1. Dr. Emad I H Shaqoura M.D, M.Sc. Anatomy Faculty of Medicine, Islamic University-Gaza Feb., 2016 Hemopoiesis

2. Hemopoiesis (or hematopoiesis) is the process of blood cell formation aiming at continual replacement of short- lived mature blood cells It first occurs in a mesodermal cell population of the embryonic yolk sac, and shifts during the second trimester mainly to the developing liver, before becoming concentrated in newly formed bones during the last 2 months of gestation. Hemopoietic bone marrow occurs in many locations through puberty, but then becomes increasingly restricted to components of the axial skeleton. Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 2

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4. Stem Cells, Growth Factors, & Differentiation Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 4 Stem cells are pluripotent cells capable of asymmetric division and self-renewal. Some of their daughter cells form specific, irreversibly differentiated cell types, and other daughter cells remain as a small pool of slowly dividing stem cells. All blood cells arise from a single major type of pluripotent stem cell in the bone marrow that can give rise to all the blood cell types.

5. Hemopoietic Stem Cells Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 5 Hemopoietic pluripotent stem cells are rare, but they proliferate and form two major lineages of progenitor cells with restricted potentials (committed to produce specific blood cells): 1. One for lymphoid cells (lymphocytes). 2. Another for myeloid cells (Gr. myelos, marrow) that develop in bone marrow. Myeloid cells include granulocytes, monocytes, erythrocytes, and megakaryocytes. The lymphoid progenitor cells migrate from the bone marrow to the thymus or the lymph nodes, spleen, and other lymphoid structures, where they proliferate and differentiate.

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7. Progenitor & Precursor Cells Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 7 The progenitor cells for blood cells are commonly called colony-forming units (CFUs), because they give rise to colonies of only one cell type when cultured or injected into a spleen. There are four major types of progenitor cells/CFUs: 1. Erythroid lineage of CFU-erythrocytes (CFU-E). 2. Thombocytic lineage of CFU-megakaryocytes (CFU-Meg). 3. Granulocyte-monocyte lineage of CFU-granulocytes- monocytes (CFU-GM). 4. Lymphoid lineage of CFU-lymphocytes of all types (CFU- L).

8. Progenitor & Precursor Cells Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 8 Each progenitor cell produces precursor cells (or blasts) that gradually assume the morphologic characteristics of the mature, functional cell types they will become. In contrast, stem and progenitor cells cannot be morphologically distinguished and simply resemble large lymphocytes. While stem cells divide at a rate only sufficient to maintain their relatively small population, progenitor and precursor cells divide more rapidly, producing large numbers of differentiated, mature cells.

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10. Progenitor & Precursor Cells Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 10 Progenitor cells, committed to forming each type of mature blood cell, proliferate and differentiate within microenvironmental niches of stromal cells, other cells, and ECM with specific growth factors. Hemopoietic growth factors, often called colony- stimulating factors (CSF) or cytokines, are glycoproteins that stimulate proliferation of progenitor and precursor cells and promote cell differentiation and maturation within specific lineages.

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12. Medical Application Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 12 Hemopoietic growth factors are important products of biotechnology companies. They are used clinically to increase marrow cellularity and blood cell counts in patients with conditions such as severe anemia or during chemo- or radiotherapy, which lower white blood cell counts (leukopenia). Such cytokines may also increase the efficiency of marrow transplants by enhancing cell proliferation, enhance host defenses in patients with infectious and immunodeficient diseases, and improve treatment of some parasitic diseases.

13. Bone Marrow Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 13 Bone marrow is found in the medullary canals of long bones and in the small cavities of cancellous bone, with two types based on their appearance at gross examination: 1. Blood-forming red bone marrow, whose color is produced by an abundance of blood and hemopoietic cells. 2. Yellow bone marrow, which is filled with adipocytes that exclude most hemopoietic cells. In the newborn all bone marrow is red and active in blood cell production, but as the child grows, most of the marrow changes gradually to the yellow variety. Under certain conditions, such as severe bleeding or hypoxia, yellow marrow reverts to red.

