Erythropoiesis

Sites of Erythropoiesis

Regulation by Erythropoietin (EPO)

• Hypoxia (from anemia, altitude, cardiopulmonary disease) stimulates HIF-2α in renal interstitial cells
• HIF-2α drives EPO gene transcription
• EPO binds EPO receptors (EPO-R) on erythroid progenitors and promotes their survival, proliferation, and differentiation via the JAK2/STAT5 signaling pathway
• Rising red cell mass corrects tissue oxygenation, completing the negative feedback loop

Progenitor Hierarchy (Pre-Morphological Stages)

HSC → CMP → MEP → BFU-E → CFU-E → Proerythroblast

• The full progenitor chain from stem cells is: Stem cell → CFU-GEMM → BFU-EMeg → BFU-E → CFU-E → Pronormoblast
• GATA-1 is the master transcription factor required for terminal erythroid differentiation

Morphologically Recognizable Stages (Maturation Series)

1. Proerythroblast (Pronormoblast)

• Size: 12-20 μm (largest erythroid precursor)
• Nucleus: large, spherical, with 1-2 nucleoli
• Cytoplasm: mild basophilia (free ribosomes beginning hemoglobin synthesis)
• Duration: ~24 hours

2. Basophilic Erythroblast

• Size: 10-16 μm (smaller than proerythroblast)
• Nucleus: smaller, progressively more heterochromatic
• Cytoplasm: strong basophilia due to abundant polyribosomes actively synthesizing hemoglobin
• Duration: ~24 hours; undergoes mitosis

3. Polychromatophilic Erythroblast

• Cytoplasm: mixed blue and pink (both ribosomes and accumulating hemoglobin present)
• Nucleus: checkerboard heterochromatin pattern; smaller than previous stage
• Duration: ~30 hours
• Last stage capable of mitosis

4. Orthochromatophilic Erythroblast (Normoblast)

• Cytoplasm: predominantly eosinophilic (large amount of hemoglobin)
• Nucleus: small, compact, pyknotic (dense, dark-staining)
• At this stage, the nucleus is extruded - this is the last nucleated stage
• Cannot undergo further division

5. Reticulocyte (Polychromatophilic Erythrocyte)

• Anucleate
• Still contains residual ribosomes and mRNA - capable of hemoglobin synthesis
• Stains with supravital dyes (e.g., new methylene blue) showing a reticulum of RNA
• Released from bone marrow into blood
• Circulates for 1-2 days; final maturation occurs in the spleen where ribosomes/mRNA are removed
• Normal reticulocyte count: ~0.5-2.5% of RBCs

6. Mature Erythrocyte

• 7.2 μm biconcave disc
• Anucleate, no organelles
• Packed with hemoglobin (~34 g/dL intracellular)
• Lifespan: ~120 days
• Removed by macrophages of spleen, bone marrow, and liver (the mononuclear phagocyte system)

RNA vs. Hemoglobin During Maturation

Kinetics Summary

• From basophilic erythroblast → circulation takes approximately 1 week
• Each proerythroblast undergoes 4 mitotic divisions (producing ~16 erythrocytes per progenitor cell)
• Mitosis occurs at: proerythroblast, basophilic erythroblast, and polychromatophilic erythroblast stages only
• Bone marrow is not a storage site for erythrocytes - cells are released as soon as formed

Nutritional Requirements

• Reduced reticulocyte count despite erythroid hyperplasia in marrow
• Elevated serum bilirubin and LDH
• Accelerated iron turnover

Iron Recycling

• Each mL of blood contains ~0.5 mg of iron
• The gut absorbs only ~1 mg iron/day (~5-10% of dietary intake)
• The majority of iron for erythropoiesis comes from recycling of iron from aged RBCs via splenic and hepatic macrophages
• Intracellular iron, heme, and globin must be precisely matched - excess of any is toxic

Red Cell Destruction (End of Lifespan)

• Globin → hydrolyzed to amino acids → recycled
• Iron from heme → stored as ferritin/hemosiderin in spleen → recycled to erythropoiesis
• Heme porphyrin ring → partially degraded to bilirubin → bound to albumin → transported to liver → conjugated to bilirubin glucuronide → excreted in bile

Clinical Relevance

• Histology: A Text and Atlas with Correlated Cell and Molecular Biology, pp. 737-746
• Basic Medical Biochemistry: A Clinical Approach 6e, pp. 1535-1536
• Brenner and Rector's The Kidney, pp. 2465-2470

Erythropoiesis - MBBS Year 1 Physiology

Definition

1. Sites of Erythropoiesis

2. Genesis of Blood Cells - Stem Cell Hierarchy

Figure: Blood cell formation from MHSC in bone marrow (Guyton & Hall, Fig. 33.2)

• Growth inducers (e.g., IL-3) - promote proliferation, not differentiation
• Differentiation inducers - cause commitment to a specific cell lineage

3. Stages of Differentiation (Maturation Series)

Figure: Genesis of RBCs and RBC appearance in different anemias (Guyton & Hall, Fig. 33.3)

CFU-E → Proerythroblast

• First morphologically recognizable stage
• Large cell with prominent nucleus
• Cytoplasm begins accumulating components for Hb synthesis

Proerythroblast → Basophil Erythroblast (1st generation)

