Erythropoiesis PDF - Medical Biology Lecture Notes

Erythropoiesis Matthew LAU (Scientific Officer) MLS 3009SEF (2025 Spring term)  Area to cover Erythropoiesis Structure of erythrocytes and precursors Metabolism and function of erythrocytes Comparative Anatomy and Histology. DOI: http://dx.doi.org/10.1016/B978-0-12-8...

Erythropoiesis Matthew LAU (Scientific Officer) MLS 3009SEF (2025 Spring term)  Area to cover Erythropoiesis Structure of erythrocytes and precursors Metabolism and function of erythrocytes Comparative Anatomy and Histology. DOI: http://dx.doi.org/10.1016/B978-0-12-802900-8.00019-1  What is Erythropoiesis? Erythropoiesis (pronounced “ur-i-throw-poy-EE-sus”) is your body’s process of making red blood cells (erythrocytes) Is a fundamental pillar of human physiology, ensuring the efficient transport of oxygen from the lungs to tissues across the body Approx. 1012 erythrocytes made each day, originating from haematopoietic stem cells (HSCs) Primitive erythroblasts, which are large nucleated cells, fulfill the oxygen requirements of early embryos. In adults, erythropoiesis predominantly occurs in the bone marrow, where the process is regulated to yield mature, enucleated red blood cells that are optimal for oxygen carriage This complex process is initiated in the embryonic stage and continues throughout an individual’s life, adapting to the body’s changing needs from early development through adulthood  Why is Erythropoiesis important? Oxygen Transport: RBCs are responsible for carrying oxygen from your lungs to tissues throughout your body. Without sufficient RBCs, your tissues wouldn't receive the oxygen they need to function properly Carbon Dioxide Removal: RBCs also help transport carbon dioxide, a waste product, from your tissues back to your lungs, where it can be exhaled Maintaining Homeostasis: Erythropoiesis ensures that your body maintains the right number of RBCs. Too few RBCs can lead to anemia, causing fatigue and weakness, while too many can lead to complications like blood clots Response to Hypoxia: When your body detects low oxygen levels (hypoxia), it stimulates erythropoiesis to increase RBC production, thereby enhancing oxygen delivery to tissues  Fetal erythropoiesis Fetal erythropoiesis is the process by which red blood cells are produced in a developing fetus. It is essential for ensuring that the growing fetus receives adequate oxygen to support its rapid development and growth. The process is tightly regulated to meet the changing needs of the fetus at different stages of development and occurs in several stages and locations throughout fetal development. Yolk Sac (Weeks 3-8): Erythropoiesis begins in the yolk sac, which is an early source of nutrients for the embryo. This stage primarily produces primitive erythroid cells Liver and Spleen (Months 2-5): As the fetus develops, erythropoiesis shifts to the liver and spleen. These organs become the primary sites for RBC production during this period Bone Marrow (Month 5 onwards): By the fifth month of gestation, the bone marrow takes over as the main site of erythropoiesis. This transition marks the beginning of definitive erythropoiesis, which continues throughout life  Adult erythropoiesis In adults, erythropoiesis primarily occurs in the bone marrow of specific bones, including the pelvis, vertebrae, ribs, sternum and proximal femur Up to 20 years old, RBCs are produced from red marrow of all the bones (long bones and flat bones) After age 20: Flat Bones: Erythropoiesis primarily occurs in the bone marrow of flat bones such as the vertebrae, sternum, ribs, scapulas and iliac bones. Long Bones: The shafts of long bones become yellow marrow due to fat deposition and lose their erythropoietic function The vertebrae, sternum, pelvis and ribs and cranial bones continues to produce RBCs throughout elderly life  Regulation of erythropoiesis Detection of Hypoxia: When oxygen levels in the blood drop (a condition known as hypoxia), the kidneys detect the change. Erythropoietin (EPO) Production: In response to hypoxia, the kidneys increase the production and release of EPO into the bloodstream. EPO Action on Bone Marrow: EPO travels to the bone marrow, where it binds to receptors on erythroid progenitor cells. Stimulation of Progenitor Cells: EPO stimulates these progenitor cells to proliferate and differentiate into erythroblasts, which are immature red blood cells. Maturation of Erythroblasts: Erythroblasts undergo several stages of maturation, eventually becoming reticulocytes (immature RBCs) and then fully mature RBCs.  