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Chapter 13. Hemopoiesis

Hemopoiesis: Introduction
Mature blood cells have a relatively short life span and must be continuously replaced with stem cell progeny produced in the hemopoietic
(Gr. haima, blood, + poiesis, a making) organs. In the earliest phase of human embryogenesis, blood cells arise from the yolk sac mesoderm.
In the second trimester, hemopoiesis (also called hematopoiesis) occurs primarily in the developing liver, with the spleen also playing a role.
Skeletal elements begin to ossify and bone marrow develops in their medullary cavities, so that, in the third trimester, bone marrow
increasingly becomes the major hemopoietic organ.
After birth and on into childhood, erythrocytes, granulocytes, monocytes, and platelets are derived from stem cells located in bone marrow.
The origin and maturation of these cells are termed, respectively, erythropoiesis (Gr. erythros, red, + poiesis), granulopoiesis,
monocytopoiesis, and thrombocytopoiesis. Development of the major types of lymphocytes by lymphopoiesis occurs in the marrow and
in the lymphoid organs to which precursor cells migrate, as discussed in Chapter 14.
Before reaching maturity and being released into the circulation, blood cells go through specific stages of differentiation and maturation.
Because these processes are continuous, cells with characteristics that lie between the various stages are frequently encountered in smears
of blood or bone marrow.

Stem Cells, Growth Factors, & Differentiation

Stem cells are pluripotent cells capable of asymmetric division and self-renewal. Some of their daughter cells form specific, irreversibly
differentiated cell types, and other daughter cells remain stem cells. A constant number of pluripotent stem cells is maintained in a pool and
cells recruited for differentiation are replaced with daughter cells from the pool.
Hemopoietic stem cells can be isolated by using fluorescence-labeled antibodies to mark specific cell-surface antigens and a fluorescenceactivated cell-sorting (FACS) instrument. Stem cells are studied using experimental techniques that permit analysis of hemopoiesis in vivo and
in vitro.

In vivo techniques include injecting the bone marrow of normal donor mice into lethally irradiated mice whose hematopoietic cells have been
destroyed. In these animals, the transplanted bone marrow cells develop colonies of hematopoietic cells in the marrow cavities and spleen.
This work led to the development of bone marrow transplants now used clinically to treat potentially lethal hemopoietic disorders.
In vitro techniques involve the use of semisolid tissue culture media made with a layer of cells derived from bone marrow stroma or purified
protein growth factors produced by marrow stromal cells. Such methods create microenvironmental conditions sufficiently similar to the normal
in vivo conditions to favor hemopoietic stem cell growth and differentiation.

Pluripotent Hemopoietic Stem Cells

It is believed that all blood cells arise from a single type of stem cell in the bone marrow called a pluripotent stem cell because it can
produce all blood cell types (Figure 131). The pluripotent stem cells proliferate and form two major cell lineages: one for lymphoid cells
(lymphocytes) and another for myeloid cells (Gr. myelos, marrow) that develop in bone marrow. Myeloid cells include granulocytes,
monocytes, erythrocytes, and megakaryocytes. Early in their development, lymphoid cells migrate from the bone marrow to the thymus or to
the lymph nodes, spleen, and other lymphoid structures, where they proliferate and differentiate (see Chapter 14).

Figure 131.

Origin and differentiative stages of blood cells. Rare pluripotent stem cells divide slowly, maintain their own population, and give rise to two major cell lineages of
progenitor cells: the myeloid and lymphoid stem cells. The myeloid lineage includes precursor cells (blasts) for erythropoiesis, thrombopoiesis, granulopoiesis, and
monocytopoiesis, all in the bone marrow. The lymphoid lineage forms lymphopoietic cells, partly in the bone marrow and partly in lymphoid organs.

Progenitor & Precursor Cells

The pluripotent stem cells give rise to daughter cells with restricted potentials called progenitor cells or colony-forming units (CFUs)
(CFUs),
since they give rise to colonies of only one cell type when cultured or injected into a spleen. There are four types of progenitors/CFUs:

Erythroid lineage of CFU-erythrocytes (CFU-E)

Thrombocytic lineage of CFU-megakaryocytes (CFU-Meg)

Granulocyte-monocyte lineage of CFU-granulocytes-monocytes (CFU-GM)

Lymphoid lineage of CFU-lymphocytes of all types (CFU-L).

