Embryonic hemopoiesis

Hemopoiesis commences from a transient population of primitive HSC generated in the yolk sac. This commences on the 14th to 19th day of embryogenesis and persists there until the end of the 12th week of gestation. The yolk sac primarily produces nucleated erythroid cells that are megaloblastic and contain embryonic hemoglobins, Gower I (ζ₂ε₂), Gower II (α₂ε₂) and Portland I (ζ₂γ₂) (Fig. 2.1). Yolk sac erythropoiesis is referred to as ‘primitive’. In the 6th and 7th weeks of gestation, the blood islands within the yolk sac also contain a few megakaryocytes. Hemopoietic activity then occurs in a region of the para-aortic splanchopleural mesoderm. This region contains the dorsal aorta, gonadal ridge and mesonephros and is known as the aorta-gonad-mesonephros or AGM region.^1,2 Hemopoiesis in the AGM region develops from ‘definitive’ HSC that eventually populate the adult BM. Some adult-type HSC also develop in the yolk sac and the placenta.

Fig. 2.1 Semithin section of a plastic-embedded chorionic villus biopsy taken at 7 weeks of gestation showing a blood vessel containing nucleated embryonic red cells. Toluidine blue stain.

HSC derived from the AGM migrate to the liver and erythropoietic foci are detectable in the 6th week of gestation. The liver remains the main site of erythropoiesis from the 3rd to the 6th month when erythroblasts account for about 50% of the nucleated cells of the liver. The erythroblasts are mainly extravascular (Fig. 2.2), located near and within Kupffer cells (emperipolesis) and continue their maturation inside sinusoids.³ Erythroblasts are initially megaloblastic but subsequently become macronormoblastic. Fetal hepatic erythropoiesis is associated with the synthesis of fetal hemoglobin (HbF; α₂γ₂) and results in the production of nucleated, macrocytic red cells. The liver continues to produce red cells in decreasing numbers after the 6th month of gestation until the end of the 1st postnatal week.

Fig. 2.2 Histologic appearances of normal fetal liver during the middle trimester of pregnancy. About 40% of the area of the section consists of erythropoietic cells which are present singly or in clusters between the plates of liver cells. Hematoxylin–eosin stain.

Small foci of erythropoietic cells are present in the vascular connective tissue of some BM cavities from 2.5 to 4 months’ gestation. From the 6th month the BM is the major site of hemopoiesis and the myeloid/erythroid (M/E) ratio is about 1 : 4. Erythropoiesis in fetal BM occurs extravascularly, is macronormoblastic and results in the production of macrocytic red cells containing HbF and HbA (α₂β₂). The mean cell volume (MCV) in the cord blood of full-term newborns is 90–118 fl. Erythropoieisis in fetal BM appears to be regulated by erythropoietin produced extrarenally, probably in the liver. From the 6th month there is also proliferation of HSC in the fetal liver which generate erythroid, myeloid and some lymphoid cells. Small foci of erythroblasts, a few granulocytopoietic cells and occasional megakaryocytes also occur in many other embryonic and fetal tissues and organs (including lymph nodes, spleen and kidneys); however, their contribution to overall hemopoietic activity is small.

Postnatal changes in the distribution of hemopoietic marrow

At birth all BM cavities contain red marrow consisting mainly of hemopoietic cells. By 1 year virtually all the hemopoietic cells in the terminal phalanges are replaced by fat cells. After the first 4 years there is an increase in fat cells amongst the hemopoietic cells of other marrow cavities. Between 10 and 14 years, the hemopoietic cells in the middle of the shafts of the long bones become virtually completely replaced by fat cells. Subsequently, these zones of non-hemopoietic yellow marrow spread proximally and distally. Distal spread is the more rapid and by about the 25th year the only regions of the long bones that contain red, hemopoietic marrow are the proximal shafts of the femora and humeri. Other sites of hemopoiesis in an adult are the skull, ribs, clavicles, scapulae, sternum, vertebrae and pelvis.

General characteristics of hemopoiesis

The formation of blood cells of all types involves two processes:

During intrauterine life and in the growing child, there is a progressive increase in the total number of hemopoietic and blood cells. In normal adults, the total number of hemopoietic and blood cells remains relatively constant. There is steady-state cell renewal with a relatively constant rate of loss of mature cells (erythrocytes, granulocytes, monocytes and platelets) balanced precisely by the production of new cells.

Hemopoietic stem cells and progenitor cells

The uncommitted HSC has the capacity for self-renewal and is pluripotent, that is, it has the ability to differentiate into lineage-committed progenitors. It is not recognizable morphologically but can be identified by antigen expression profile. The differentiation of HSC generates multipotent myeloid progenitors and lymphoid progenitors (Fig. 2.3).⁴ Evidence for the presence of pluripotent HSC has come from a number of pieces of evidence including:

Fig. 2.3 Schematic diagram showing the differentiation of hemopoietic stem cells to mature blood cells. B-cell P, B-cell precursor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; HSC, hemopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; T-cell P, T-cell precursor; TSP, thymus-seeding progenitor.

