Hematopoiesis

Pluripotential hematopoietic stem cells first develop from endothelial cells within the embryonic yolk sac , where they form erythroid blood islands. Embryonic red cells are large nucleated cells that contain embryonic hemoglobin ( hemoglobin Gower 1 , hemoglobin Gower 2 , and hemoglobin Portland ). Embryonic erythropoiesis in the yolk sac is followed by the appearance of nonerythroid progenitors in both the yolk sac and the aorto-gonado-mesonephros (AGM) region of the embryo. Progenitor cells from the yolk sac and AGM colonize the hepatic cords of the fetal liver and later the red pulp of the spleen. Erythropoiesis predominates in the fetal liver and is associated with a switch from production of embryonic hemoglobin to fetal hemoglobin ( hemoglobin F ). The higher oxygen affinity of embryonic hemoglobin and hemoglobin F compared with adult hemoglobin A facilitates oxygen transport in the placenta from maternal blood to fetal blood.

Vascularization of intraosseous cartilage leads to formation of a well-vascularized cavity in the bones, known as the bone marrow . By week 20, hematopoietic cells from the fetal liver and spleen have migrated to the marrow. By birth, virtually all hematopoiesis takes place within the marrow, and hemoglobin F is steadily replaced by adult hemoglobin A and A ₂ . In young children, marrow production is found throughout the entire marrow space, including the long bones. In adults, marrow production is limited to the marrow space of the central axial skeleton, with the marrow space in peripheral bones of the extremities occupied primarily by fatty tissue.

The marrow contains many nonhematopoietic cells and a specialized extracellular matrix (ECM) that is collectively referred to as the marrow stroma . The stroma provides an environment conducive to stem cell growth and differentiation. Stromal cells include endothelial cells, adventitial cells, adipocytes, osteoblasts, osteoclasts, mast cells, and macrophages. The stromal ECM provides a physical site for binding of marrow stem cells. Stromal cells produce many of the growth factors required for marrow cell growth, including stem cell factor (SCF) , FMS-like tyrosine kinase 3 ligand (Flt-3 ligand), interleukin 6 (IL-6), interleukin 11 (IL-11), granulocyte colony-stimulating factor (G-CSF), and monocyte colony-stimulating factor (M-CSF). Growth factors produced by non-stromal cells include IL-1 (by monocytes and granulocytes); IL-3, IL-5, and granulocyte-monocyte colony stimulating factor (GM-CSF) (by T cells); erythropoietin (EPO) (by renal peritubular cells); and thrombopoietin (TPO) (by hepatocytes). These growth factors seldom act individually, instead acting synergistically to induce marrow cell growth and differentiation. Many growth factors bind to membrane receptors with inducible tyrosine kinase activity that trigger cell proliferation, activation, and differentiation. An example is SCF receptor (CD117, c-kit) , which is expressed by all hematopoietic stem cells. Many of these growth factors act not only to stimulate proliferation but also to inhibit programmed cell death ( apoptosis ).

Not all cytokines act on marrow cells to promote growth and differentiation. Instead, some cytokines inhibit hematopoiesis. Examples of inhibitory cytokines include IL-1, tumor necrosis factor (TNF), transforming growth factor beta (TGF- β ), and interferon gamma (IFN- γ ). These proinflammatory cytokines contribute to the marrow suppression seen in chronic inflammatory conditions.

Pathologic conditions caused by imbalances in hematopoietic cytokine production include anemia of renal failure caused by EPO deficiency, anemia of chronic disease (inflammation) caused by excess IL-1 and hepcidin, aplastic anemia caused by gamma interferon–mediated marrow suppression, and thrombocytopenia caused by TPO deficiency in chronic liver disease.

Hematopoietic stem cells undergo a process of stepwise differentiation from undifferentiated (multipotential) cells to fully differentiated (unipotential) cells. Multipotential stem cells can give rise to any marrow cell lineage. This process is largely controlled by differential binding of exogenous growth factors (secreted by numerous cell types, including marrow stromal cells, T cells, renal tubular cells, and hepatocytes) to growth factor receptors expressed by hematopoietic progenitor cells. Binding of growth factor ligands to cellular growth factor receptors leads to expression of nuclear transcription factors that activate lineage-specific gene expression. In addition to differentiation, growth factors ( cytokines ) acting at early stages of marrow cell differentiation induce cell proliferation. Thus, in general, progressive maturation is accompanied by a progressive increase in cell number.