14. Bone Marrow Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 14 Red bone marrow contains a reticular connective tissue stroma, hemopoietic cords or islands of cells, and sinusoidal capillaries. The stroma is a meshwork of specialized fibroblastic cells called stromal cells (also called reticular or adventitial cells) and a delicate web of reticular fibers supporting the hemopoietic cells and macrophages. The matrix of bone marrow also contains collagen type I, proteoglycans, fibronectin, and laminin, the latter glycoproteins interacting with integrins to bind cells to the matrix. Red marrow is also a site where older, defective erythrocytes undergo phagocytosis by macrophages, which then reprocess heme-bound iron for delivery to the differentiating erythrocytes.

15. Bone Marrow Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 15 The hematopoietic niche in marrow includes the stroma, osteoblasts, and megakaryocytes. Between the hematopoietic cords run the sinusoids, which have discontinuous endothelium, through which newly differentiated blood cells and platelets enter the circulation.

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18. Medical Application Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 18 Red bone marrow also contains stem cells that can produce other tissues in addition to blood cells. These pluripotent cells may make it possible to generate specialized cells that are not rejected by the body because they are produced from stem cells from the marrow of the same patient. The procedure is to collect bone marrow stem cells, cultivate them in appropriate medium for their differentiation to the cell type needed for transplant, and then use the resulting cells to replace defective cells. These studies in regenerative medicine are at early stages, but results with animal models are promising.

19. Maturation of Erythrocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 19 Several major changes take place during erythropoiesis: 1. Cell and nuclear volumes decrease. 2. The nucleoli diminish in size and disappear. 3. Chromatin density increases until the nucleus presents a pyknotic appearance and is finally extruded from the cell. 4. There is a gradual decrease in the number of polyribosomes (basophilia), with a simultaneous increase in the amount of hemoglobin (a highly eosinophilic protein). 5. Mitochondria and other organelles gradually disappear.

20. Maturation of Erythrocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 20 There are three to five intervening cell divisions between the proerythroblast and the mature erythrocyte. The development of an erythrocyte from proerythroblast to the release of reticulocytes into the blood takes approximately 1 week. The glycoprotein erythropoietin, a growth factor produced by cells in the kidneys, stimulates production of mRNA for globins, the protein components of hemoglobin, and is essential for the production of erythrocytes. Reticulocytes pass to the circulation (where they may constitute 1% of the red blood cells), quickly lose the polyribosomes, and mature as erythrocytes.

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23. Maturation of Granulocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 23 Granulopoiesis involves cytoplasmic changes dominated by synthesis of proteins for the azurophilic granules and specific granules. These proteins are produced in the rough ER and Golgi apparatus in two successive stages: 1. Made initially are the azurophilic granules, which contain lysosomal hydrolases, stain with basic dyes, and are basically similar in all three types of granulocytes. 2. Golgi activity then changes to produce proteins for the specific granules, whose contents differ in each of the three types of granulocytes.

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25. Medical Application Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 25 Before its complete maturation, the neutrophilic granulocyte passes through an intermediate stage, the band cell (or stab cell), in which the nucleus is elongated but not yet polymorphic. The appearance of large numbers of immature neutrophils (band cells) in the blood, sometimes called a “shift to the left,” is clinically significant, usually indicating a bacterial infection.

26. Maturation of Granulocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 26 The vast majority of granulocytes are neutrophils and the total time required for a myeloblast to produce mature, circulating neutrophils ranges from 10 to 14 days. Developing and mature neutrophils exist in four functionally and anatomically defined compartments: 1. The granulopoietic compartment in active marrow. 2. Storage as mature cells in marrow until release. 3. The circulating population. 4. A population undergoing margination, a process in which neutrophils adhere loosely and accumulate transiently along the endothelial surface in venules and small veins. Margination can persist for several hours and is not always followed by emigration from the vessels. 5. Inflamed connective tissue.

