Haemopoiesis

• Haemopoiesis involves identifying the different haematopoietic sites from fetal to adult life.
• It includes describing the pluripotential stem cell, and the the functional capabilities of haematopoietic growth factors by target cells
• The understanding and evaluation of erythropoiesis is an important factor
• Describe the site of production for erythropoietin and what stimulates its synthesis to allow understanding of this process
• Defining and explaining the significance of a reticulocyte

Key Terms

• Haemopoiesis is the process of blood cell formation.
• Erythropoiesis regulates haemopoiesis and early formation of red cells.
• Myelopoiesis is the formation of granulocytes and monocytes.
• Thrombopoiesis is the formation of platelets.

Sites of Haemopoiesis

• The yolk sac is the main site of haemopoiesis during the first few weeks of gestation.
• The first haematopoietic stem cells are observed on the Aorta-Gonads-Mesonephros (AGM) region.
• The liver and spleen take over from about 6 weeks to 6-7 months of fetal life and continue until about two weeks after birth.
• Bone marrow takes over the process by 5 months BM.
• Bone marrow becomes the only site of haemopoiesis during childhood and adult life.
• Developing cells are located outside the bone marrow sinus.
• Mature cells are released into the sinus spaces marrow microcirculation general circulation.
• All of the bone marrow is haemopoietic during infancy.
• Progressive fatty replacement of marrow occurs throughout the long bones during childhood.
• In adult life, haemopoietic marrow is confined to the central skeleton and proximal ends of the femurs and humeri.
• 50% of marrow consists of fat.
• Sites slowly replaced with yellow inactive marrow during old age.
• The Liver and spleen can resume haemopoietic function, being extramedullary haemopoiesis
• Intramedullary refers to haemopoiesis within the bone marrow.
• Extramedullary refers to haemopoiesis activity in sites other than the bone marrow.
• In adulthood, extramedullary is present only in pathological conditions like when Bone Marrow is infiltrated with fibrous tissues (myelofibrosis)

Origin of Haemopoiesis the Stem Cell

• Haemopoiesis starts with a pluripotential stem cells.
• It's able to self-renew by asymmetric cell division and give rise to separate cell lineages.
• Cell differentiation happens from stem cell via committed progenitor (multipotent) cells.
• An example of a progenitor cell is the mixed myeloid precursor which gives rise to granulocytes, erythrocytes, monocytes and megakaryocytes.
• This is termed as CFU (colony forming unit)-GEMM
• The bone marrow is also the primary site of lymphocytic origin
• Colony Forming Units (CFU) are colonies formed from multipotential stem cells.
• Colony Forming Units (CFU) are capable of developing into specific mature cells. -CFUEo creates Eosinophils -CFUBaso creates Basophils -CFUMeg creates Megakaryocyte (Platelets) -CFUGM creates Neutrophils and Monocytes -CFUE creates Erythroid (red cells)

Bone Marrow Components

• The bone marrow is the site of erythropoiesis.
• At birth, all marrow is red, but with age, more of it is converted to yellow marrow.
• In adults, red active marrow is mainly found in the flat bones and at the epiphysial ends of long bones such as femur and humerus.
• Bone marrow comprises soft tissue referred to as red and yellow marrow.
• Red marrow is haemopoietic tissue.
• Yellow marrow is fatty cells.
• The bone marrow forms a suitable environment for the survival of stem cells.
• It's composed of stroma cells (stroma) and a microvascular network.
• Stroma cells consist of Mesenchymal cells, adipocytes, fibroblasts, endothelial cells and macrophages.
• Stroma cells secrete extracellular molecules such as collagen, glycoproteins and glycosaminoglycans which form extracellular matrix.
• Growth factors or cytokines are also secreted from the stroma and are necessary for regulation and cell survival.
• Mesenchymal cells are critical in stromal cell formation.
• Niches are local tissue environments that maintains and regulates stem cells.
• HSCs and hematopoietic progenitors reside in these microenvironments.
• The niche is perivascular, created partly by mesenchymal stromal cells and endothelial cells and often, near trabecular bone
• This is present in diverse tissues throughout development beginning in the aorta-gonad-mesonephros (AGM) region and the yolk sac
• There are three types of niches including
  • Endosteal
  • Reticular
  • Vasicular