Steps of erythropoiesis There are four main cell stages: Stem cells Progenitor cells Precursor cells Mature cells Distinctive characteristics: The size of the cell decreases The cytoplasm volume increases As the cell matures the size of the nucleus decreases until it vanishes with the condensation of the chromatin material Duration of erythropoiesis from proerythroblast to erythrocyte is 6 – 8 days Proerythroblast to Reticulocyte = 4 days (1 day for each) Reticulocyte to Erythrocyte = 2 to 4 days (reticulocyte spends 1-2 days in marrow and circulates for 1-2 days in bloodstream before maturing to erythrocyte) Bhoopalan SV, Huang LJs and Weiss MJ. Erythropoietin regulation of red blood cell production: from bench to bedside and back [version 1; peer review: 4 approved]. F1000Research 2020, 9(Faculty Rev):1153 (https://doi.org/10.12688/f1000research.26648.1) Differentiation of HSC to erythroid progenitor Lineage commitment: HSCs first differentiate into MultiPotent Progenitor cells (MPP) Have limited self-renewal capacity but can give rise to all blood types Lineage-specific progenitors: MPPs further differentiate into lineage-specific progenitor cells, such as Common Myeloid Progenitor (CMP) CMP differentiate into bi-potential Megakaryocyte-Erythroid Progenitors (MEPs) Have the ability to further develop into either megakaryocytes or erythroid cells MEPs that commit to the erythroid lineage begin to express specific transcription factors such as GATA-1 and FOG-1, which are crucial for erythroid differentiation Key Factors in Erythroid Lineage Commitment GATA-1: A master regulator of erythropoiesis, GATA-1 is essential for the differentiation of progenitor cells into erythroid cells. It activates the transcription of erythroid-specific genes and represses genes associated with other lineages FOG-1 (Friend of GATA-1): This cofactor works in conjunction with GATA-1 to regulate erythroid differentiation. FOG-1 enhances the function of GATA-1 by stabilizing its binding to DNA KLF1 (Kruppel-like factor 1): KLF1 is another critical transcription factor that works downstream of GATA-1. It is involved in the activation of genes necessary for erythroid maturation and haemoglobin production TAL1 (T-cell acute lymphocytic leukemia 1): TAL1 is involved in the early stages of erythroid differentiation. It forms complexes with other transcription factors to regulate the expression of erythroid-specific genes BACH1 and BACH2: These transcription factors play roles in repressing the myeloid program, thereby promoting erythroid and lymphoid differentiation. They help maintain the balance between different haematopoietic lineages  Generation of BFU-E and CFU-E The burst-forming unit-erythroid (BFU-E) and colony-forming unit- erythroid (CFU-E) are crucial stages in the process of erythropoiesis, which is the production of red blood cells. MEPs give rise to both megakaryocytes and erythroid cells, with the BFU-E and CFU-E stages representing more committed steps towards becoming mature red blood cells Involves several key factors and stages Burst-forming unit-erythroid (BFU-E) Influenced by Stem Cell Factor, IL-3 and specific transcription factors such as GATA-1 and FOG-1 MEPs proliferate and differentiate into BFU-E cells. BFU-E are characterized by their ability to form large colonies or "bursts" of erythroid cells in culture Known for its ability to proliferate extensively Colony-forming unit-erythroid (CFU-E) Influenced by Erythropoietin (EPO) which responds to hypoxia condition and binds to EPO receptors expressed on BFU-E cell surface BFU-E subsequently differentiates into CFU-E. This involves a reduction in cell size, chromatin condensation and the initiation of haemoglobin synthesis CFU-E cells undergo rapid division and further maturation over a short period, typically 2-3 days. They form smaller colonies compared to BFU-E cells and are characterized by high responsiveness to EPO  Erythropoietin (EPO) A glycoprotein that regulates erythropoiesis 90% produced in kidney; 10% in liver No preformed storage, production in response to oxygen tension in kidney tissue Responds to hypoxia by Hypoxia-Inducible Factors (HIFs), which changes according to oxygen levels. HIF stabilizes and promote expression of EPO gene EPO response can be impaired by iron deficiency Watts, D., Gaete, D., Rodriguez, D., Hoogewijs, D., Rauner, M., Sormendi, S., & Wielockx, B. (2020). Hypoxia Pathway Proteins are Master Regulators of Erythropoiesis. International Journal of Molecular Sciences, 21(21), 8131. https://doi.org/10.3390/ijms21218131 Generation of ProErythroblasts (ProE) After the CFU-E stage, the next step in erythropoiesis is the differentiation into proerythroblasts (ProE) ProE are the earliest recognizable precursors in the erythroid lineage. They are highly mitotic and have high expression of EPO receptors Key features: Nucleus: centrally located, has a fine chromatin pattern and is almost perfectly round. High N:C ratio Cytoplasm: deeply basophilic and has a dark border. May contain cytoplasmic projections (ears). Overall size is 12 – 20µm. Generation of ProErythroblasts (ProE) After the CFU-E stage, the next step in erythropoiesis is the differentiation into proerythroblasts (ProE; sometimes called pronormoblast / rubriblast) ProE are the earliest recognizable precursors in the erythroid lineage. They are highly mitotic and have high expression of EPO receptors. Roughly 1% in bone marrow Key features: Nucleus: centrally located, has a fine chromatin pattern and is almost perfectly round. Very high N:C ratio (8:1). 0 – 2 nucleoli may be present. Cytoplasm: deeply basophilic and has a dark border. May contain cytoplasmic projections (ears). Overall size is 12 – 20µm.  Generation of Basophilic erythroblasts (BasoE) As ProE cells differentiate, they become smaller and start to accumulate ribosomes This gives the cytoplasm a basophilic (blue) appearance when stained and generating Basophilic Erythroblast (sometimes called prorubricyte) This stage is characterized by the condensation of chromatin and the beginning of haemoglobin synthesis. Roughly 1-5% in bone marrow Key features: Nucleus: dark round nucleus, chromosome is coarser and slightly clumped. High N:C ratio (6:1). 0 – 1 nucleoli may be present. Cytoplasm: very dark blue. May see a perinuclear halo. Overall size is 10 – 15µm.  Generation of Polychromic erythroblasts (PolyE) As BasoE cells mature, they start to accumulate more haemoglobin. This causes the cytoplasm to change color, appearing gray-green due to the mix of ribosomes (blue) and haemoglobin (pink) staining, generating polychromic erythroblasts (sometimes called rubricyte) The chromatin continues to condense, making the nucleus smaller and more compact. Roughly 5-30% in bone marrow. Normally NOT present in the peripheral blood but some may be seen in the peripheral blood smears of newborns Key features: Nucleus: round eccentric nucleus, chromatin is coarse and irregularly clumped. N:C ratio (4:1). Nucleoli absent. Cytoplasm: gray-blue to pink color. Abundant Overall size is 10 – 12µm.  Generation of Orthrochromic erythroblasts (OrthoE) As PolyE cells mature, they accumulate more haemoglobin This causes the cytoplasm to become uniformly pink color, generating Orthrochromic erythroblasts (sometimes called metarubricyte) Smallest RBC precursor and incapable of further DNA synthesis. Nucleus is dying and will be expelled from cell in the next stage (enucleation) Roughly 5-10% in bone marrow. Normally NOT present in the peripheral blood but some may be seen in the peripheral blood smears of newborns Key features: Nucleus: round, dark eccentric nucleus, fully condensed chromatin with pyknotic features. Low N:C ratio (1:1). Nucleoli absent. Cytoplasm: pink or salmon color. May appear slightly blue. Overall size is 8 – 10µm.  Generation of Orthrochromic erythroblasts (OrthoE) As PolyE cells mature, they accumulate more haemoglobin This causes the cytoplasm to become uniformly pink color, generating Orthrochromic erythroblasts (sometimes called metarubricyte) Smallest RBC precursor and incapable of further DNA synthesis. Nucleus is dying and will be expelled from cell in the next stage (enucleation) Roughly 5-10% in bone marrow. Normally NOT present in the peripheral blood but some may be seen in the peripheral blood smears of newborns Key features: Nucleus: round, dark eccentric nucleus, fully condensed chromatin with pyknotic features. Low N:C ratio (1:1). Nucleoli absent. Cytoplasm: pink or salmon color. May appear slightly blue. Overall size is 8 – 10µm.  Generation of Reticulocytes (Retics) The next step after OrthoE is the formation of reticulocytes (Retics; sometimes called Polychromatic Erythrocyte) Nucleus has now been expelled from the cell leaving only residual RNA, giving it a polychromatic appearance The use of supravital stains (eg. new methylene blue or brilliant cresyl blue) can help to identify and enumerate Retics by visualizing reticular inclusions Roughly 1% in bone marrow and 0.5 – 2% in peripheral blood Key features: Nucleus: Absent Cytoplasm: Light blue-purple to pink. Overall size is 8 – 8.5µm. Maturation into normochromic erythrocytes (RBC) Retics enter the bloodstream and finally matures into an erythrocyte (RBC) within one to two days Each RBC contains approximately 270 million haemoglobin molecules RBC have a biconcave shape, which means they are disc-shaped with a central indentation on both sides. This shape increases their surface area for gas exchange and allows them to deform as they pass through narrow capillaries Predominant in peripheral blood Key features: Nucleus: Absent Cytoplasm: Salmon pink. Contains a central pallor which is normally one-third of the cell’s diameter. Overall size is 7 – 8µm.  