All pluripotent HSC express CD34 antigen and are usually AC133⁺, CD59⁺; Thy1^low; CD38⁻; C-kit^−/low; CD33⁻; lineage⁻. By flow cytometry (FCM) CD34⁺ HSC constitute most cells of the CD45^dim/SSC^low (blast) region and are a heterogeneous cell population. A small fraction of pluripotent HSC with long-term repopulating cell activity have been associated with the CD34⁺/CD38⁻ phenotype.^5,6 These cells are very rare in normal BM (usually <0.1%), but may increase in regenerating BM and in myelodysplastic syndromes.^7,8 CD34⁺/CD45^dim cells include a major fraction of progenitor cells that are already committed to specific hematopoietic lineages (erythroid, neutrophil, monocytic, dendritic cell (DC), basophil, mast cell (MC), eosinophil and megakaryocytic) and variable numbers of CD34⁺ B-cell precursors (BCP).⁹

The multipotent (or pluripotent) HSC undergo gradual restriction in their hemopoietic potential as they eventually give rise to unipotent progenitor cells^10,11 (Fig. 2.4). The progeny of HSC therefore become progressively restricted to one cell lineage and they lose the capacity to self-renew. The earliest branching between myeloid and lymphoid development is to committed progenitor cells of myeloid or lymphoid types, as follows:^11,12

Fig. 2.4 Normal adult hemopoiesis in a bone marrow aspirate showing a range of normal erythroid and granulocytic progenitors at different stages of differentiation. May–Grünwald–Giemsa stain. × 400.

The complex mechanisms involved in the regulation of hemopoietic stem cells and progenitor cells are described in Chapter 4. Growth-promoting cytokines, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF), other cytokines and transcription factors are key regulators of hemopoiesis and may also enhance some functions of the mature cells.

Regulation of erythropoiesis

A key factor determining the rate of red cell production is the glycoprotein hormone erythropoietin (EPO) which, in the adult, is produced mainly by the peritubular cells of the kidney. EPO receptors are expressed on erythroid progenitor cells and the binding of EPO to its receptor results in the activation of JAK2 tyrosine kinase, which causes tyrosine phosphorylation in a number of proteins and triggers the activation of several signal transduction pathways involved in proliferation and in the prevention of apoptosis.¹⁸ An important effect of EPO is, therefore, to maintain the viability and proliferation of erythroid progenitor cells, by preventing apoptosis. In synergy with stem cell factor (SCF), GM-CSF, interleukin (IL)-3 and insulin-like growth factor-1 (IGF-1), EPO stimulates the rate of differentiation of CFU-E to pronormoblasts. EPO also stimulates terminal differentiation and decreases the time taken for the maturation of a pronormoblast to a marrow reticulocyte and its release into the circulation. The plasma level of EPO is inversely related to the capacity of the blood to deliver oxygen to the kidneys and other tissues. Reduction of the oxygen supply to the kidney results in enhanced EPO gene expression via a hypoxia-regulated transcription factor, HIF (hypoxia-inducible factor).¹⁹ Thus, in most anemic states there is an EPO increased level in the plasma, which in turn causes an enhancement of the rate of erythropoiesis.

Erythropoiesis is also influenced by the secretions of various endocrine glands. For example, hypofunction of the thyroid, testes, adrenal glands or anterior lobe of the pituitary gland result in mild to moderate anemia. Erythropoiesis is stimulated by thyroxine (stimulates terminal differentiation of erythroid progenitor cells), androgens (proliferation and expansion of erythroid progenitors) and growth hormone partly by an effect on the kidneys resulting in increased EPO production.²⁰ Corticosteriods enhance erythropoiesis by stimulating EPO production and by a direct effect on progenitor cells. There is some evidence that estrogens may inhibit erythropoiesis;²¹ the sex difference in the hemoglobin levels of adults appears to be largely due to the higher androgen levels in males.

Light microscope cytology

The term erythroblast is used to define erythroid progenitors. When these appear normal the term normoblast is applied. In Romanowsky-stained normal BM smears morphologically identifiable erythroid cells have the following features (Figs 2.5 and 2.6):

Fig. 2.6 (A, B) Erythroblasts in a normal bone marrow aspirate showing all stages of erythropoiesis from pronormoblast to late polychromatic normoblasts. May–Grünwald–Giemsa stain. × 400. (A) Two pronormoblasts and many late polychromatic normoblasts. (B) Early and late polychromatic normoblasts.

Cytochemistry

Perls’ acid ferrocyanide stain identifies one small blue–black granule in up to 30% of polychromatic erythroblasts. These iron-containing siderotic granules are randomly distributed in the cytoplasm, and erythroblasts containing such granules are termed sideroblasts. Abnormalities of siderotic granulation include increased numbers of sideroblasts, increased granulation or abnormal granule localization. The term ring sideroblasts is used when there are five or more perinuclear siderotic granules. Erythroid cells at all stages of maturation frequently contain coarse acid phosphatase-positive paranuclear granules. Normal erythroblasts are periodic acid-Schiff (PAS)-, Sudan black- and myeloperoxidase (MPO)-negative. Occasional erythroblasts contain a few alpha-naphthol AS-D chloroacetate esterase-positive granules.

