Hemopoiesis

Introduction to Hemopoiesis

• Definition: Hemopoiesis (or Hematopoiesis) is the physiological process of blood cell (blood formed element) production.

• Mechanisms: It involves the multiplication of stem cells and their subsequent differentiation into specific lineages.

• Objective: The primary objective is to maintain a constant, homeostatic level of circulating blood cells.

Chronological Phases of Hemopoiesis During the Human Lifespan

• Early Embryo (Yolk Sac Phase):

  • Blood cells arise from the yolk sac.

  • These cells have a mesodermal origin.

• Second Trimester (Hepatic Phase):

  • Hemopoiesis occurs mostly in the liver.

  • The spleen plays a minor role during this phase.

• Third Trimester and Postnatal (Bone Marrow Phase):

  • The bone marrow becomes the major hematopoietic organ.

Anatomical Localization of Cell Lineage Differentiation

• Lineages occurring strictly in the Bone Marrow:

  • Erythropoiesis (production of red blood cells).

  • Granulopoiesis (production of granulocytes).

  • Monocytopoiesis (production of monocytes).

  • Thrombocytopoiesis (production of platelets).

• Lineages occurring in both Bone Marrow and lymphatic organs:

  • Lymphopoiesis (production of lymphocytes).

Monophyletic Theory of Hemopoiesis

• Hematopoietic Stem Cell (HSC):

  • The HSC is a pluripotent cell.

  • It exhibits the property of self-renewal (dividing to yield more HSCs).

  • It exhibits the property of differentiation (dividing to yield progenitor cells).

• Multipotential Progenitor Cells: Pluripotent progenitor cells differentiate into two major colonies:

  • Common Myeloid Progenitor (CMP) Cells: These differentiate into lineage-restricted progenitors.

  • Common Lymphoid Progenitor (CLP) Cells: These differentiate into lymphocytes.

Lineage-Restricted Progenitors

Common Myeloid Progenitor (CMP) Lineages

1. Megakaryocyte/Erythrocyte Progenitor (MEP) Cells: Bipotential stem cells that further divide into:

   • Monopotent Megakaryocyte-Committed Progenitor Cells: Also known as MKP or CFU-Meg.

   • Monopotent Erythrocyte-Committed Progenitor Cells: Also known as ErP or CFU-E.

2. Granulocyte/Monocyte Progenitor (GMP or CFU-GM): Differentiation requires high levels of the transcription factor $PU.1$. Subtypes include:

   • Neutrophil Progenitors (NoP or CFU-G): Produce Neutrophils.

   • Eosinophil Progenitors (EoP or CFU-Eo): Produce Eosinophils.

   • Basophil/Mast Cell Progenitors (BMCP):

     • Basophil progenitor cells (BaP or CFU-Ba) in the bone marrow produce Basophils.

     • Mast progenitor cells (MCPs) in the gastrointestinal mucosa produce Mast cells.

   • Monocyte Progenitors (MoP or CFU-M): Produce Monocytes.

Common Lymphoid Progenitor (CLP) Lineages

1. B cells

2. T cells

3. NK cells

Morphological Changes During Development

• Stem and Progenitor Cells: These cells cannot be morphologically distinguished from one another using standard microscopy. They generally resemble large lymphocytes.

• Precursor Cells (Blasts): These derive from specific progenitor cells. Blasts gradually assume the morphological characteristics defining the mature, functional cell types they will become.

Hemopoietic Growth Factors (Cytokines)

• Definition: Also known as colony-stimulating factors (CSF) or cytokines. They are glycoproteins that stimulate the proliferation of progenitor and precursor cells, as well as differentiation and maturation within specific lineages.

Detailed Cytokine Function and Sources

• Interleukin-1 ($IL-1$): Regulates activities and cytokine secretion of many leukocytes; sourced from Macrophages and T helper cells.

• Interleukin-2 ($IL-2$): Mitogen for activated T and B cells; promotes NK cell differentiation; sourced from T helper cells.

• Interleukin-3 ($IL-3$): Mitogen for all granulocyte and megakaryocyte progenitor cells; sourced from T helper cells.

• Interleukin-4 ($IL-4$): Promotes development of basophils and mast cells and B-lymphocyte activation; sourced from T helper cells.

