HEMATOPOIESIS

Hematopoiesis (from Greek haima for blood and poiein, to make) is the dynamic and
complex developmental process of the formation of new blood cells. Understanding the
biology of hematopoietic stem cells as well as hematopoiesis is important to developing
improved treatments for hematologic malignancies, congenital disorders, chemotherapyrelated cytopenias, and blood and marrow transplants.

I. Introduction
Each day the human body produces billions of new white blood cells, red blood cells, and
platelets to replace blood cells lost to normal cell turnover processes as well as to illness
or trauma. A variety of homeostatic mechanisms allow blood cell production to respond
quickly to stresses such as bleeding or infection and then return to normal levels when the
stress is resolved. The highly orchestrated process of blood cell production and
homeostasis is termed hematopoiesis.
An understanding of the principal mechanisms in hematopoiesis, as well as our
current understanding of the processes central to hematopoiesis, is important to the
practice of oncology for a variety of reasons. Disorders of hematopoiesis underlie a
number of hematologic malignancies and other disorders such as leukemia, aplastic
anemia, lymphoma, myelodysplasia, myeloproliferative disorders, and inborn errors of
metabolism. Chemotherapy-induced cytopenia is one of the primary causes of morbidity
and mortality in the treatment of cancer.

II. Embryology of Hematopoiesis

Hematopoietic stem cells originate from mesenchymal tissue in the yolk sac during very
early embryonic life. In the human, at some point during the first six weeks of gestation,
primitive blood islands appear in the yolk sac, which represent the earliest sites of stem
cells proliferation and differentiation. At about week six, the stem cells migrate from the
yolk sac into the substance of the embryo where they proliferate first in the liver (from
about the sixth through the tenth week of gestation), next in the spleen (weeks 10-15),
and finally in the bone marrow, where they take up residence by about the 15th week of
fetal life. From about the fifteenth week of gestation through the death of the individual,
the bone marrow is the primary site of blood cell production.
Age of animal
Site of hematopoiesis
Embryo
rd

th

yolk sac then liver

3 to 7 month

spleen

4th and 5th months

marrow cavity - esp. granulocytes and platelets

th

7 month

marrow cavity - erythrocytes

Birth

mostly bone marrow; spleen and liver when needed

Birth to maturity

number of active sites in bone marrow decreases but

retain ability for hematopoiesis

Adult

bone marrow of skull, ribs, sternum, vertebral column,

pelvis, proximal ends of femurs

In healthy persons, the marrow is for practical purposes the only site of
hematopoiesis. However, during periods of hematopoietic stress, the liver and spleen may
revert to their fetal role and produce blood cells in the adult. Examples of this event may
be seen, for example, when the marrow is replaced by metastatic breast cancer or by
fibrosis.

III. Structure and function of bone marrow

Bone marrow has a vascular compartment and an extravascular compartment.
The vascular compartment is supplied by a nutrient artery which branches into
central longitudinal arteries which send out radial branches that eventually open
into sinuses. These sinuses converge into a central vein that carries the blood out of
the bone marrow into the general circulation. Hematopoiesis takes place in the
extravascular compartment.
The extravascular compartment consists of a stroma of reticular connective tissue
and a parenchyma of developing blood cells, plasma cell, macrophages and fat
cells.
The high activity of the bone marrow is demonstrated by its daily output of
mature blood cells: 2.5 billion erythrocytes, 2.5 billion platelets, 50-100 billion
granulocytes. The numbers of lymphocytes and monocytes is also very large.
Bone marrow is the site for other important activities in addition to
hematopoiesis. These include the removal of aged and defective erythrocytes and the
differentiation of B lymphocytes. It is also the site of numerous plasma cells.

IV. The process of hematopoiesis

All the cells that circulate in the peripheral blood are derived from primitive
mesenchymal cells referred to as pluripotential hematopoietic stem cells.
In the adult, most of these cells are located in the bone marrow, but they are
capable of circulating freely in the peripheral blood. Indeed, small numbers of these cells
are always present in the peripheral bloodstream in transit from one site in the marrow to
another via hematogenous spread; in this manner, the marrow is continuously "seeded"
with these cells in a homogenous fashion. It has been estimated that there is
approximately 1 stem cell per 104 bone marrow cells. These stem cells represent a selfrenewing population of cells. These cells also must have the potential to differentiate and
to become committed to a particular blood cell lineage.
In the marrow of a healthy person :
most stem cells are neither dividing nor differentiating. These cells are considered to
be in a prolonged intermitotic interval and comprise the reserve stem cell pool that
can be called into division during times of hematopoietic stress.
another smaller fraction of the total population hematopoietic stress cells is
composed of cells that are dividing but not differentiating. Through this mechanism,
the hematopoietic stem cell pool maintains its own numbers throughout the life-span
of the individual.
finally, at any given moment in time, a relatively tiny portion of the total stem cell
pool in a healthy person is made up of cells that are beginning to differentiate along
one the several pathways of hematopoietic development through a process that
ultimately gives rise to the wide diversity of cells that circulate in the peripheral

blood. This elegant process, which generally is restricted to the bone marrow, is
referred to as hematopoiesis.
The very first step in hematopoietic differentiation involves a commitment of the
stem cell to either one of two large pathways, myeloid or lymphoid. Subsequently,
progeny of cells committed to each of theses two pathways may become obligated to
specific sublines of development. After becoming committed to a specific pathway, or
lineage, the cells are referred to as committed progenitor cells. These cells have lost the
capacity for self-renewal and are committed to a given cell lineage.
Hematopoietic ontogeny can be represented by a tree whose trunk divides early
into two large branches and some of whose smaller branches divide many times.

