Normal Hemopoiesis Ahmad Sh Silmi Msc Haematology FIBMS

Normal Hemopoiesis Ahmad Sh. Silmi Msc Haematology, FIBMS

Lifespan and production of blood cells Cell type Red Cells Neutrophils Platelets Lymphocytes Approximate lifespan Production rate cells/day Production rate cells/sec Production rate Kg/year 100 days 2 x 1011 2. 3 million 7. 3 t½ 6 hours 3 x 1010 350, 000 10. 9 7 days 1 x 1011 1. 2 million 4. 6 t½ 10 days 1 x 1010 116, 000 3. 7 Annual total 26. 5 Kg

So our body is in a continuous dynamic and a very rapid cell turnover to be able to live. HOW ? It’s the Stem Cell !

HEMOPOIESIS: INTRO Hemo: Referring to blood cells Poiesis: “The development or production of” The word Hemopoiesis refers to the production & development of all the blood cells: n n n Erythrocytes: Erythropoiesis Leucocytes: Leucopoiesis Thrombocytes: Thrombopoiesis.

HEMOPOIESIS Hemopoiesis depends on 3 important components: § the bone marrow stroma (Local control) § the hemopoietic stem and progenitor cells § the hemopoietic growth factors (Humoral control)

STEM CELL THEORY The dazzling array of all the blood cells are produced by the bone marrow. They all come from a single class of primitive mother cells called as: PLURIPOTENT STEM CELLS. These cells give rise to blood cells of: n n Myeloid series: Cells arising mainly from the bone marrow. Lymphoid series: cells arising from lymphoid tissues.

STEM CELLS These cells have extensive proliferative capacity and also the: n n Ability to give rise to new stem cells (Self Renewal) Ability to differentiate into any blood cells lines (Pluripotency) They grow and develop in the bone marrow. The bone marrow & spleen form a supporting system, called the “hemopoietic microenvironment”

STEM CELLS: Types Pluripotent Stem cells: n n n Has a diameter of 18 – 23 μ. Giving rise to: both Myeloid and Lymphoid series of cells Capable of extensive self-renewal. Myeloid Stem cells: Generate myeloid cells: n n n Erythrocytes Granulocytes: PMNs, Eosinophils & Basophils. Thrombocytes. Lymphoid Stem cells: Giving rise only to: n Lymphocytes: T type mainly.

Self renewal of stem cells Symmetric cell division Asymmetric cell division Stem cells divide asymmetrically Non-stem cells divide symmetrically

Rules of Normal Cell Proliferation 1 2 3 Stem cells self renew--immortal non-stem cells have finite life span

But also when it is required : Stem cells divide symmetrically to restore stem cell quantity Stem cells symmetric cell division

CLONAL HEMOPOIESIS MULTIPLICATION STEM CELL COMMITTMENT MULTIPLICATION COMMITTED STEM CELL CFU: COLONY FORMING UNIT

CLONAL HEMOPOIESIS: (Contd) COLONY FORMING UNIT (CFU) MORPHOLOGICALLY RECOGNIZABLE MATURE BLOOD CELLS END CELLS: FINITE LIFE SPAN INTERMEDIATE BLAST CELLS

Properties of stem cells 1. Self renewal 2. Hierarchy 3. Extensive proliferative capacity 4. Cell cycle status 5. Surface Markers 6. Interact with microenvironment

Stem Cell Hierarchy

Stem Cell Progression Is Associated By Changes In: - Specific cell markers - Receptors - Adhesion molecules - Chromatin openness - Access to epigenetic transcription factors and loss of proliferative potential.

SITES OF HEMOPOIESIS n. Yolk n. Liver sac and spleen n. Bone marrow –Gradual replacement of active (red) marrow by inactive (fatty) tissue –Expansion can occur during increased need for cell production

SITES OF HEMOPOIESIS Active Hemopoietic marrow is found, in children throughout the: n Axial skeleton: n n n Cranium Ribs. Sternum Vertebrae Pelvis Appendicular skeleton: • Bones of the Upper & Lower limbs In Adults active hemopoietic marrow is found only in: • The axial skeleton • The proximal ends of the appendicular skeleton.