All four progenitor/CFUs produce precursor cells or blasts in which the cells' morphologic characteristics begin to differentiate, suggesting
the mature cell types they will become (Figure 131). In contrast, stem and progenitor cells cannot be morphologically distinguished and

resemble large lymphocytes. Stem cells divide at a rate only sufficient to maintain their relatively small population. The rate of cell division is
accelerated in progenitor and precursor cells, and large numbers of differentiated, mature cells are produced (3 x 109 erythrocytes and 0.85 x
109 granulocytes/kg/day in human bone marrow). Whereas progenitor cells divide asymmetrically to produce both progenitor and precursor
cells, precursor cells produce only cells on the path to differentiation.

Hemopoiesis depends on favorable microenvironmental conditions and the presence of paracrine or endocrine growth factors. This
microenvironment in hematopoietic organs is furnished largely by stromal cells and their extracellular matrix (ECM), which together create the
niche in which stem cells are maintained. A general view of hemopoiesis shows that during this process, both the potential for various
outcomes of differentiation and the self-renewing capacity of the cells gradually decrease. In contrast, the mitotic response to growth factors
increases, attaining its maximum in the middle of the process. From that point on, mitotic activity decreases, morphologic characteristics and
functional activity develop, and mature cells are formed (Table 131).

Table 131. Changes in properties of hematopoietic cells during differentiation.

Hemopoietic growth factors, called colony-stimulating factors (CSF

CSF) or hematopoietins (poietins
poietins), are proteins with complex,
overlapping functions in stimulating proliferation (mitogenic activity) of immature (mostly progenitor and precursor) cells, supporting
differentiation of maturing cells, and enhancing the functions of mature cells. These three functions may all occur in the same growth factor or
they may be expressed with different levels of intensity in different growth factors. The isolation and cloning of genes for several growth factors
has permitted both the mass production of these proteins and tremendously advanced study of their effects in vivo and in vitro. The main
characteristics of five well-characterized growth factors are presented in Table 132.

Table 132. Main characteristics of five hemopoietic growth factors (colony-stimulating factors, CSF).

Name
Granulocyte (G-CSF)

Human Gene Location and

Producing Cells

Main Biologic Activity

Chromosome 17 macrophages

Stimulates formation (in vitro and in vivo) of granulocytes.

Endothelium fibroblasts

Enhances metabolism of granulocytes.

Stimulates malignant (leukemic) cells.
Stimulates in vitro and in vivo production of granulocytes and
macrophages.

Granulocyte + macrophage
(GM-CSF)

Chromosome 5 T lymphocytes

Macrophage (M-CSF)

Chromosome 5 macrophages

Stimulates formation of macrophages in vitro.

Endothelium fibroblasts

Increases antitumor activity of macrophages.

Endothelium fibroblasts

Interleukin 3 (IL-3)

Chromosome 5 T lymphocytes

Stimulates in vivo and in vitro production of all myeloid cells.

Erythropoietin (EPO)

Chromosome 7 renal interstitial

Stimulates red blood cell formation in vivo and cells (outer

cortex) in vitro.

MEDICAL APPLICATION
Growth factors have been used clinically to increase marrow cellularity and blood cell counts. Potential therapeutic uses of growth factors
include increasing the number of blood cells in diseases or induced conditions (eg, chemotherapy, irradiation) that result in low blood counts;
increasing the efficiency of marrow transplants by enhancing cell proliferation; enhancing host defenses in patients with malignancies and
infectious and immunodeficient diseases; and enhancing the treatment of parasitic diseases.