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28. Maturation of Granulocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 28 Neutrophilia, an increase in the number of circulating neutrophils, does not necessarily imply an increase in granulopoiesis. 1. Transitory neutrophilia:  Intense muscular activity or the administration of epinephrine can cause the marginating neutrophils to move into the circulating compartment.  Glucocorticoids increase the mitotic activity of neutrophil precursors. Transitory neutrophilia is typically followed by a recovery period during which no neutrophils are released. 2. Prolonged neutrophilia:  Bacterial infections is due to an increase in production of neutrophils and a shorter duration of these cells in the medullary storage compartment. In such cases, immature forms such as band or stab cells, neutrophilic metamyelocytes, and even myelocytes may appear in the bloodstream.

29. Maturation of Agranulocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 29 The precursor cells of monocytes & lymphocytes do not show specific cytoplasmic granules or nuclear lobulation. The monoblast is a committed progenitor cell that is virtually identical to the myeloblast morphologically. Promonocytes divide twice as they develop into monocytes. Monocytes circulate in blood for several hours and enter tissues where they mature as macrophages & function for up to several months.

30. Maturation of Agranulocytes Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 30 The first identifiable progenitor of lymphoid cells is the lymphoblast, a large cell capable of dividing two or three times to form lymphocytes. In the bone marrow and in the thymus, these cells synthesize the specific cell surface proteins that characterize B or T lymphocytes, respectively.

31. Medical Application Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 31 Abnormal proliferation of stem cells in bone marrow can produce a range of myeloproliferative disorders. Leukemias are malignant clones of leukocyte precursors. They can occur in both lymphoid tissue (lymphoblastic leukemias) and bone marrow (myelogenous leukemias). In these diseases, there is usually a release of large numbers of immature cells into the blood and an overall shift in hemopoiesis, with a lack of some cell types and excessive production of others. The patient is usually anemic and prone to infection. Diagnosis of leukemias and other bone marrow disturbances involves bone marrow aspiration. A needle is introduced through the compact bone, typically at the iliac crest, and a sample of marrow is withdrawn. Immunocytochemistry with labeled monoclonal antibodies specific to membrane proteins of precursor blood cells contributes to a more precise diagnosis of the leukemia.

32. Origin of Platelets Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 32 Platelets originate in the red bone marrow by dissociating from mature megakaryocytes, which in turn differentiate from megakaryoblasts in a process driven by thrombopoietin. The megakaryoblast is 25 to 50 μ m in diameter and has a large ovoid or kidney-shaped nucleus, often with several small nucleoli. Before differentiating, these cells undergo endomitosis, with repeated rounds of DNA replication not separated by cell divisions, resulting in a nucleus that is highly polyploid (i.e, 64N or >30 times more DNA than in a normal diploid cell). The cytoplasm of this cell is homogeneous and highly basophilic.

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34. Origin of Platelets Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 34 Megakaryocytes are giant cells, up to 150 μ m in diameter, with large, irregularly lobulated polyploid nuclei, coarse chromatin, and no visible nucleoli. Their cytoplasm contains numerous mitochondria, a well- developed RER, and an extensive Golgi apparatus from which arise the conspicuous specific granules of platelets. They are widely scattered in marrow, typically near sinusoidal capillaries. To form platelets, megakaryocytes extend several long(>100 μ m), wide (2-4 μ m) branching processes called proplatelets. These cellular extensions penetrate the sinusoidal endothelium and are exposed in the circulating blood of the sinusoids

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38. Origin of Platelets Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 38 Internally proplatelets have a framework of actin filaments and microtubules along which membrane vesicles and specific granules are transported. A loop of microtubules forms a teardrop-shaped enlargement at the distal end of the proplatelet, and cytoplasm within these loops is pinched off to form platelets with their characteristic marginal bundles of microtubules, vesicles, and granules.

39. Origin of Platelets Hemopoiesis, Dr. Emad I Shaqoura, IUG Faculty of Medicine, 2016 39 Mature megakaryocytes have numerous invaginations of plasma membrane ramifying throughout the cytoplasm, called demarcation membranes, which were formerly considered “fracture lines” or “perforations” for the release of platelets but are now thought to represent a membrane reservoir that facilitates the continuous rapid proplatelet elongation. Each megakaryocyte produces a few thousand platelets, after which the remainder of the cell shows apoptotic changes and is removed by macrophages.

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