Sites of Hemopoiesis starts as early as yolk sac development. 2 -3 weeks after fertilization 3 layers are developed (ecto, meso, and endoderm) Hemoangioblast which is derived from the mesoderm Hemoangioblast Endothelial stem cell Hemopoietic stem cell Will develop to Blood vessels will develop to Blood cells

In the embryo § 2 week old embryo, hematopoiesis begins in yolk sac. THE 1 ST CELL TO BE PRODUCED IS erythrocytes § By 2 month old fetus, granulocyte and megakaryocyte production. § 4 th month, lymphocytes production. § 5 th month, monocytes produced.

CONTINUED…. . In the 3 rd to 7 th month of fetal life Hemopoietic stem cells will migrate to the liver and spleen, where hemopoiesis starts there and hemopoiesis is still mainly erythropoietic in nature, with minimal granulopoiesis

The bone marrow (BM) The stem cells then migrate to the bone marrow (BM) where hemopoiesis starts and continue all over the life. In the bone marrow all types of blood cells are formed which include: RBCS Granulocytes: Neutrophils, Eosinophils, Basophils Lymphocytes Monocytes and macrophages Platelets

Extramedullary Hemopoiesis When required, yellow marrow can be replaced by red marrow. Liver & spleen can aslo resumed. This will multiply the production by 6. Remark that Hemopoiesis within the marrow is called intramedullary or medullary hemopoiesis

Stages in haemopoietic cell development

Cell hierarchy (Haemopoiesis schematic representation)

Hematopoietic Inductive Microenvironment

Why hemopoietic cells reside in the bone marrow The stromal matrix plays an important role in presenting growth factors and nutrients to developing blood cells. The most immature cells have receptors which bind them to proteoglycan molecules on the matrix and to receptors on the stromal cells (i. e. macrophages, fibroblasts, fat cells and endothelial cells) There are lineage specific regions ( "niches" ) which provide the molecular basis for homing of transplanted stem cells. The unique supportive microenvironment stem cell niche - regulates proliferation and differentiation - supports survival and inhibits apoptosis Similar principles apply to malignant stem cells in myeloid leukemias The sinusoids are lined with specialized endothelial cells which play an important role by producing factors which regulate growth and differentiation.

Interaction of stromal cells, growth factors and haemopoietic cells

Stromal Cells of BM Endothelial cells Fat cells Fibroblasts Lymphocytes Macrophage

Haemopoietic growth factors

Haemopoietic growth factors The haemopoeitic growth factors are glycoprotein hormones that regulate the proliferation and differentiation of haemopoietic progenitor cells and the function of mature blood cells. T lymphocytes, monocytes, marcrophages and stromal cells are the major sources of growth factors except for erythropoietin, 90% of which is synthesized in the kidney and thrombopoietin, made largely in liver.

Haemopoietic growth factors Overview Regulate growth and differentiation - a family of glycoproteins with clinical utility - with different specificities - with different origins - bind to cognate receptors on progenitor cells - act locally or at a distance - regulate proliferation and differentiation - prevent apoptosis - enhance function of some end stage cells eg. GM-CSF enhances PMN function. . , Adhesion molecules /integrins

Haemopoietic growth factors GM-CSF § Granulocyte-Macrophage colony stimulating factor M-CSF § Macrophage colony stimulating factor Erythropoietin § Erythropoiesis stimulating hormone (These factors have the capacity to stimulate the proliferation of their target progenitor cells when used as a sole source of stimulation) Thrombopoietin § Stimulates megakaryopoiesis

Haemopoietic growth factors Cytokines § § § § § IL 1 (Interleukin 1) IL 3 IL 4 IL 5 IL 6 IL 9 IL 11 TGF-β SCF (Stem cell factor, also known as kit-ligand) Cytokines have no (e. g IL-1) or little (SCF) capacity to stimulate cell proliferation on their own, but are able to synergise with other cytokines to recruit nine cells into proliferation

Role of growth factors in normal haemopoiesis

Order of blood cell formation 1: STEM CELLS 2: Progenitor cells may be mutli-potential, bipotential or uni-potential 3: Precursor cells, or also called maturing cells 4: Terminally differentiated cells Mature cells