Bone Marrow
Under normal conditions, the production of blood cells by the bone marrow is adjusted to the body's needs, increasing its activity several-fold
in a very short time. Bone marrow and adipocytes are found in the medullary canals of long bones and in the cavities of cancellous bones.
There are two types of bone marrow based on their appearance at gross examination: blood-forming red bone marrow, whose color is
produced by an abundance of blood and hemopoietic cells, and yellow bone marrow
marrow, which is filled with adipocytes and essentially
excludes hemopoietic cells. In the newborn, all bone marrow is red and active in blood cell production, but as the child grows most of the
marrow changes gradually to the yellow variety. Under certain conditions, such as severe bleeding or hypoxia, yellow marrow reverts to red.
Red bone marrow (Figure 132) is composed of a stroma (Gr: stroma, bed), hemopoietic cords or islands of cells,, and sinusoidal
capillaries. The stroma is a meshwork of specialized fibroblastic cells called reticular or adventitial cells and a delicate web of reticular fibers
supporting hemopoietic cells and macrophages. The matrix of bone marrow also contains collagen type I, proteoglycans, fibronectin, and
laminin, the latter glycoproteins interacting with integrins to bind cells to the matrix.

Figure 132.

Red bone marrow (active in hemopoiesis).

Red bone marrow contains adipocytes but is also active in hemopoiesis, with several cell lineages usually present. It can be examined histologically in sections of bones or in
biopsies, but its cells can also be studied in smears. Marrow consists of capillary sinusoids running through a stroma of specialized, fibroblastic reticular cells and an ECM
meshwork. Reticular cells secrete various colony-stimulating factors and the stroma forms the microenvironment for hemopoietic stem cell maintenance, proliferation, and
differentiation. This section of red bone marrow shows some of its components. Sinusoid capillaries (S) containing erythrocytes are surrounded by stroma containing
adipocytes (A) and islands or cords (C) of hemopoietic cells. Sinusoidal endothelial cells (one nucleus at E) are very thin. Most reticular cells and cells of the hemopoietic
lineages are difficult to identify with certainty in routinely stained sections of marrow. X400. H&E.

Sinusoids are formed by a thin layer of endothelial cells. Differentiated blood cells from the hemopoietic cords enter the circulation by passing
through openings in the endothelium (Figure 133). Red bone marrow is also a site where macrophages phagocytose worn-out erythrocytes
and store iron derived from hemoglobin breakdown.

Figure 133.

Sinusoidal endothelium in active marrow.

Diagram shows that mature, newly formed erythrocytes, leukocytes, and platelets in marrow enter the circulation by passing through the sinusoid capillary endothelium.
Because erythrocytes (unlike leukocytes) cannot migrate through the wall of the sinusoid actively, they are believed to enter the sinusoid by a pressure gradient across its
wall. Leukocytes cross the wall of the sinusoid by their own activity. All blood cells apparently penetrate through apertures and between the endothelial cells. Megakaryocytes
form thin processes (proplatelets) that also pass through such apertures and liberate platelets at their tips.

MEDICAL APPLICATION
Red bone marrow also contains stem cells that can produce other tissues in addition to blood cells. With their great potential for differentiation,
these cells make it possible to generate specialized cells that are not rejected by the body because they are produced from stem cells from
the marrow of the same person. The procedure is to collect bone marrow stem cells, cultivate them in appropriate medium to direct their
differentiation to the cell type needed for transplantation, and then use the cells originating in tissue culture to replace certain defective cells. In
this case the donor and the recipient are the same individual and histocompatibility is complete, excluding the possibility of rejection. These
studies are at early stages, but results with animal models so far are promising.

Maturation of Erythrocytes
A mature cell is one that has differentiated to the stage at which it can carry out all its specific functions. Erythrocyte maturation involves
hemoglobin synthesis and formation of a small, enucleated, biconcave corpuscle. Several major changes take place during erythrocyte
maturation (Figure 134). Cell and nuclear volume decrease, and the nucleoli diminish in size and disappear. The chromatin becomes
increasingly denser until the nucleus presents a pyknotic appearance and is finally extruded from the cell. There is a gradual decrease in the
number of polyribosomes (basophilia decreases), with a simultaneous increase in the amount of hemoglobin (an acidophilic protein) within the
cytoplasm. Mitochondria and other organelles gradually disappear.

Figure 134.

Summary of erythrocyte maturation.

The color change in the cytoplasm shows the continuous decrease in basophilia and the increase in hemoglobin concentration from proerythroblast to erythrocyte. There is
also a gradual decrease in nuclear volume and an increase in chromatin condensation, followed by extrusion of a pyknotic nucleus. The times are the average duration of
each cell type. In the graph, 100% represents the highest recorded concentrations of hemoglobin and RNA.