• Interleukin-5 ($IL-5$): Also known as eosinophil differentiation factor (EDF). Promotes development and activation of eosinophils; sourced from T helper cells.

• Interleukin-6 ($IL-6$): Mitogen for many leukocytes; promotes B cell and regulatory T cell activation; sourced from Macrophages, neutrophils, and local endothelial cells.

• Interleukin-7 ($IL-7$): Major mitogen for all lymphoid stem cells; sourced from stromal cells of the bone marrow.

Specific Lineage Stimulators

• Erythropoietin ($EPO$): Specifically stimulates the erythroid lineage.

• Thrombopoietin: Specifically stimulates the thrombopoietic lineage.

• Granulocyte/Monocyte-CSF ($GM-CSF$): Stimulates granulocytic and monocytic lineages (affects all myeloid progenitor cells).

• Granulocyte-CSF ($G-CSF$): Specifically stimulates the granulocytic lineage.

• Monocyte-CSF ($M-CSF$): Specifically stimulates the monocytic lineage.

Histology of the Bone Marrow

• Localization:

  • Medullary canals of long bones (Yellow bone marrow).

  • Cavities of cancellous bone (Red bone marrow).

• Yellow Bone Marrow (BM): Composed primarily of adipose tissue.

• Red Bone Marrow (BM): Composed of hematopoietic tissue.

  • In newborns, all bone marrow is red.

  • As an organism grows, red BM is replaced by yellow BM.

  • Yellow BM can revert to red BM in cases of severe hypoxia.

Microscopic Structure of Red Bone Marrow

• Stroma:

  • Consists of reticular connective tissue formed by stromal cells (specialized fibroblastic cells) and reticular or adventitial cells.

  • Matrix components: Collagen type I, Proteoglycans, Fibronectin, and Laminin (which interacts with integrins to bind cells to the matrix).

• Parenchyma:

  • Consists of hemopoietic cords (islands) of cells.

  • Surrounded by sinusoids.

  • Hemopoietic cells and macrophages are supported by reticular fibers.

Erythropoiesis

• Timeline and Magnitude: Approximately one week duration involving three cell divisions.

• Regulatory Factors: $EPO$ stimulates transcription of mRNA. Expression of transcription factor $GATA-1$ is required for the definite differentiation of MEP into the erythroid lineage.

Major Morphological Changes during Erythropoiesis

• Cell and nuclear sizes decrease.

• Nucleoli decrease in size and eventually disappear.

• Nucleus becomes pyknotic (chromatin density increases) and is eventually extruded.

• Cytoplasmic Color Shift: Initial basophilia (high polyribosome count) gradually decreases and shifts to acidophilia (increased hemoglobin concentration).

• Organelles: Mitochondria and other organelles disappear.

Stages of Erythroid Precursors

1. Proerythroblast: The first recognizable precursor. Large cell with a spherical nucleus and lacy chromatin. Contains 1-2 nucleoli and mild cytoplasmic basophilia (free polyribosomes).

2. Basophilic Erythroblast: Smaller than the proerythroblast. Exhibits intense cytoplasmic basophilia with many free polyribosomes. Heterochromatin increases as mitosis occurs.

3. Polychromatophilic Erythroblast: Size continues to reduce. Cytoplasm shows mixed basophilia and acidophilia. The nucleus is heterochromatic with a characteristic checkerboard pattern.

4. Orthochromatophilic Erythroblast (Normoblast): Reduced cell and nuclear size; slightly larger than a mature erythrocyte. Cytoplasm is acidophilic with very few polyribosomes. The nucleus is very heterochromatic and incapable of cell division. The nucleus is extruded late in this stage.

5. Reticulocyte: An anucleate cell that retains some polyribosomes and mitochondria. Ready to leave the BM. They constitute approximately $1\%$ of red blood cells in peripheral blood. They lose remaining organelles while in the bloodstream.

6. Mature Erythrocyte: Final stage lacking ribosomes and mitochondria.

Granulopoiesis

• Synthesis Stages:

  1. Azurophilic Granules (Promyelocyte stage): Contain lysosomal hydrolases. They exhibit cytoplasmic basophilia and are similar in all three granulocyte types.

  2. Specific Granules (Myelocyte stage): The Golgi apparatus begins packaging proteins for specific granules, found closer to the Golgi.

