Red bone marrow, erythropoiesis. Formation of leukocytes Dr. Zita Puskár EM I. (Gr 1-8) 21/11/2016

Formed elements of the blood Red blood cells (erythrocyte) White blood cells (leukocytes) Agranulocytes Lymphocytes Monocytes Granulocytes Neutrophils Eosinophils Basophils Platelets (thrombocytes) Scanning electron microscop image from normal circulating human blood Red blood cells (4-6 million/mm3)>Platelets (250 000-400 000/mm3)>white blood cells (5000-9000/mm3)

Name, (functions,) diameter, N or % and lifetime Monocyte D≈ 15-20 µm, % = 4-6 T= months Platelet D≈ 2-3 µm, N=250-400 thousands T=7-11 days Small lymphocyte D≈ 5-7 µm, % = 20-30 T= depending on the type 3 2 4 1 Red blood cell/erythrocyte D≈ 7.5 µm, N=4-6 million T= 120 days Basophil granulocyte D≈ 10-14 µm, %= 0.01-0.5 T= 3-10 days Eosinophil granulocyte D≈ 12-17 µm, %= 2-4 T= 6 days Neutrophil granulocyte D≈ 12-14 µm, %=60-70 T= 3 days 5 6 7

Hemopoiesis/Hematopoiesis Hemopoiesis: the process by which blood cells are formed. Adult myelopoiesis – bone marrow Erythropoiesis: formation of erythrocytes Granulo(cyto)poiesis: formation of granulocytes Monocytopoiesis: formation of monocytes Thrombocytopoiesis: formation of thrombocytes (platelets) Adult lymphopoiesis - bone marrow and lymphatic tissues Lymphopoiesis: formation of lymphocytes

What we need for this? A STEM CELL What is a Stem Cell?

Hierarchy of Stem Cells Totipotens stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. „They have potential to create an entire organism.” Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. „They can give rise to any cell, without the potential to create an entire organism.” Multipotent stem cells can produce only cells of a closely related family of cells (e.g. stem cells differentiate into red blood cells, white blood cells, platelets, etc.). „They give rise to cells with a specific purpose and function.” Unipotent cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells. Multipotent

Hematopoietic Stem Cell (HSC) HSC possess the ability of : multi-potency (is the ability to differentiate into all functional blood cell) and self-renewal (is the ability to give rise to HSC itself without differentiation). Niche

Hematopoietic Stem Cell (HSC)

Formation of blood cells Hematopoietic Stem Cell (HSC) Pluripotential (true) stem cells: self-renewal, enormous proliferation, differentiation into multiple cell lineage Myeloid SC Lymphoid SC multipotential stem cells CFU-Gemm CFUs progenitor cells B T Colony Forming Unit Cells: proliferate and differentiate into precursor cells in the presence of appropriate growth factors, cytokines, morphologically indistinguishable from stem cells BLASTs precursor cells proB T NK Specific Colony Forming Cells display different morphological characteristics

Synthesis and accumulation of hemoglobin Basophilia → Eosinophilia Erythropoiesis Synthesis and accumulation of hemoglobin ↓ Basophilia → Eosinophilia Extrusion of the nucleus → Reticulocyte

Erythroid island

Regulating factors Decrease of O2 → Erythropoietin (EPO, Kidney, Liver), Growth/Differentation IL-1, IL-3, IL-6 CSF-E (Colony Stimulating Factor- Erythro) Maturation Vitamin B12 Folic acid Hemoglobin synthesis Fe, Cu, Zn, Co Vitamin C Hormones

Megakaryopoesis - thrombopoesis nuclear endomitosis → 64N DNA Megakaryoblast ↓ Promegakaryocyte Megakaryocyte Thrombocytes (platelets) diploid 2n poliploid 4n, 8n, 16n, 32n, 64n Platelet: irregularly shaped fragments of megakaryocytes Regulating factor : Thrombopoietin (Liver)

http://italianolab.bwh.harvard.edu/research/

Megakaryopoesis - thrombopoesis Megakaryoblast ↓ Promegakaryocyte Megakaryocyte Thrombocytes

Granulopoiesis Myeloblast ↓ Promyelocyte (azurophilic granules) (specific granules→neurophil, basophil, eosinophil) Metamyelocyte (beggining the formation of lobulated nucleus, increased number of specific granules) mature immature mature basophil neutrophil eosinophil „band” (segmentation of the nucleus) mature neutrophil

Granulopoiesis Myeloblast ↓ Promyelocyte (azurophilic granules) (specific granules→neurophil, basophil, eosinophil) Metamyelocyte (beggining the formation of lobulated nucleus, increased number of specific granules) mature immature mature basophil neutrophil eosinophil „band” (segmentation of the nucleus) mature neutrophil

Monopoesis

Lymphopoiesis

Embryonic hemopoiesis Intrauterine – fetal hemopoiesis First or prehepatic phase – blood islands in the wall of yolk sac Second or hepatosplenothymic phase – liver and lymphatic tissues Third or medullolymphatic phase – bone marrow and lymphatic tissues Blood development: Pimitive wave (1) → Definitive wave (2-3)

Primitive wave in extra embryonic yolc sac erythrocyte tissue oxygenazition Erythroid progenitor not pluripotent do not have renewal capability macrophage

Definitive wave in embryo proper 19-day: the splanchnopleura is hemogenic 27-40-day: the aorta-gonad-mesonephros (AGM) region

Multipotent lymphomyeloid stem cells in the ventral site of the aorta Tavian et al: Int J Dev Biol. 2010; 54(6-7):1061-5.  Fig. 1. Hematopoietic stem cell clusters inside human embryonic arteries. (A) Cross sections of a 34-day human aorta (Ao) stained with the anti-CD34 (green) and anti-CD45 (red) antibodies. Arrows indicate the clusters of hematopoietic stem cells adhering to the ventral aspect of the aortic endothelium (white arrowhead). (B) Confocal images at higher magnificationof the region included in the dotted white line in panel A show the changed morphology of endothelial cells underlying hematopoietic clustered progenitors (arrow). Fig. 2. Expression of BB9/angiotensin-converting-enzyme (BB9/ACE) in the human embryo. Cross sections through the dorsal aorta (Ao) in a 34-day embryo. BB9/ACE is expressed by hematopoetic CD34+CD45+ cell clusters associated with the endothelium on the ventral site of the aorta (arrowheads), as well as by underlying endothelial CD34+ cells (white arrowheads).

Migration to the fetal liver and the spleen

…finally to the red bone marrow

1. articular cartilage 2. spongy bone 3. compact bone 4 1. articular cartilage 2. spongy bone 3. compact bone 4. medullary/marrow canal 5. yellow bone marrow 6. periosteum 7. proximal epiphysis 8. diaphysis 9. distal epiphysis

Bone marrow aspirate

Red bone marrow A M S

Composition of red bone marrow Specialized blood vessels sinuses (diameter: 50 –70 μm) which are interposed between arteries and veins Reticular fibers Sponge like network of hemopoetic, reticular and other cells Sinuses: endothelial lining basal lamina (barely) outer (adventitial) reticular cell layer support for the developing blood cells reticular fiber production stimulating the differentiation of stem cells into blood cells Cell types: developing blood cells, megakaryocytes, macrophages, mast cells, plasma cells, fat cells Transitory opening of the sinusoids – aperture – basal lamina and the adventitial cells become displaced

Hematopoietic Stem Cell Transplantation For patients with life threatening diseases (eg. multiple myeloma, leukemia) HSC can be derived from: bone marrow peripheral blood umbilical cord blood Autologus - the patient’s own HSCs Allogenic - the HSCs from a donor