There are three to five intervening cell divisions between the proerythroblast and the mature erythrocyte. The development of an erythrocyte
from the first recognizable cell of the series to the release of reticulocytes into the blood takes approximately a week. The glycoprotein
erythropoietin (Epo)
(Epo), a growth factor produced in the kidneys, stimulates production of mRNA for globin, the protein component of
hemoglobin and is essential for the production of erythrocytes.
The first recognizable cell in the erythroid series (Figure 135) is the proerythroblast, a large cell with loose, lacy chromatin, nucleoli, and
basophilic cytoplasm. The next stage is represented by the basophilic erythroblast
erythroblast, with more strongly basophilic cytoplasm and a
condensed nucleus with no visible nucleolus. The basophilia of these two cell types is caused by the large number of polyribosomes
synthesizing hemoglobin. During the next stage cell volume is reduced, polyribosomes decrease and some cytoplasmic areas begin to be
filled with hemoglobin, producing regions of both basophilia and acidophilia in the cell, now called a polychromatophilic erythroblast. In the
next stage, the cell and nuclear volumes continue to condense and no basophilia is evident, resulting in a uniformly acidophilic cytoplasmthe
orthochromatophilic erythroblast
erythroblast. Late in this stage, this cell ejects its nucleus which is phagocytosed by macrophages. The cell still has a
small number of polyribosomes that, when treated with the dye brilliant cresyl blue, form a faintly stained network and the cell is called the
reticulocyte
reticulocyte. Reticulocytes pass to the circulation, where they may constitute 1% of the red blood cells, lose the polyribosomes and quickly

mature as erythrocytes.

Figure 135.

Erythropoiesis: Major erythrocyte precursors.

(a): Micrographs showing a very large and scarce proerythro-blast (P), a slightly smaller basophilic erythroblast (B) with very basophilic cytoplasm, typical and late
polychromatophilic erythroblasts (Pe and LPe) with both basophilic and acidophilic cytoplasmic regions, and a small orthochromatophilic erythroblast (Oe) with cytoplasm
nearly like that of the mature erythrocytes in the field. All X1400. Wright. (b): Micrograph containing reticulocytes (arrows) that have not yet completely lost the polyribosomes
used to synthesize globin, as demonstrated by a stain for RNA. X1400. Brilliant cresyl blue.

Maturation of Granulocytes
Granulopoiesis involves cytoplasmic changes dominated by synthesis of proteins for the azurophilic granules and specific granules.
These proteins are produced in the rough endoplasmic reticulum and the prominent Golgi apparatus in two successive stages (Figure 136).
The azurophilic granules, which contain lysosomal hydrolases, stain with basic dyes, and are somewhat similar in all three types of
granulocytes, are made first. Golgi activity then changes to produce proteins for the specific granules, whose contents differ in each of the
three types of granulocytes and endow each type with certain different properties. In sections of bone marrow, cords of granulopoietic cells
can be distinguished by their granule-filled cytoplasm from erythropoietic cords (Figure 137).

Figure 136.

Granulopoiesis: Formation of granules.

Diagram illustrating the sequence of cytoplasmic events in the maturation of granulocytes from myeloblasts. Modified lysosomes or azurophilic granules form first at the
promyelocyte stage and are shown in blue; the specific granules of the particular cell type form at the myelocyte stage and are shown in pink. All granules are fully
dispersed at the metamyelocyte stage, when indentation of the nucleus begins.

Figure 137.

Developing erythrocytes and granulocytes in marrow.

Precursor cells of different hemopoietic lineages develop side by side with some intermingling as various cell islands or cords in the bone marrow. Plastic section of red bone
marrow showing mitotic figures (arrows), a plasma cell (arrowhead), and fairly distinct regions of erythropoiesis and granulopoiesis. Most immature granulocytes are in the
myelocyte stage: their cytoplasm contains large, dark-stained azurophilic granules and small, less darkly stained specific granules. X400. Giemsa.