Membrane of RBC Membrane lipids Red cell membrane is a lipid bilayer, molar ratio of cholesterol to phospholipid molecules is 1:1 Principle of red cell membrane structure, disturbance of this ratio may affect its deformability Membrane proteins Transmembrane (integral) proteins provide anion channel through the cell membrane, and oligosaccharide chain attached to external surface provide negative charge surface Band 3 protein (anion exchanger), glycophorin A Cytoskeletal (skeletal, peripheral) proteins provide structural network on the inner surface of the membrane, giving it the biconcave structure Spectrin, protein 4.1, actin, ankyrin  Permeability of RBC membrane Red cell membrane Freely permeable to water and anions [bicarbonate (HCO3-) and chloride (Cl-)] Impermeable to monovalent and divalent cations (Na+, K+, Ca2+ and Mg2+) Take up glucose by glucose transporter which does not require ATP Na+/K+ cation pump and Ca2+-ATPase pump Maintain cations levels different between erythrocyte and plasma Maintain erythrocyte osmotic equilibrium If red cell membrane permeability to cations increase or if cation pumps fail, Na+ accumulates in the cells in excess of K+ loss, water moves in, cell swells and results in osmotic haemolysis  RBC metabolism Binding, transport, and release of oxygen and carbon dioxide are not required energy Essential energy-dependent metabolic process require for erythrocyte viability Cation pumps to move cations against electrochemical gradients, Haemoglobin iron in reduced state (Fe2+), Reduce sulfhydryl groups in haemoglobin and other proteins, Red cell membrane integrity and deformability Mature red cells has no mitochondria for citric acid cycle, energy source (ATP) rely solely on anaerobic glycolysis Four important metabolic pathways for red cell function: Glycolytic pathway: anaerobic pathway of glucose metabolism for ATP production Hexose-Monophosphate (HM) pathway: protect erythrocytes from oxidant damage Methemoglobin reductase pathway: methemoglobin reduction Rapoport-Luebering shunt: generates 2,3-DPG to alter haemoglobin-oxygen affinity  Glucose hexokinase Glycolytic Pathway Glucose 6-phosphate Fructose 6-phosphate An anaerobic pathway of glucose metabolism (formerly called Embden-Meyerhof pathway) depends on plasma glucose Glyceraldehyde 3-phosphate 90 – 95% red cell glucose metabolized in this pathway from glucose to pyruvate with G3P dehydrogenase a net gain of 2 moles of ATP per mole of glucose 1,3-diphosphoglycerate ATP is necessary to maintain erythrocytes shape, flexibility, and membrane integrity, and to regulate intracellular cation concentration Phosphoglycerate kinase Abnormal cation permeability and/or decrease ATP production results in change 3-diphosphoglycerate from biconcave disc to sphere shape Production of glucose-6-phosphate (Hexose Monophosphate Pathway) Phosphoenolypyruvate Maintain pyridine nucleotide in a reduced state (NADH) [MetHb reductase pathway] Pyruvate kinase Production of 1,3-DPG (Rapoport-Luebering shunt) Pyruvate  Key enzymes of RBC Glycolytic Pathway Hexokinase: Catalyzes the phosphorylation of glucose to glucose-6-phosphate, the first step in glycolysis. This step traps glucose in the cell and commits it to the glycolytic pathway Phosphofructokinase-1: Catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. This is a key regulatory step in glycolysis, often considered the rate-limiting step Aldolase: Splits fructose-1,6-bisphosphate into two three-carbon molecules, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate Glyceraldehyde-3-phosphate Dehydrogenase: Catalyzes the oxidation and phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. This step produces NADH, which is crucial for maintaining the redox balance in RBCs Phosphoglycerate Kinase: Converts 1,3-bisphosphoglycerate to 3-phosphoglycerate, generating ATP in the process Pyruvate Kinase: Catalyzes the final step of glycolysis, converting phosphoenolpyruvate to pyruvate and producing ATP. This enzyme is another