• Stains strongly with basic dyes (deeply basophilic cytoplasm)
• Rich in ribosomes (polyribosomes) actively synthesizing hemoglobin
• Undergoes mitosis → divides several times

Basophil Erythroblast → Polychromatophil Erythroblast (2nd-3rd generation)

• Hemoglobin first appears in the cytoplasm at this stage
• Mixed staining: both basophilic (ribosomes) and acidophilic (hemoglobin)
• Nucleus becomes progressively smaller and denser

Polychromatophil → Orthochromatic Erythroblast (4th generation)

• Cytoplasm becomes predominantly eosinophilic (filled with Hb ~34%)
• Nucleus condenses to small pyknotic remnant
• Nuclear remnant is absorbed or extruded from cell
• Endoplasmic reticulum also reabsorbed at this stage

Orthochromatic Erythroblast → Reticulocyte

• Anucleate - no longer has a nucleus
• Still contains small amounts of basophilic material: remnants of Golgi apparatus, mitochondria, and cytoplasmic organelles
• Named "reticulocyte" because of this reticular (net-like) basophilic material seen with supravital stains
• Passes from bone marrow into blood capillaries by diapedesis (squeezing through capillary pores)

Reticulocyte → Mature Erythrocyte

• Basophilic material disappears within 1-2 days in circulation
• Normal reticulocyte count = slightly less than 1% of all RBCs
• Mature RBC: biconcave disc, ~7-8 μm diameter, no nucleus or organelles, packed with hemoglobin

Memory tip: Pro → Baso → Poly → Ortho → Retic → RBC ("Please Be Polite Or Remain Respectful")

4. Regulation of RBC Production - Erythropoietin

Figure: Erythropoietin feedback loop (Guyton & Hall, Fig. 33.4)

The Key Hormone: Erythropoietin (EPO)

• A glycoprotein, molecular weight ~34,000
• Produced: 90% kidneys (peritubular fibroblast-like interstitial cells in cortex and outer medulla), 10% liver
• EPO stimulates production of proerythroblasts from hematopoietic stem cells, and speeds up all subsequent maturation steps

The Feedback Loop:

Hypoxia / Low O₂ delivery
        ↓
Kidney senses ↓ O₂ → produces EPO
        ↓
EPO acts on bone marrow CFU-E
        ↓
↑ Proerythroblast formation
        ↓
↑ RBCs released into circulation
        ↓
Tissue oxygenation restored
        ↓
EPO production falls (negative feedback)

Molecular Mechanism:

• Hypoxia → ↑ HIF-1 (Hypoxia-Inducible Factor-1) in renal tissue
• HIF-1 binds to hypoxia response element in the EPO gene
• → Transcription of EPO mRNA → ↑ EPO synthesis

Timeline:

• EPO production begins within minutes to hours of hypoxia
• Reaches maximum within 24 hours
• New RBCs appear in blood only after ~5 days

Factors that DECREASE tissue oxygenation → INCREASE EPO → INCREASE RBC production:

5. Maturation Factors - Nutritional Requirements

A. Vitamin B12 (Cyanocobalamin)

• Essential for DNA synthesis
• Deficiency → cells cannot divide normally → abnormally large cells (megaloblasts)
• Absorbed in terminal ileum with the help of Intrinsic Factor (produced by gastric parietal cells)
• Deficiency causes: Megaloblastic (Pernicious) Anemia

B. Folic Acid

• Also essential for DNA synthesis (one-carbon transfer reactions)
• Deficiency produces identical blood picture to B12 deficiency
• Megaloblastic anemia - large oval macrocytes, hypersegmented neutrophils

C. Iron

• Essential for hemoglobin synthesis (incorporated into heme)
• Total body iron: 4-5 g
  • ~65% in hemoglobin
  • ~4% in myoglobin
  • ~15-30% stored as ferritin/hemosiderin
  • ~0.1% in plasma as transferrin-bound iron

• Absorbed from small intestine as Fe²⁺ (ferrous form)
• Transported in plasma bound to transferrin (beta-globulin)
• Stored in cells as ferritin (soluble) or hemosiderin (insoluble)
• Transferrin delivers iron to erythroid precursors by binding to receptors on erythroblasts → endocytosis → iron delivered to mitochondria for heme synthesis

Figure: Iron transport and metabolism (Guyton & Hall, Fig. 33.7)

6. Life Span and Destruction of RBCs

• Life span: ~120 days
• Old RBCs become fragile because metabolic enzymes become progressively less active
• Self-destruct mainly in the spleen (red pulp spaces only 3 μm wide vs. 8 μm RBC diameter)
• Destroyed RBCs are phagocytized by macrophages (Kupffer cells of liver, splenic macrophages, bone marrow macrophages)

7. Anemias - Applied Physiology

Quick Revision Summary

SITES:  Yolk sac → Liver → Bone marrow (adult)

STAGES: CFU-E → Proerythroblast → Basophil → Polychromatophil
        → Orthochromatic → Reticulocyte (1-2 days) → RBC (120-day lifespan)

REGULATION: Hypoxia → HIF-1 → Kidney → EPO → Bone marrow → ↑ RBCs

MATURATION FACTORS: Iron (Hb synthesis) + B12 + Folate (DNA synthesis)

DESTRUCTION: Spleen (mainly) + Macrophages
             → Iron recycled; Heme → Bilirubin → Bile