The myeloblast is the most immature recognizable cell in the myeloid series (Figure 138). It has a finely dispersed chromatin, and faint
nucleoli. In the next stage, the promyelocyte is characterized by its basophilic cytoplasm and azurophilic granules containing lysosomal
enzymes and myeloperoxidase. Different promyelocytes activate different sets of genes, resulting in lineages for the three types of

granulocytes. The first visible sign of differentiation appears in the myelocytes (Figure 139), in which specific granules gradually increase in
number and eventually occupy most of the cytoplasm at the metamyelocyte stage. These neutrophilic, basophilic, and eosinophilic
metamyelocytes mature with further condensations of the nuclei. Before its complete maturation the neutrophilic granulocyte passes through
an intermediate stage, the stab or band cell
cell, in which its nucleus is elongated but not yet polymorphic.

Figure 138.

Granulopoiesis: Major granulocyte precursors.

Two micrographs from smears of bone marrow showing the major cells of the neutrophilic granulocyte lineage. Typical precursor cells shown are labeled as follows:
myeloblast (MB); promyelocyte (1); myelocytes (2); late myelocyte (3); metamyelocytes (4); stab or band cells (5); nearly mature segmented neutrophil (6). Some of the early
stages show faint nucleoli (N). Inset: Eosinophilic myelocytes (EM) and metamytelocytes (EMm) with their specific granules having distinctly different staining. These and
cells of the basophilic lineage are similar to developing neutrophils, except for their specific staining granules and lack of the stab cell form. Also seen among the erythrocytes
of these marrow smears are some orthochromatophilic erythroblasts (Oe), a small lymphocyte (L), and a cell in mitosis (arrow). All X1400. Wright.

Figure 139.

Neutrophilic myelocyte.
At the myelocyte stage lysosomes (azurophilic granules) have formed and production of specific secretory granules is underway. This micrograph shows ultrastructurally a
peroxidase-stained section of a neutrophilic myelocyte with cytoplasm containing both large, peroxidase-positive azurophilic granules (AG) and smaller specific granules
(SG), which do not stain for peroxidase. The peroxidase reaction product is present only in mature azurophilic granules and is not seen in the rough ER (RER) or Golgi
cisternae (GC), which are located around the centriole (C) near the nucleus (N). X15,000. (With permission, from Dr. Dorothy F. Bainton, Department of Pathology, University
of California at San Francisco.)

MEDICAL APPLICATION
The appearance of large numbers of immature neutrophils (band cells) in the blood, sometimes called a "shift to the left", is clinically
significant, usually indicating bacterial infection.
The vast majority of granulocytes are neutrophils. The total time taken for a myeloblast to emerge as a mature, circulating neutrophil is about
11 days. Under normal circumstances, five mitotic divisions occur in the myeloblast, promyelocyte, and neutrophilic myelocyte stages.
Developing and mature neutrophils can be considered to exist in four functionally and anatomically defined compartments: the granulopoietic
compartment in marrow; storage as mature cells in marrow until release; the circulating population; and a marginating population of cells
adhering to endothelial cells of postcapillary venules and small veins (Figure 1310). Margination of neutrophils in some organs can persist for
several hours and is not always immediately followed by the cells' emigration from the microvasculature.

Figure 1310.

Functional compartments of neutrophils.

Neutrophils exist in at least four anatomically and functionally distinct compartments, the sizes of which are proportional to the number of cells. (1) Granulopoietic
compartment subdivided into a mitotic part and a maturation part. (2) Storage (reserve) compartment, also in red marrow, acts as a buffer system, capable of releasing large
numbers of mature neutrophils on demand. (3) Circulating compartment throughout the blood. (4) Marginating compartment, in which cells temporarily do not circulate, but
rather adhere via selectins to vascular endothelial cells of postcapillary venules, particularly in the lungs. The marginating and circulating compartments are of about equal
size, and there is a constant interchange of cells between them. The half-life of a neutrophil in these two compartments is less than 10 hours. The granulopoietic and storage
compartments together include cells in the first 11 days of their existence and are about 10 times larger than the circulating and marginating compartments.

Neutrophils and other granulocytes enter the connective tissues by migrating through intercellular junctions between endothelial cells of
postcapillary venules in diapedesis. The connective tissues thus form a fifth terminal compartment for neutrophils, where the cells reside for a
few days and then die by apoptosis, regardless of whether they have performed their major function of phagocytosis.