Stem cells under the influence of growth factors SCF and TPO, and transcription factor Hox differentiate into common myeloid progenitors (CMPs), and stem cells under the influence of IL-7 and the transcription factor Ikaros differentiate into common lymphoid progenitors (CLPs). CMP respond to cytokines GM-CSF and G-CSF, GM-CSF, and M-CSF, SCF, or IL-5 by further differentiating into granulocytes, monocytes, mast cells, or eosinophils, respectively. Under the influence of cytokines IL-3 and TPO, CMP undergo differentiation into bilineal erythroid–megakaryocyte precursors that further differentiate into unilineal erythroid or megakaryocyte precursors induced by EPO or TPO, respectively. CLP respond to cytokines IL-2, IL-4, or IL-15 (among other factors), with further differentiation into T-cell, B-cell, or natural killer (NK) cell precursors, respectively ( Figs. 2.1 and 2.2 ).

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Hematopoietic cytokines and transcription factors.

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Hematopoiesis. See text for abbreviations.

Hematopoietic stem cells are rare cells with the ability to self-renew and give rise to multilineage and unilineage progenitor cells. Multilineage progenitor cells , such as colony-forming unit–granulocytic-erythroid-monocytic-megakaryocytic cells (CFU-GEMM), can give rise to more than one type of lineage-committed precursor cell, whereas unilineage progenitor cells , such as colony-forming unit–erythroid (CFU-E) cells, give rise to only one type of precursor cell. Stem cells and progenitor cells are primitive undifferentiated cells that display no identifiable morphologic features. Stem cells express specific cell surface proteins, including CD34, which mediates adhesion to marrow stroma; CD117 (c-kit), the SCF receptor that induces stem cell proliferation when bound by SCF (kit ligand, produced by endothelial cells); CD133, which induces development of cell membrane protrusions; and c-mpl , the TPO receptor that promotes stem cell growth.

In contrast to progenitor cells, precursor cells display lineage-specific morphologic and phenotypic features. For example, erythroid precursor cells contain hemoglobin-rich cytoplasm, myeloid precursor cells contain myeloperoxidase (MPO)–positive cytoplasmic granules, and megakaryocyte precursors display enlarged hyperlobated nuclei and cytoplasmic buds. Precursor cells also express lineage-specific molecules that can be exploited as phenotype markers when detected with monoclonal antibodies by flow cytometry, immunohistochemistry, or cytochemistry. Examples include glycophorin A, CD71 (transferrin receptor), hemoglobin, and e-cadherin for erythroid precursors; CD13, CD33, MPO, and alpha naphthyl acetate esterase (ANAE) for myeloid precursors; CD41, CD61, and von Willebrand factor (VWF, factor VIII–related antigen) for megakaryocyte precursors; cytoplasmic CD3 (cCD3), CD7, and terminal deoxynucleotidyl transferase (TdT) for T cell precursors; CD19, paired box (PAX) protein 5 (PAX-5), cCD22, and TdT for B cell precursors; CD14, CD68, CD163, and alpha naphthyl butyrate esterase (ANBE) for monocyte precursors; and CD117 and mast cell tryptase for mast cell precursors ( Table 2.1 ).

The normal bone marrow contains both stromal and hematopoietic elements ( Fig. 2.3 ). Bone marrow cellularity can be determined by examination of a bone marrow biopsy and aspirate. Bone marrow cellularity is calculated as the ratio of cellular marrow volume to fatty marrow volume. Normal iliac crest marrow cellularity in a newborn is 90%, with a steady reduction to 30% to 40% in older adults. The normal marrow is populated by myeloid and erythroid cells in approximately a 3:1 (myeloid predominant) ratio ( Fig. 2.4 ). Most myeloid cells are neutrophils, with scattered eosinophils, basophils, and mast cells ( Fig. 2.5 ). In the normal marrow, most neutrophilic cells are mature (metamyelocytes, bands, and segmented neutrophils), with lesser numbers of myelocytes and promyelocytes. Myeloblasts (and stem cells) are rare cells in the normal marrow, accounting for no more than 3% of the marrow cell count. Lymphocytes account for 10% to 15% of the marrow cellularity in adults but in young children may account for up to 50%. Monocytes and promonocytes account for 2% to 3% of the marrow cellularity. Relatively few megakaryocytes (0.1%) are scattered throughout the normal marrow, often in proximity to vascular sinuses.

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Bone marrow structure.

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Normal adult marrow biopsy (hematoxylin and eosin stained). Note the clear spaces that represent adipocytes removed by tissue processing and the large megakaryocyte at the left center. In this field, there is a predominance of myeloid cells with relatively few erythroid cells (small cells with dense basophilic nuclei).

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Adult marrow aspirate smear with an eosinophil (upper middle) , mast cell (center) , and basophil (lower middle) (Wright-Giemsa stain).