Regulation of Haemopoiesis

• Transcription factors regulate gene expression by controlling transcription of specific genes of gene families, where mutations lead to cancers.
• Growth factors and hormones play a huge roll
• Adhesion molecules meditate attachment of marrow precursors, leucocytes and platelets to various extracellular cells and are important in inflammatory and immune responses.
• The cell cycle regulates and dysregulation of proliferation prevents cancers.
• Epigenetics: Changes in DNA and chromatin affect gen expression, without affecting DNA sequence.
• Apoptosis happens as group activity
• Several transcription factors (TFs) such as Scl/Tal1, Gata2, Tel, Fli1, or Runx1 are critical for the specification and/or biological function of HSC's.
• Growth factors/Hormones can regulate proliferation and differentiation of progenitor cells and mature blood cells
• They can stimulate cell maturation and prevents apoptosis

Types of Growth Factors

• Factors acting on stromal cells include IL-1 and TNF.
• Factors acting on pluripotential stem cells include SCF, FLT3-L, and VEGF.
• Factors acting on multipotential progenitor cells include IL-3, GM-CSF, IL-6, G-CSF, and Thrombopoietin.
• Factors acting on committed progenitor cells contain G-CSF, M-CSF, IL-5 (eosinophil-CSF), Erythropoietin, Thrombopoietin

Erythropoiesis

• Erythropoiesis is a term used to describe red cell production.
• The Greek prefix erythro- meaning red and suffix- poiesis meaning production.
• Erythrocyte production takes place in tissues collectively known as haematopoietic tissue, that contain mesynchymal cells
• During fetal stages, erythropoiesis happens in the yolk sac and then transfer to the liver and spleen before localizing to the bone marrow after birth

Regulation and Action of Erythropoiesis

• Regulation happens in blood stream by stimulated oxygen tension due to hormones as required
• Erythropoiesis is regulated by the hormone erythropoietin.
• Erythropoietin is a glycoprotein mainly produced in the kidney (90%) and in the liver (10%).
• Erythropoietin is not stored but mainly stimulated by Oxygen (O2) tension.
• Hypoxia stimulates the production of erythropoietin through the hypoxia inducible factor (HIF-1a and B).
• Erythropoietin increases in anaemic states or any case that leads to dysfunction in hemoglobin unable to give up O2 to the cells (cardiac or pulmonary disorders or renal functions)
• Erythropoietin stimulates erythropoiesis by increasing progenitor cells committed to erythropoietin, and are activated by erythropoietin
• GATA-2 is a transcription factor involved in initiating erythroid differentiation from stem cells
• GATA-1 and FOG are activated by erythropoietin enhancing expression of erythroid specific genes and anti-apoptotic genes
• Erythroid Colony forming units(CFU (E)) are stimulated to proliferate, differentiate and produce haemoglobin
• Increased in anemic states, when HB is defective to give up O2, when atmospheric O2 us low,
• And when Cardiac, Pulmonary function or Renal circulation is impaired due defective renal function.
• Conversely, the body decreases erythropoietin when there is an increase in O2 supply.
• There are no stores for erythropoietin
• Its Production stimulated by oxygen tension in the tissues of the kidney, which can be therapeutically used like.
• For Anaemia in chronic renal disease. and Anaemia associated with malignancy and chemotherapy.
• Recombinant erythropoietin use, such as, Aids, Preoperative uses and Anaemia of prematurity.

Other Factors Regulating Erythropoiesis

• Protein in nutritional factor aspect
• Iron, Cu, Zn, Co- Hb synthesis in minerals content
• Testosterone, the hormones Thyroxine and Adrenal hormones content. and Pituitary hormones stimulate the release of Erythropoietin in hormones content
• Vitamins such as B12 & folic acid - for synthesis of DNA.
• Riboflavin as it normalises bone marrow division to create cells
• Pyridoxine for heme synthesis
• Vitamin C -Absorption of Fe from gut
• Neural such as Stimulation of Hypothalamus ↑ RBC production

Erythropoiesis

• Erythroid stem cells go through maturation to become a red cell that contains mesynchymal cells
• Erythropoiesis passes from stem cells, through progenitor cells, to pronormobalst as it synthesises and prepares
• Pronormoblasts mature to reticulocytes in 6 days
• Reticulocytes are retained in the bone marrow for 2 to 3 days depending on RBC demand before release of mature RBC,
• Reticulocytes which takes the release bone Marrow one day to mature into fully developed red blood cell
• Cell changes occur where there are more cells from pronormoblasts
• Cells decrease in cell diameter and the nucleus to cytoplasm ratio is decreased, as there in synthesis
• Chromatin condensation and gradual increase in Synthesis
• Cytoplasmic changes where and increase in deep blue to basophilic and pink colour can be seen in acidophilic process
• When the erythropoiesis matures, reports on nomenclature of blood cells were issued between 1948 and 1950
  • Rubriblast (Pronormoblast or Proerythroblast)= First red cell precursor,
  • Prorubricyte (Basophilic normoblast or early erythroblast)
  • Rubricyte (polychromatophilic normoblast or intermediate erythroblast)
  • Metarubricyte (orthochromatic or acidophilic normoblast or late erythroblast)
  • Reticulocyte
  • Erthrocyte