• Regulators: $GM-CSF$, $G-CSF$, and $IL-3$.

Stages of Neutrophil Development

• Process: Takes $10$ to $14$ days with five mitotic divisions.

• Stages:

  1. Myeloblast: Most immature recognizable cell. Large spherical euchromatic nucleus with $3$ to $5$ nucleoli. Large nuclear-to-cytoplasm ratio. Golgi area is seen as an unstained cytoplasmic region.

  2. Promyelocyte: Large spherical nucleus. Differentiation into subtypes is impossible at this stage. First synthesis of azurophilic granules (lysosomal enzymes, myeloperoxidase).

  3. Myelocyte: First sign of differentiation. Specific granules appear (emerging from the convex surface of Golgi), and azurophilic granules are seen on the concave side. Nucleus becomes heterochromatic.

  4. Metamyelocyte: Specific granules outnumber azurophilic granules. Golgi apparatus reduces. The nucleus becomes indented and heterochromatic.

  5. Band (Stab) Cell: Precedes the development of nuclear lobes. Nucleus is elongated with uniform width (horseshoe appearance). Normally up to $3\%$ of circulating neutrophils; increases during infection/inflammation.

  6. Mature Neutrophil: Characterized by the presence of $2$ to $4$ nuclear lobules.

Eosinophils and Basophils

• GMP differentiates into eosinophil progenitor cells (EoP) via $GM-CSF$, $IL-3$, and $IL-5$.

• In the absence of $IL-5$, GMP differentiates into basophil progenitor cells (BoP).

• Differentiation is not morphologically possible until the myelocyte stage.

Monopoiesis

• Differentiation: CMPs to GMPs (influenced by $IL-3$), then into Monocyte progenitors (MoP or monoblasts) stimulated by $IL-3$ and $GM-CSF$.

• Timeline: MoPs to monocytes takes $55$ hours. Monocytes circulate for $16$ hours before entering tissues.

• Stages:

  • Monoblast: Identical to myeloblast.

  • Promonocyte: Large cell with basophilic cytoplasm. Divides twice before becoming a monocyte.

  • Differentiating Monocytes: Feature well-developed RER, large Golgi complexes, and lysosome formation (fine azurophilic granules).

Lymphopoiesis

• Morphological Changes: Two or three divisions of lymphoblasts. Marked by nuclear and cellular size reduction, disappearance of nucleoli, and synthesis of specific cell surface proteins ($CD$ or Clusters of Differentiation).

• T Cell Development:

  • Influenced by the Ikaros family of transcription factors.

  • Transcription factor $GATA-3$ leads to T lymphocyte identity.

  • Cells leave the BM as pre-T lymphocytes and travel to the thymus to become immunocompetent.

• B Cell Development:

  • Transcription factor $Pax5$ determines the B lymphocyte lineage from the CLP.

  • Originate and mature entirely within the bone marrow before entering lymphoid organs.

• NK Cell Development:

  • $IL-2$ and $IL-5$ induce differentiation of lymphoblasts into immature pre-NK cells, which later become mature NK cells (cytotoxicity and interferon secretion).

Thrombopoiesis

• Pathway: CMP $\rightarrow$ MEP $\rightarrow$ MKP (or Meg-CFU).

• Thrombopoietin (TPO):

  • A glycoprotein hormone from the liver and kidney.

  • Stimulates endomitosis (DNA replication without karyokinesis or cytokinesis) in megakaryoblasts.

  • Results in giant cells with polyploid nuclei (ranging from $8n$ to $64n$).

• Stages:

  1. Megakaryoblast: Large cell ($~30\,μm$) with a non-lobed nucleus and very basophilic cytoplasm.

  2. Megakaryocyte: Giant polyploid cell ($50-70\,μm$ up to $150\,μm$). Features a complex multilobed nucleus and cytoplasm with scattered azurophilic granules. Mature cells have cytoplasmic expansions called proplatelets.

  3. Platelet Formation:

     • Megakaryocytes extend proplatelets into sinusoidal epithelium.

     • Proplatelets consist of an actin framework and microtubules that transport membrane vesicles and specific granules.

     • A microtubule loop forms at the tip; a teardrop-shaped enlargement distal to the proplatelet is pinched off to form a platelet.