Med

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

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, alpha2, 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 -, SO

, etc.)

-2

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 Circulation

Diff. Compartment

<------ Proliferation ---------------------> Differentiation --------------------> Maturation -----------------------> Release -------------------------> PHSC = Pluripotent hemopoietic stem cells CSC = Committed stem cells

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

Stem Cells and their Therapeutic 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 Plasmopoiesis 3. Factors affecting leukopoiesis

Classification of WBC 1. Shape of the nucleus 1.1 mononuclear : Lymphocyte, Monocyte 1.2 Polymorphonuclear : PMN, Eos, Baso 2. Granules 2.1 Granulocytes : PMN, Eos, Baso 2.2 Agranulocyte : Lymphocyte, Monocyte 3. Site of origin Myeloid cells / Lymphoid cells 4. Function / Phagocyte : Macrophage - Monocyte

Leukopoiesis Study 1. Morphology 2. Biochemical markers 3. Immunological markers 4. Gene markers

Stage of Granulopoiesis Stem cell Myeloblast Promyelocyte Basophilic Eosinophilic Myelocyte Myelocyte Metamyelocyte Metamyelocyte Band

Neutrophilic Myelocyte

Metamyelocyte

Band

Band

Regulation of Neutrophil Production Bone Marrow

Blood stream

Proliferatrion freely compartment

Circulating compartment

Storage compartment

Marginating compartment

infection) Release control

Tissue Move

Move restricted (Area of

Neutropenia : PMN decrease Neutrophilia : PMN increase Cytokine

:

Multi-CSF (IL-3) GM-CSF G-CSF IL-6 Granulopoietin

(Granulocytic growth factor)

Eosinophilia : Eos. increase Cytokine

:

IL-3 IL-5 GM-CSF ECF-A (Eosinophilic

chemotactic factor of anaphylaxis)

Basophilia : Baso. increase Cytokine

: GM-CSF IL-4 IL-9

Monopoiesis Stem cell Monoblast Promonocyte Monocyte

Monocytosis : Monocyte increase Cytokine

:

IL-3 GM-CSF M-CSF

Tissue macrophage Osteoclast

: Bone

Macrophage : Lymphoid tissue Kupffer cell

: Liver

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 of antigen by RE cells - proliferation of thymus-derived cells - thymic humoral factors

Central Lymphoid Lymphoid Thymus Stem

Peripheral

T-lymph Th

CMI Ts

cells Bursal

B-lymph

Plasma

cell equivalent HMI

Stage of T-cell differentiation Thymic cortex - Stage I (Early thymocyte) thymocyte)

- Stage II (Common

Thymic medulla - Stage III (Late thymocyte) Peripheral blood - Mature cell - Activated T-cell

(See picture of Immunophenotype of Ontogeny T-

Biochemical marker Thymocyte Stage cell

I

II

III

TdT -

++

++

+

ADA ++

++

++

++

PNP ++

+

+

++

dCK +

+++

+

+

5’NT

+

+/-

++

T-

Molecular Marker Thymocyte Stage cell TCR- beta rearr. TCR- beta mRNA

I

II

III

T-

--------------------------------------------

TCR-gamma gene rearr. -------------------------TCR-gamma mRNA TCR-alpha mRNA CD3 mRNA Cytoplasmic CD3 Surface CD3/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 Early

Proerythroblast (Pronormoblast)

Intermediate Late

Polychromatophilic Normoblast

Basophilic Normoblast

Reticulocyte

Orthochromatophilic Normoblast

Erythrocyte

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

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-beta-5 - 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 on 1. Nature cells 2.Humoral factors : EPO, Granulopoietin, thrombopoietin, CSF, GM-CSF, IL-1, Cytokines, etc. 3. Cell-cell interaction 4. Pharmacologic substances : Chalone (inhibit stem cells)