key regulatory point in glycolysis  Hexose Monophosphate Pathway 5 – 10% red cell glucose metabolized in this oxidative pathway, an ancillary pathway to produce reducing substance. Also called pentose phosphate pathway (PPP) H2O2 H20 Functionally dependent on G6P dehydrogenase (G6PD) G6PD oxidize glucose-6-phosphate (G6P) to 6-phosphogluconate (6PG), NADP is Glutathione peroxidase reduced to NADPH NADPH converts GSSG (glutathione disulfide; oxidized form) back to GSH GSH GSSG (glutathione; reduced form), which is necessary to maintain haemoglobin in the reduced functional state Glutathione reductase Defect causes haemoglobin sulfhydryl groups (-SH) to be oxidized resulting in Hb denature and precipitate (Heinz body) which attached to inner surface of red cell NADP NADPH membrane causing decrease cell flexibility leading to prematurely removal by macrophages in spleen Glucose 6-phosphate 6-phosphogluconate G6PD  Key enzymes of HMP Glucose-6-Phosphate Dehydrogenase: Catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone, producing NADPH in the process. This enzyme is crucial for protecting RBCs from oxidative damage by maintaining the supply of NADPH 6-Phosphogluconolactonase: Hydrolyzes 6-phosphoglucono-δ-lactone to 6-phosphogluconate. Ensures the efficient progression of the oxidative phase by preventing the accumulation of the lactone intermediate 6-Phosphogluconate Dehydrogenase: Catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribulose-5-phosphate, producing NADPH and CO2. Further contributes to the cell’s pool of reducing agents and provides ribulose-5- phosphate for nucleotide biosynthesis  Methaemoglobin Reductase Pathway Methaemoglobin (haeme iron in oxidized state, ferric, Fe3+) unable to bind oxygen MetHb reductase pathway is an offshoot of the glycolytic pathway to maintain haeme iron in the reduced state (ferrous, Fe2+). Also known as Cytochrome b5 Reductase Pathway In the absence of MetHb reductase pathway, 2% of methaemoglobin formed daily eventually builds up to 20-40%, this may severely limit the blood’s oxygen-carrying capacity resulting in cyanosis Glyceraldehyde 3-phosphate NAD MetHb reductase +H Methaemoglobin G3P dehydrogenase NADH MetHb reductase Haemoglobin 1,3-diphosphoglycerate  Key enzymes of MRP Cytochrome b5 Reductase (Methaemoglobin Reductase): This enzyme transfers electrons from NADH to cytochrome b5, which in turn reduces methaemoglobin (MetHb) back to haemoglobin (Hb). It is essential for converting methaemoglobin, which cannot bind oxygen, back to functional haemoglobin, ensuring efficient oxygen transport1 Cytochrome b5: Acts as an electron carrier, transferring electrons from cytochrome b5 reductase to methaemoglobin. Facilitates the reduction of methaemoglobin to haemoglobin, playing a critical role in maintaining the proper function of RBCs  Rapoport-Luebering shunt It is a part of the glycolytic pathway, which 1,3-diphosphoglycerate bypass the formation of 3-phosphoglycerate and ATP from 1,3- diphosphoglycerate (1,3- Diphosphoglycerate DPG) mutase Instead, 2,3-DPG (also known as 2,3- bisphosphoglycerate) formed from 1,3-DPG Phosphoglycerate kinase ADP ATP and sacrifices one ATP-producing step 2,3- DPG 2,3-DPG binds to hemoglobin, and facilitate the release of oxygen by decreasing the oxygen affinity Diphosphoglycerate phosphatase 3-diphosphoglycerate  Key enzymes of MRP Bisphosphoglycerate Mutase (also known as Diphosphoglycerate mutase): Catalyzes the conversion of 1,3-bisphosphoglycerate (1,3-BPG) to 2,3-bisphosphoglycerate (2,3-BPG). This enzyme is essential for the production of 2,3-BPG, which decreases haemoglobin's affinity for oxygen, facilitating oxygen release to tissues Bisphosphoglycerate Phosphatase (also known as Diphosphoglycerate phosphatase): Hydrolyzes 2,3-BPG to 3-phosphoglycerate (3-PG). This step allows the pathway to integrate back into the glycolytic pathway, ensuring the continuation of glycolysis and energy production  Erythrocyte Metabolic Pathways Hexose Monophosphate Pathway Glycolytic Pathway Rapoport-Luebering shunt Methaemoglobin Reductase Pathway  Further reading Cha, H.J. Erythropoiesis: insights from a genomic perspective. Exp Mol Med 56, 2099–2104 (2024). https://doi.org/10.1038/s12276-024-01311-1 Hoffbrand AV and Moss PAH. (2016). Hoffbrand’s Essential Hematology, 7th ed. Chichester: Wiley-Blackwell. Turgeon ML. (2018). Clinical Hematology: theory & procedures, 6th ed Philadelphia: Wolters Kluwer. Keohane EM, Otto CN, & Walenga JM. (2020). Rodak’s Hematology: Clinical Principles and Applications. 6th Edition. St Louis: Elsevier.