MEDICAL APPLICATION
Changes in the number of neutrophils in the blood must be evaluated by taking all these compartments into consideration. Thus,
neutrophilia
neutrophilia, an increase in the number of neutrophils in the circulation, does not necessarily imply an increase in neutrophil production.
Intense muscular activity or the administration of epinephrine can cause neutrophils in the marginating compartment to move into the
circulating compartment, producing an apparent neutrophilia even though neutrophil production has not increased. However, glucocorticoids
(adrenal gland hormones) increase the mitotic activity of neutrophil precursors in the marrow and do increase the blood count of neutrophils.
Neutrophilia may also result from liberation of greater numbers of neutrophils from the medullary storage compartment. This type of
neutrophilia is transitory and is followed by a recovery period during which no neutrophils are released.
The neutrophilia that occurs during bacterial infections is due to an increase in production of neutrophils and a shorter duration of these cells
in the medullary storage compartment. In such cases, immature forms such as band cells, neutrophilic metamyelocytes, and even myelocytes
may appear in the bloodstream. The neutrophilia that occurs during infection is of longer duration than that which occurs as a result of intense
muscular activity.

Maturation of Agranulocytes
Study of the precursor cells of monocytes and lymphocytes is difficult, because these cells do not show specific cytoplasmic granules or
nuclear lobulation, both of which facilitate the distinction between young and mature forms of granulocytes. Monocytes and lymphocytes in
smear preparations are discriminated mainly on the basis of size, chromatin structure, and the presence of nucleoli.

Monocytes
The monoblast is a committed progenitor cell that is virtually identical to the myeloblast in its morphologic characteristics. Further
differentiation leads to the promonocyte, a large cell (up to 18 m in diameter) with basophilic cytoplasm and a large, slightly indented
nucleus. The chromatin is lacy and nucleoli are evident. Promonocytes divide twice as they develop into monocytes (Figure 131). A large
amount of rough ER is present, as is an extensive Golgi apparatus in which granule condensation occurs. These granules are primary
lysosomes, which are observed as fine azurophilic granules in blood monocytes. Mature monocytes enter the bloodstream, circulate for about
eight hours, and then enter tissues where they mature as macrophages (or other phagocytic cells) and function for several months.

Lymphocytes
As discussed in Chapter 14, circulating lymphocytes originate mainly in the thymus and the peripheral lymphoid organs (eg, spleen, lymph
nodes, tonsils etc.). However, all lymphocyte progenitor cells originate in the bone marrow. Some of these lymphocytes migrate to the thymus,
where they acquire the full attributes of T lymphocytes. Subsequently, T lymphocytes populate specific regions of peripheral lymphoid organs.
Other bone marrow lymphocytes differentiate into B lymphocytes in the bone marrow and then migrate to peripheral lymphoid organs, where
they inhabit and multiply in their own special compartments.
As lymphocytes mature, their chromatin becomes more compact, nucleoli become less visible, and the cells decrease in size. In addition,
subsets of the lymphocyte series acquire distinctive cell-surface receptors during differentiation that can be detected by immunocytochemical
techniques. The first identifiable progenitor of lymphoid cells is the lymphoblast, a large cell capable of dividing two or three times to form
prolymphocytes. Prolymphocytes are smaller and have relatively more condensed chromatin but none of the cell-surface antigens that mark
T or B lymphocytes. In the bone marrow and in the thymus, these cells synthesize cell-surface receptors characteristic of the B or T
lymphocyte lineages. In routine histological procedures B and T lymphocytes cannot be distinguished; immunocytochemical techniques using
cell-specific markers are required to make the distinction.

MEDICAL APPLICATION
Abnormal stem cells in bone marrow can produce diseases based on cells derived from that tissue. Leukemias are malignant clones of
leukocyte precursors. They occur in lymphoid tissue (lymphocytic
lymphocytic leukemias
leukemias) and in bone marrow (myelogenous
myelogenous and monocytic
leukemias
leukemias). In these diseases, there is usually a release of large numbers of immature cells into the blood. Some symptoms of leukemias are
a consequence of this shift in cell proliferation, with a lack of some cell types and excessive production of others. The patient is usually anemic
and prone to infection.
A clinical technique that is helpful in the study of leukemias and other bone marrow disturbances is bone marrow aspiration
aspiration. A needle is
introduced through compact bone and a sample of marrow is withdrawn. The use of labeled monoclonal antibodies specific to membrane
proteins of precursor blood cells aids in identifying cell types derived from these stem cells and contributes to a more precise diagnosis of the
leukemia.