Precursor cells

Stem Cells § Stem Cells: undifferentiated cells that give rise to all of the bone marrow cells. § Only 0. 5% of all marrow nucleated cells. § Multipotential precursors. § High self-renewal – give rise to daughter stem cells that are exact replicas of the parent cell. § Not morphologically distinguishable At any time, the majority of stem cells (95%) are out of the cell cycle (they are in G 0 mode/phase, also called quiescent). § The current phenotype is: CD 34+CD 38 -Lin-HLADR-Rh 123 Dull

Progenitor cells of the BM § Stem cells which undergo differentiation. § Limited self-renewal ability. § Multipotential but Gradually they become unilineage or committed progenitor cell. § ~3% of total nucleated hematopoietic cells of bone marrow

Progenitor cells of the BM § Form colonies of cells in semisolid media in vitro – described as colony forming units (CFU). § CFU-GEMM (granulocytic, erythrocytic, monocytic, megakaryocytic, CFU-GM, CFU-Mk, etc. ) § Survival and differentiation of progenitor cells influenced by growth regulatory glycoproteins, called cytokines – include interleukins, colony stimulating factors

Morphology of stem cells and progenitor cells Stem cells & progenitor cells are not recognized morphologically but all look like small mononuclear lymphocytes

Maturing blood cells 1 - Majority of cells (>95%) 2 - lose adherence receptors, become deformable. 3 - migrate through cytoplasm of lining endothelial cell to enter sinusoids. 4. Platelets are the exception. Megakaryocytes form part of the sinusoidal wall. They form long processes of proplatelets which fragment into nascent platelets.

Regulation of Haemopoiesis n. There should be a balance between cell production and cell death except at the times of requirement

Regulation of Haemopoiesis Local environmental control Stromal cell mediated Haemopoiesis Apoptosis Haemopoietic growth factors (Humoral regulation)

Regulation of Haemopoiesis § Immunoglobulin superfamily - Growth Factor Receptors (GFr) bind to cognate "ligand" to initiate signal transduction ( GF binds to extracellular domain of GFr, alters configuration of cytoplasmic domain, causes phosphorylation of a tyrosine kinase and initiates the signal that is ultimately transmitted to the nucleus. ) § Selectins are involved in: a) inflammatory and immune responses b) platelet adhesion and activation § Integrins - control traffic and specific cell functions - adhere to matrix niches - release from marrow - homing of lymphocytes and stem cells etc.

Apoptosis important for homeostasis Hematopoietic cells are programmed to self destruct but can be rescued from apoptosis by: specific growth factors certain gene products specific antigen some viruses eg EBV

Mechanism of apoptosis

Apoptosis, Continue Defects in the apoptotic pathway are very important in causing diseases such as chronic lymphocytic leukemia (CLL), and in enhancing resistance to chemotherapy in malignancy. Induction of apoptosis is a therapeutic strategy in resistant CLL Inappropriate apoptosis is a disease mechanism in myelodysplasyic syndromes. Apoptosis is induced by chemotherapy, and resistance to chemotherapy is associated with blocked apoptosis.

Assessment of hemopoiesis Hemopoiesis can be assessed clinically by; 1 - (FBC, CBC= complete blood count) on peripheral blood. 2 - Bone marrow Aspiration also allows assessment of the later stages of maturation of hemopoietic cells. 3 - Bone marrow Trephine Biopsy provides a core of bone and bone marrow to show architecture.