Under normal circumstances, only fully mature (enucleated) erythroid cells and myeloid cells are released into the bloodstream from the bone marrow ( Fig. 2.6 ). Under stress conditions (e.g., infection, inflammation, blood loss, trauma), less mature cells are released into the blood. For example, acute bacterial infection leads to release of immature myeloid cells (band neutrophils, metamyelocytes, and myelocytes), and blood loss leads to release of reticulocytes and nucleated red cells. While mature erythrocytes remain in the bloodstream, myeloid cells (neutrophils, eosinophils, and basophils) and monocytes are recruited to inflamed tissues under the influence of a closely related group of chemoattractant cytokines known as chemokines . Chemokines are produced by a range of cell types, including endothelial cells, macrophages, T cells, fibroblasts, keratinocytes, and stromal cells.

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Peripheral blood smear (Wright stained) with six leukocytes: band neutrophil (upper left) , eosinophil (upper right) , basophil (lower right) , monocyte (lower left) , lymphocyte (middle left) , and segmented neutrophil (middle right) . A single platelet is noted to the right of the band neutrophil. The remaining cells in the field are red blood cells.

Under normal conditions, neutrophils migrate from the blood to bronchial and intestinal submucosa, where they serve as first responders to infection. Blood neutrophils also rapidly migrate to localized sites of acute infection or injury. Intravascular neutrophils reside in two freely exchangeable pools: the circulating pool and the marginal pool . At any time, most intravascular neutrophils are not circulating, instead marginating along capillary and venular walls (the marginal pool) in the spleen and lungs. In response to infection or inflammation, cells within the marginal pool rapidly enter the circulating pool. The total intravascular granulocyte pool (circulating and marginal) is supported by the marrow granulocyte reserve. This reserve, primarily composed of mature myeloid cells, is approximately 20 times larger than the blood granulocyte pool and capable of rapidly repleting the blood granulocyte pool in the face of infection or inflammation. The rapid migration of blood neutrophils into sites of inflammation is mediated by the chemokine IL-8 (CXCL-8) produced by activated macrophages.

Eosinophils and basophils in blood migrate to the submucosa of the aerodigestive tract ( Fig. 2.7 ) and, like neutrophils, can migrate to other sites in response to inflammatory chemokines. Eosinophils are recruited to inflamed tissues by IL-5, eotaxin, and chemokine ligand 5 (CCL-5), and basophils are recruited by CCL-2 and CCL-5. Mast cells, unlike basophils, are not typically found in peripheral blood, instead homing to perivascular sites within a variety of connective tissues, including marrow stroma. Mast cells maintain vascular integrity by secretion of heparin , and like basophils, release the vasoactive factor histamine in response to allergens.

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Eosinophils and mast cells in the intestinal submucosa. Note the eosinophils with bright red cytoplasmic granules and the mast cells, small mononuclear cells with purple cytoplasm.

Many blood monocytes, like neutrophils, reside in the marginal pool and rapidly enter tissues to undergo further differentiation into several specialized cell types of the mononuclear phagocyte system, including histiocytes (macrophages), dendritic cells, osteoclasts, and microglial cells. Blood monocyte–derived macrophages are particularly numerous in organs such as the liver, spleen, lymph nodes, and lungs that capture and process antigen ( Fig. 2.8 ). To replenish macrophages in inflamed tissues, blood monocytes are recruited to areas of inflammation by the chemokine CCL-2 (macrophage chemoattractant protein-1).

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Hemophagocytic histiocyte (macrophage) with abundant vacuolated cytoplasm and ingested cellular debris in hemophagocytic syndrome (bone marrow aspirate).

Megakaryocytes remain in the marrow in the vicinity of vascular sinuses, producing platelets by cytoplasmic budding and release directly into the bloodstream ( Fig. 2.9 ).

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A megakaryocyte is noted in the center of the field (marrow aspirate). Note the cytoplasmic blebs that will give rise to budding platelets.

In response to the chemokine IL-13 (CXCL-13), naïve immunoglobulin M (IgM) and/or IgD-positive B cells enter peripheral lymphoid tissues via high endothelial venules and home in on lymphoid follicles in lymph nodes and spleen to await antigen-driven germinal center maturation ( Fig. 2.10 ). Naïve CD7+, CD3− T cells home to the thymic cortex to begin the complex process of T-cell maturation ( Fig. 2.11 ). NK cells are released into the bloodstream as fully mature and functional cells, homing primarily to lymphoid tissues and submucosal sites. Circulating NK cells also enter sites of inflammation in response to the cytokine IL-12 released by activated macrophages.

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Secondary lymphoid follicle with a large central germinal center composed of activated B cells surrounded by a cuff of small resting mantle zone B cells.

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Thymus with central medullary region and peripheral cortical zone. Note the pink epithelial whorls (Hassall corpuscles) in the medullary region.