Origin of Platelets
In adults, the membrane-enclosed cell fragments called platelets originate in the red bone marrow by dissociating from mature
megakaryocytes (Gr. megas, big, + karyon, nucleus, + kytos), which in turn differentiate from megakaryoblasts in a process driven by
thrombopoietin. The megakaryoblast is 2550 m in diameter and has a large ovoid or kidney-shaped nucleus (Figure 1311) with
numerous small nucleoli. Before differentiating, these cells undergo endomitosis, with repeated rounds of DNA replication not separated by
cell divisions, resulting in a nucleus that is highly polyploid (ie, 64N or more than thirty times more DNA than a normal cell). The cytoplasm of
this cell is homogeneous and intensely basophilic.

Figure 1311.

Megakaryoblast and megakaryocytes.

(a): Megakaryoblasts (Mb) are very large, fairly rare cells in bone marrow, with very basophilic cytoplasm. X1400. Wright. (b): Megakaryoblasts undergo endomitosis (DNA
replication without intervening cell divisions), becoming polyploid as they differentiate into megakaryocytes (M). These cells are even larger, but with cytoplasm that is less
intensely basophilic. X1400. Wright. (c): Micrograph section of bone marrow megakaryocyte (M) shown near sinusoids (S). X400. Giemsa. Megakaryocytes produce all the
characteristic components of platelets (membrane vesicles, specific granules, marginal microtubule bundles, etc) and in a complex process extend many long, branching
pseudopodia-like projections called proplatelets, from the ends of which platelets are pinched off almost fully formed.

Megakaryocytes are giant cells, 35150 m in diameter, with irregularly lobulated polyploid nuclei, coarse chromatin, and no visible nucleoli.
Their cytoplasm contains numerous mitochondria, a well-developed rough ER, and an extensive Golgi apparatus from which arise the
conspicuous specific granules of platelets, or thrombocytes (Chapter 12). They are widely scattered in marrow, typically near sinusoidal
capillaries.
To form platelets, megakaryocytes extend several long (>100 m), wide (24 m), branching processes called proplatelets
proplatelets. These extending
proplatelets penetrate the sinusoidal endothelium and appear as long processes disposed lengthwise with the blood flow in these vessels
(Figure 133). The proplatelet framework consists of actin filaments and a loose bundle of mixed polarity microtubules along which organelles,
membrane vesicles, and specific granules are transported. A loop of microtubules forms a teardrop-shaped enlargement at the distal end of
the proplatelet and cytoplasm within these loops is pinched off to form platelets with their characteristic marginal bundles of microtubules,
vesicles and granules (Figure 1213b).

During proplatelet growth microtubules polymerize in both directions. Proplatelet elongation does not depend on this polymerization, but on a
dynein-based microtubule sliding mechanism similar to that of extension ladders. Mature megakaryocytes have numerous invaginations of
plasma membrane ramifying throughout the cytoplasm, called demarcation membranes (Figure 1312) which were formerly considered
"fracture lines" or "perforations" for release of platelets, but are now thought to represent a membrane reservoir that facilitates continuous
rapid proplatelet elongation. Each megakaryocyte produces a few thousand platelets, after which the remainder of the cell shows apoptotic
changes and is removed by macrophages.

Figure 1312.

Megakaryocyte ultrastructure.
TEM analysis of a megakaryocyte during platelet formation showing a lobulated nucleus (N), numerous cytoplasmic granules (G), and an extensive system of demarcation
membranes (D) through the cytoplasm. Once believed to be perforations along which platelets were shed from the cells, this membrane system is now considered a reservoir
of membrane used during elongation of the numerous proplatelets which extend from the megakaryocyte surface. X10,000.

MEDICAL APPLICATION
In certain forms of thrombocytopenic purpura
purpura, a disease in which the number of blood platelets is reduced, the platelets appear to be
bound to the cytoplasm of the megakaryocytes, indicating a defect in the liberation mechanism of these corpuscles. The life span of platelets
is approximately 10 days.