Bone marrow Aspiration

Bone marrow aspiration

Hypercellular Normocellular Hypocellular

The M: E ratio Myeloid to Erythroid ratio in the bone marrow Is called M: E ratio Normally it is 3 -4: 1

Normal Blood Cells

ERYTHROPOIESIS PROERYTHROBLAST BASOPHILIC ERYTHROBLAST POLYCHROMATOPHILIC ERYTHROBLAST ORTHOCHROMATIC ERYTHROBLAST RETICULOCYTE MATURE ERYTHROCYTES

Stages in Red cell (erythroid) Maturation Proerythroblast Basophilic erythroblast Polychromatic erythroblast (two examples) Orthochromic erythroblast

ERYTHROID PROGENITOR CELLS BFU-E: Burst Forming Unit – Erythrocyte: n Give rise each to thousands of nucleated erythroid precursor cells, in vitro. n n Undergo some changes to become the Colony Forming Units-Erythrocyte (CFU-E) Regulator: Burst Promoting Activity (BPA)

ERYTHROID PROGENITOR CELLS CFU-E: Colony Forming Unit- Erythrocyte: n n Well differentiated erythroid progenitor cell. Present only in the Red Bone Marrow. Can form upto 64 nucleated erythroid precursor cells. Regulator: Erythropoietin. Both these Progenitor cells cannot be distinguished except by in vitro culture methods.

Normoblastic Precursors PROERYTHROBLAST: n n Large cell: 15 – 20 Microns in diameter. Cytoplasm is deep violet-blue staining Has no Hemoglobin. n Large nucleus 12 Microns occupies 3/4 th of the cell volume. n Nucleus has fine stippled reticulum & many nucleoli. n

Normoblastic Precursors EARLY NORMOBLAST: (BASOPHILIC NORMOBLAST) – – Smaller in size. Shows active Mitosis. No nucleoli in the nucleus. Fine chromatin network with few condensation nodes found. – Hemoglobin begins to form. – Cytoplasm still Basophilic.

Normoblastic Precursors INTERMEDIATE NORMOBLAST: (POLYCHROMATOPHILIC NORMOBLAST) – Has a diameter of 10 – 14 Microns. – Shows active Mitosis. – Increased Hemoglobin content in the cytoplasm – Cytoplasm is Polychromatophilic.

Normoblastic Precursors LATE NORMOBLAST: (ORTHOCHROMIC NORMOBLAST) – Diameter is 7 – 10 Microns. – Nucleus shrinks with condensed chromatin. – Appears like a “Cartwheel” – Cytoplasm has a Eosinophilic appearance.

RETICULOCYTE – The penultimate stage cell. – Has a fine network of reticulum like a heavy wreath or as clumps of dots – This is the remnant of the basophilic cytoplasm, comprising RNA. – In the Neonates, Count is 2 – 6/Cu. mm. – Falls to <1 in the first week of life. – Reticulocytosis is the first change seen in patients treated with Vit B 12

MATURE ERYTHROCYTE – Biconcave disc. – No nucleus. – About One-third filled with Hemoglobin.

Erythropoiesis § 4 mitotic divisions between pronormoblast and orthochromic normoblast stage. § Thus giving rise 16 RBCs. § But not all of the 16 will be good RBCs, some are bad and will be destroyed, these destroyed cells is called ineffective erythropoiesis. § 2 – 7 days for pronormoblast to mature into orthochromic normoblast § 1 more day to extrude the nucleus from the orthochromic normoblast. § Reticulocyte further matures for 2 – 3 days in bone marrow before it is released into the peripheral blood. § Red cell has life span of 120 days in peripheral blood.

Phases of Erythropoiesis

FACTORS REGULATING ERYTHROPOIESIS SINGLE MOST IMPORTANT REGULATOR: “TISSUE OXYGENATION” BURST PROMOTING ACTIVITY ERYTHROPOIETIN IRON VITAMINS: – Vitamin B 12 – Folic Acid MISCELLANEOUS

ERYTHROPOIETIN A hormone produced by the Kidney. A circulating Glycoprotein Nowadays available as Synthetic Epoietin Acts mainly on CFU – E. Increases the number of: – Nucleated precursors in the marrow. – Reticulocytes & Mature Erythrocytes in the blood.

VITAMINS B 12: Cyanocobalamine & Folic Acid: – Is also called Extrinsic Factor of Castle. – Needs the Intrinsic Factor from the Gastric juice for absorption from Small Intestine. – Deficiency causes Pernicious (When IF is missing) or Megaloblastic Anemia. – Stimulates Erythropoiesis – Is found in meat & diary products.

IRON Essential for the synthesis of Hemoglobin. Deficiency causes Microcytic, Hypochromic Anemia. The MCV, Color Index & MCH are low.