Antigen expression

Early erythroblasts have weak CD45 expression, are CD44 (strong), CD71, CD36, HLA-DR and CD117 positive. Glycophorin A (CD235a) is expressed at a low level at this stage. Maturation to the basophilic erythroblast is accompanied by a decrease in CD44, CD45 and acquisition of CD235a antigen. Transition to polychromatic erythroblast shows a further loss of CD45, HLA-DR and CD44, a mild decrease in CD36 expression and presence of hemoglobin.^22,23

Ultrastructure

Electron microscope studies show that all erythroblasts contain characteristic surface invaginations which develop into small intracytoplasmic vesicles (rhopheocytotic vesicles) (Fig. 2.5B). Morphological changes can be seen with maturation from pronormoblasts to mature late polychromatic normoblasts. The major changes are an increase in the amount of heterochromatin, decrease in the number of ribosomes, increase in the electron-density of the cytoplasm due to the accumulation of hemoglobin, a decrease in the number and size of mitochondria and aggregation of ferritin molecules into siderosomes. A Golgi apparatus persists in polychromatic normoblasts.

Light microscope cytology

Early megakaryoblasts are difficult to distinguish from BM myeloblasts (see later) but do have a distinct ultrastructural appearance and phenotype. Megakaryoblasts (20–30 µm diameter) have a single large oval, kidney-shaped or lobed nucleus with several nucleoli, a very high nucleus to cytoplasm ratio and deeply basophilic agranular cytoplasm. Promegakaryocytes are usually larger than megakaryoblasts and have a lower nucleus to cytoplasm ratio and less basophilic cytoplasm. They have overlapping nuclear lobes and the cytoplasm may contain azurophilic cytoplasmic granules. The granular megakaryocytes (Fig. 2.7) are up to 70 µm in diameter and possess abundant pale-staining cytoplasm and numerous azurophilic cytoplasmic granules. The nucleus has coarsely granular chromatin and multiple lobes which extend through much of the cell. Prior to the formation of platelets by the fragmentation of cytoplasmic processes, the nuclear lobes become fairly tightly packed together. Following completion of platelet formation, a ‘bare’ nucleus remains (Fig. 2.7B).

Antigen expression

Cells committed to the megakaryocyte lineage express platelet glycoproteins. The earliest is platelet glycoprotein IIIa (CD61; integrin αIIβ3) followed by glycoprotein IIb (CD41; integrin αIIb), glycoprotein Ib (CD42), glycoprotein V and factor VIII-related antigen. Thrombospondin receptor (CD36; glycoprotein IIIb) and platelet-endothelial cell adhesion moleculae (PECAM-1; CD31) are also expressed.

Ultrastructure

Extensive studies have been performed of megakaryocytes throughout differentiation; the reader is referred to the previous edition of this book and other references for details of the ultrastructural features.^25,26 Ultrastructural cytochemical studies have demonstrated platelet peroxidase (PPO), distinct from myeloperoxidase (MPO), in the endoplasmic reticulum and perinuclear space of promegakaryoblasts, megakaryoblasts and megakaryocytes. PPO is also present in the dense bodies and dense tubular system of platelets.

Emperipolesis

Emperipolesis describes the presence and movement of one cell within the cytoplasm of another; the ‘engulfed’ cell can subsequently leave the ‘engulfing’ cell and appears morphologically unaltered by the interaction.²⁷ Emperipolesis is most commonly seen within megakaryocytes and sometimes one megakaryocyte may contain several cells ‘inside’ it (Fig. 2.8). The engulfed cells may be neutrophils, eosinophils and their precursors, lymphocytes, erythroblasts or red cells. Megakaryocytic emperipolesis is of uncertain significance but may represent a transmegakaryocytic route for the entry of blood cells into the circulation; it has been suggested that some of the intramegakaryocytic cells may enter the circulation via the processes of megakaryocytic cytoplasm which protrude into adjacent marrow sinusoids. Emperipolesis has also been described in non-hemopoietic cells and malignant cells, including blast cells in the blast phase of chronic myelogenous leukemia.

Fig. 2.8 (A, B) Megakaryocytes showing emperipolesis. (A) Granulated megakaryocyte showing emperipolesis of a neutrophil granulocyte. May–Grünwald–Giemsa stain. × 400. (B) Electron micrograph of a megakaryocyte which is apparently showing an unusual degree of emperipolesis. Six cells (two eosinophil granulocytes, two neutrophil granulocytes and a monocyte) can be seen within the megakaryocyte profile. It is possible that at least some of these cells are not completely within the megakaryocyte but merely protruding into it. Uranyl acetate and lead citrate. × 6800.