Stages of Maturation

• There are seven stages of maturation, but only six are morphologically distinct
• Classification of each stage is shown from oldest to youngest for earliest stage to mature cells -[root]blast -Pro[root]cyte -[root]cyte -Meta[root]cyte -Mature Cell -The root for CFU-E is which is "rubri", and CFU-GM which is "Granulo" followed by CFU-L is "Lympho” and CFU-Me is “Megakaryo"

Erythroblast: Stage 1 cells

• These cells Contains large Nucleoli
• 80% Nuclei with dispersed and and distinct Chromatin which stains in reddish and white
• Cytoplasms is stained in dark Blue with a dimeter of 14 um

Stages E2 & E3: Prorubricyte-Prerubioblast

• Size diameter 12 to 17 um
• Shapre is irregular to oval
• Nucleus with is compact condensed 75% the size of total Cell , the appearance of Card wheel as 2 nucleoli
• Cytoplasm is blue but during hemoglobin synth it stains sometimes to reddish and occurs around 1 to 2 days

Stages E4: Rubricyte or E4

• Size 10 to 15 m
• Shape with Irregular round
• 25 % is Nucleaus and the chormarin appears darker
• Retains its Card wheel shape at its typical eccentric location
• Its no have nucleoli with round an colour
• Cytopasm is blue with a pink tint with rapid color change
• The total Matuartion take at about 24 hours

Stage E5: Metarubricyte

• Its 10 to 15 um diameter with a slight oval shape
• With the eccentric nucleus with 25 % of dark shade
• The chormatin has an limited structure
• It usually round or 3 leaf clover shape and doens not a hve nucleoli
• At this states the nuclei is ejected and divide so it takes 30 hours
• With a slight blue to light pink in perinuclear area

Stage E6 - Reticulocyte

• They contains are diameters of 0 to 10 um
• With Irregular shape when exposed to supravital stain and a wet environment
• Its shows that in younger than normal erythrocytes
• It contain parts of Organelles , ribosomes ,mitochondrial visualized in supravital as it maturation time
• It exist for 2 to 3 hours

Stage E7 Erthrocyte

• Diameter 7.4 to 8.2 m
• Can Be deformed in to concave disc
• The cytoplasm transition Centre and has hemoglobin
• Its area of thinnest area can show that hase transition from its center its Life is 120 Days

Reticulocytes

• Immature(stage just before the mature red), non-nucleated red blood cells.
• Has it contain remaining ribosomal RNA that is visible under the microscope with supravital stains.
• Develop in bone marrow and circulate in the blood stream for bone marrow and bout a day before developing into a mature erythrocyte
• They made from when the normoblast's nucleus is violently ejected
• It stays in marrow for 2 to 3 till enter in to blood and matures in 1 day and after release

Ineffective Erythropoiesis

• 10-15 %developing erythroblasts die within the marrow without developing into mature cells
• Where the bilirubin (breakdown of HB) and lactate dehydrogenase (breakdown of cells) will be raised
• The count is low in due to anemia and of of eyrthroblast
• With steady state Proliferation, differentiation, apoptosis
• With ineffective it leads to increased Proliferation, differentiation and apoptosis

Assessment of Erythropoiesis

• total and it can assessed examine that with bone marrow and reticulocyte count its Feedback the problems such the stem cell's the to Bone Marrow
• it The that assessment total Effective that cellularity in is in

erythropoiesis

Factors anemia Cancers VitB12 Reduced infections

• Oxygen erythrocyte

Reticulocyte count

• It must present Retics
• To that amount it is 50×10^9
• Response

Retictulocyte response to anemia

• Marrow iron
• Erythropoietin infections

Reticulocytes production index

• Its More than in stem cell

Normal vs Accelerated Erythropoiesis

• accelerated
• Normal cells

Reticulocytes

• Normal

Erythropoietin

• Wet high losses