Regulation of Erythropoiesis Stimulates CFU – E Proerythroblasts ERYTHROPOIETIN Mature Erythrocytes De c es s rea Tissue Oxygenation Factors decreasing: Hypovolemia n n. Anemia Poor blood flow n. Pulmonary Disease n An example of a Negative feed back mechanism

Erythropoietin (Epo) As its name suggests, EPO stimulates growth of Erythrocytes, and its function include: § Activates stem cells of bone marrow to differentiate into pronormoblasts. § Shortening cell cycle. § Decrease maturation time. § Increases rate of mitosis and maturation process. § Increases rate of hemoglobin production. § Causes increased rate of reticulocyte release into the peripheral blood, (normally the reticulocyte when it is released to the peripheral blood it need only one day to mature to RBC, but here they will be released prematurely into peripheral blood, thus they need more than one day to mature RBC. § Prevent apoptosis.

EPO receptors §Found on surface on bone marrow erythroid progenitor and precursor cells. §The highest number of EPO receptors is seen on the CFU-E and the pronormoblasts. §The number of EPO receptors per cell gradually decreases during erythroid cell differentiation, and studies have shown that the reticulocyte and mature erythrocyte do not contain EPO receptors

EPO Receptor Divided into extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain has terminal that contains both positive and negative growth-regulatory domains. After EPO binding, EPOR homodimarizes and JAK-2 phosphorylates itself, so EPOR and other proteins like STAT-5 initiate the cascade of proliferative signals.

Granulopoiesis E Granulocytes Neutrophils Eosinophils Basophils Only mature cells are present in peripheral blood N B

Stages in Granulocyte Maturation Blast cell Promyelocyte Metamyelocyte Band cell Segmented Neutrophil

Neutrophil Maturation Myeloblast Cells in the BM proliferation pool take 24 -48 hours for a single cell cycle Less than 1% of the normal BM compartment is composed of myeloblasts Large, 15 -20 um in size Delicate nucleus with prominent nucleoli Small amount of cytoplasm with rough endoplasmic reticulum, a developing Golgi apparatus and an increasing number of azurophilic granules

Neutrophil Maturation Myeloblast Cytochemical staining shows presence of myeloperoxidase which is required for intracellular kills Killing function is the first to be operational in the neutrophil cell line Myeloblast is incapable of motility, adhesion and phagocytosis and is therefore nonfunctional

Neutrophil Maturation Promyelocyte After a few days in the blast stage, the cell becomes a promyelocyte 1 -5% of BM compartment composed of promyelocytes Size is variable and may exceed 20 um, so may be larger than myeloblast Nuclear chromatin may be delicate or may show slight clumping Nuceloli begin to fade

Stages in Granulocyte Maturation Blast cell Promyelocyte Metamyelocyte Band cell Segmented Neutrophil

Neutrophil Maturation Production and accumulation of neutrophilic Myelocyte granules is characteristic of the myelocyte The myelocyte is the last cell of the BM compartment capable of mitosis Myelocytes demonstrate morphologic variability as this development stage lasts from 4 -5 days and cause alterations in the staining characteristics of the cell

Neutrophil Maturation Smaller in size than. Myelocyte the promyelocyte (12 -18 um) Less than 10% of BM compartment is made up of myelocytes Nucleus is round to oval with a flattened side near the now well-developed Golgi apparatus Nuclear chromatin shows clumping Nucleoli no longer visible

Stages in Granulocyte Maturation Blast cell Promyelocyte Metamyelocyte Band cell Segmented Neutrophil

Neutrophil Maturation Myelocyte Secondary granules stain pink causing a “dawn of neutrophilia” or pink blush within the cytoplasm Compounds such as alkaline phosphatase begin to concentrate in the cell The cell acquires some motility

Granulopoiesis Notice change in granules color

Neutrophilic Maturation Metamyelocyte 13 -22 % of BM compartment 10 -15 um in size Not seen in normal PB Not fully functional, part of the maturation component of the marrow

