal deoxytransferase; ADA = Adenosine deaminase; PNP = Purine nucleoside phosphorylase

; -dCK = Deoxycytidine kinase; 5’NT = 5’ Nucleotidase

Molecular Marker

Thymocyte

-Stage I II III T cell

-TCR beta rearr. --------------------------- TCR beta mRNA -------------------

TCR-gamma gene rearr. --------------------------TCR-gamma mRNA ---------TCR-alpha mRNA ------------------- 3 mRNA --------------------------Cytoplasmic --------------------3

Surface -----------3CD /TCR

Stage of Human B-cell

Stem cell

Pre-B

Resting

Activated

Proliferating

Differentiating

Secretory

Plasmopoiesis

B-cell

Plasmoblast (Plasmablast)

Proplasmocyte (Proplasmacyte)

Plasma cell

Lymphocyte Growth Factor

Cytokine Cell Function

IL-1 Monocyte T-cell activation

IL-2 T-cell Proliferation of activated T-cell

IL-3 T-cell For T-cell subset and myeloid cell

BGCF T-cell For B-cell

BCDF T-cell For B-cell Ig synthesis

Factors Affecting Lymphopoiesis

• Age

• Diet

• - Steroid Testosterone

• Thyroxine

• Thymosine

• - Infection Viral

- - Bacterial Pertussis

- - Chronic disease TB

Factors Affecting Lymphopoiesis

• Age

• Diet

• - Steroid Testosterone

• Thyroxine

• Thymosine

• - Infection Viral

- - Bacterial Pertussis

- - Chronic disease TB

Erythroid Maturation Sequence

Proerythroblast(Pronormoblast)

BasophilicNormoblast

PolychromatophilicNormoblast

OrthochromatophilicNormoblast

Reticulocyte

Erythrocyte

Early Intermediate Late

Hemopoietic progenitor cells

CFU-GEMM (Granulocyte, erythrocyte, monocyte, megakaryocyte) CFU-GM (Granulocyte, monocyte) CFU-Eo (Eosinophil) CFU-Baso (Basophil) CFU-Meg (Megakaryocyte) BFU-E (Erythrocyte) CFU-E (Erythrocyte) CFU = Colony forming unit; BFU = Burst forming unit

Hemopoietic growth factors

• A family of hormones• As cytokines• Regulatation - proliferation - differentiation - maturation of progenitor cells - survival and function of mature cells• Hemopoietin

Cytokines

• Produced by - monocyte - fibroblast - lymphocyte - endothelial cells• Act several target cells - fibroblast - hemopoietic cell - lymphoid cell - hepatocyte - endocrine cell

Cytokines Family

• Interferons - - - -IFN alpha, IFN beta, IFN gamma• Interleukins - - - - - -IL 1, IL 2, IL 3, IL 4, ……. IL 15• CSF (Colony stimulating factors) - - - - G CSF, GM CSF, M CSF, EPO• Growth factors - - - - - - -TGF beta 1 , TGF beta 2 , …. TFG beta5

- EGF, FGF ( T = transforming, E = epidermal, F = fibroblast)

Cellular Cytokines Production (1)

• Endothelial cells - GM-CSF, G-CSF, M-CSF, IL-6• Macrophage - GM-CSF, G-CSF, M-CSF, IL-3, IL-6, EPO, SCF • T cells - GM-CSF, IL-2, IL-3, IL-4, IL-5• Fibroblast - GM-CSF, G-CSF, M-CSF, IL-6, SCF

Cellular Cytokines Production (2)

• NK cells - GM-CSF• PMN - G-CSF, GM-CSF • B cells - GM-CSF, M-CSF, IL-2, IL-5, IL-6 • Marrow stroma - GM-CSF, G-CSF, M-CSF, IL-6, IL-3, SCF, IL-11• Renal parenchyma, Liver, Bone marrow - EPO

Control Mechanism of Hemopoiesis

Depend on1. Nature cells2.Humoral factors : EPO, Granulopoietin, thrombopoietin, CSF, GM-CSF, IL-1, Cytokines, etc.3. Cell-cell interaction4. Pharmacologic substances : Chalone (inhibit stem cells)