Neutrophilic Maturation Band The band is a transitional form that exists in both the PB and the BM and considered part of both the maturation and storage pools Up to 40% of the WBCs of the BM are bands Represents the “almost mature” neutrophil having full motility, active adhesion properties, and some phagocytic ability

Neutrophilic Maturation Band forms begin to produce tertiary granules Membrane maturity shows changes in cytoskeleton, surface charge and presence of receptors for complement Once entered into the PB, account for less than 6% of circulating WBCs 10 -15 um in size Found in marginating and circulating pools of the PB

Neutrophilic Maturation PMN This cell’s nucleus continues to indent until thin strands of membrane and heterochromatin form into segments, hence it is also called a “seg” Polymorphonuclear means “many-shaped nucleus”, describing the varied nuclear shapes Cell is completely functional and spend time in the storage pool of the BM as well as marginating and circulating pools of the PB 50 -70% of circulating WBCs of PB

Neutrophilic Maturation PMNs spend their life performing phagocytosis and pinocytosis Phagocytosis involves larger material and can be observed with light microscopy, pinocytosis involves small material (liquids) and is observed with EM Both of these function can be performed in the circulation of the blood stream or in the tissues

Neutrophil kinetics

Monocyte/Macrophage Maturation Monocyte/Macrophage cells mature from monoblast to promonocyte to blood monocyte to free and fixed macrophages, but the mechanism of commitment is not well understood. Granular content vary considerably with more than 50 secretory compounds having been identified. PB monocytes demonstrate morphologic variability Aggressive motility and adherence may distort the monocytes during PB smear preparation

Monocyte/Macrophage Maturation Monocyte nucleus is indented or curved with chromatin that is lacy with small clumps Typically the largest cell in the PB Cytoplasm is filled with minute granules that produce a cloudy appearance Cytoplasmic membrane may be irregular, pseudopods and phagocytic vacuoles may be evident

Monocyte/Macrophage Maturation

Lymphocytes The only human WBCs whose site of development is not just BM, but also tissues referred to as primary and secondary lymphoid organs In humans, the primary lymphoid organs are thymus and bone marrow, the secondary organs include the spleen, Peyer’s patches of the GI tract, the Waldermyer ring of the tonsils and adenoids, the lymph nodes and modules scattered throughout the body

Lymphocytes circulate throughout the body in both PB and lymph which act as carrier streams to bring the lymphocytes to sites of activity Lymphocytes migrate from thoracic duct through vessel endothelium to lymph nodes to blood stream and back. Lymphocytes are categorized in a variety of ways and may be short-lived or long-lived cells Lymphocytes may produce antibodies or lymphokines and have different surface charges, densities and antigen receptors.

Lymphocytes Development The PSC results in a stem cell for the lymphoid cell (CFU-L) as a result of hormonal stimuli The CFU-L matures in several environments Thymus and BM give rise to lymphocytes, foster differentiation and are indepentendent of antigenic stimulation

Lymphocytes Development Lymphocyte % in the PB varies, depending on age. Children under the age of 4 have a higher proportion of lymphocytes in the PB than do adults Lymphocytes are the second most common WBC of the PB making up 20 -40% of WBCs. 20 -35% of circulating lymphocytes are B cells

Lymphocytes - Maturation Lymphoblast to prolymphocyte Lymphoblast is small, 10 -18 um Round to oval nucleus Loose chromatin with one or more active nucleoli Scanty cytoplasm Prolymphocyte difficult to distinguish, subtle changes, more clumped chromatin, lessening nucleolar priminence, change in thickness of the nuclear membrane

Lymphopoiesis

Thrombopoiesis Platelet play a major role in primary hemostasis Life span 7 -10 days Production, fragmentation of cytoplasm Megakaryocytes undergoes endomitotic division

The role of cytokines in Megakaryocytopoi esis

The effect of IL-1 on target cells & Tissues

Summary Normal haemopoiesis is necessary for the survival It is under the control of multiple factors Normal bone marrow environment is necessary for normal haemopoiesis Decreased production results in cytopenias

Hematopoiesis Just notice general trends, Don’t memorize

Maturation Sequence Notice general trends, Don’t memorize

Monophyletic theory of cell formation