There are three major branches of the myeloid pathway, erythroid,

megakaryocytic, and phagocytic.
The erythroid line, which does not branch further, gives rise to red blood cells which
carry oxygen from the lungs to the tissues.
The megakaryocytic line, which also does not undergo further subdivision, provides
blood platelets which are the primary defense against hemorrhage and which play a
major role in maintaining and repairing the endothelium.
The phagocytic pathway, however, undergoes complex subdivisions, first into the
monocytic and the granulocytic pathways.
o Blood monocytes, which mature in the tissues to various classes of
macrophages, derive from the monocytic pathway.
o The granulocyte pathway divides into the neutrophilic, eosinophilic, and
basophilic pathways.
Neutrophils are the body's main defense against bacterial infections.
Eosinophils play an important role in allergic states and in combating
various parasitic infestations.
Basophils are probably the precursors of tissue mast cells, which provide an
important tripartite link among the hemapoietic, immune, and nervous

systems through the generation of a variety of vasoactive amines and

neurotransmitters.
The lymphoid pathway subdivides into the B cell pathway; the T cell pathway;
and the "null" cell, or "non-B, non-T' cell pathway. B lymphocytes differentiate further
into plasma cells which secrete immunoglobulins. T lymphocytes play critical roles in
cell-mediated immunity. The null cell pool in the peripheral blood is actually quite
heterogeneous, containing highly cytotoxic natural killer (NK) cells and a variety of
immature lymphoid cells. Since hematopoietic stem cells and committed progenitor cells
from the bone marrow cannot be morphologically distinguished from lymphocytes, they
masquerade as null cells when they are in transit in the peripheral blood.
The monophyletic theory of hematopoiesis states that pluripotent stem cells multiply to
produce more pluripotent stem cells, thus ensuring the steady and lasting supply of stem
cells. Some of the pluripotent stem cells differentiate into precursor cells that are at least
partially committed to become one type of mature blood cell.
Pluripotent stem cells multiply slowly into one of five possible unipotential stem
cells which then multiply rapidly into the precursor of the specific mature blood cell for
which they are destined.
Although the pluripotent stem cells and the unipotential stem cells cannot be
distinguished from one another histologically, the precursor cells can be distinguished
with a trained and practiced eye. The precursor cells suffer different changes from the
most immature cell (headline) to the mature one which is sent into the peripheral blood,
changes which can be distinguished on the blood marrow smear.
Since structure is (always) related to function, the structure of the precursor cell
changes as it goes from making more protein to making less protein. Thus :
a cell that is making a lot of protein will have a nucleus containing dispursed or
active chromatin, i.e., that is being transcribed actively when this cell is making
less protein, the chromatin is condensed or clumped because it is not being
transcribed.
a cell that is making a lot of protein will have many and large nucleoli, the site of
ribosomal RNA synthesis and assembly as protein secretion decreases there are
smaller and fewer nucleoli.
cells with high protein synthetic activity have more ribosomes in their cytoplasm and
consequently the cytoplasm stains more basophilic (hematoxylin staining of the RNA
in ribosomes) cells with lower protein synthetic activity have fewer ribosomes,
thus less basophilic staining with hematoxylin leaving the cytoplasm appearing more
acidophilic due to the eosin staining of cytoplasmic proteins.
cells with high protein synthetic activity the Golgi apparatus is highly developed,
occupies much of the cytoplasm thus pushing the nucleus off to one side (acentric
nucleus) cells with low protein synthetic activity have a smaller Golgi and the
nucleus tends to be more centrally located.

V. Erythropoiesis
As the cells are maturing In the erythrocytic series, the cells are usually getting smaller,
the nucleus is becoming smaller and more condensed and is eventually lost, and the
cytoplasm is becoming more pink rather than blue.

The cells in the developing erythrocyte series are as follows:

Stem cell compartment :
Unipotent stem cell: not shown, cannot be distinguished from other unipotent
stem cells histologically
Progenitor cells compartment :
BFUE Burst forming units erythroid
CFU-E Colony forming units erythroid
Precursor cells compartment :
Proerythroblast: nucleus still rather large, taking up most of the cell; nucleus not
condensed; cytoplasm still very blue or basophilic
Basophilic erythroblast: not shown; very difficult to distinguish from the
proerythroblast
Polychromatophilic erythroblast: nucleus is more condensed than that of the
proerythroblast; cytoplasm less blue, more grayish
Orthochromatophilic erythroblast: nucleus more condensed, smaller than that
of previous cells and looks pyknotic by comparison; cytoplasm beginning to take
on a more pinkish cast
Reticulocyte: no nucleus; cytoplasm still stains somewhat bluish due to presence
of remnants of polyribosomes
Erythrocyte: mature erythrocyte has no nucleus (in mammals); cytoplasm stains
very pink due to lack of ribosomes and presence of high amounts of protein, i.e.,
hemoglobin

VI. Granulopoiesis

Unipotent stem cell: not shown, cannot be distinguished from other unipotent
stem cells histologically
Myeloblast: large cell with blue-staining cytoplasm; large nucleus; often as in
this example, a clear area near the nucleus can be seen - this is where the rather
large Golgi is located
Promyelocyte: example not shown; still a rather large cell with azurophilic (not
specifically stained) granules
Myelocyte: example not shown; overall cell still rather large; nucleus still round
without indentation; granules staining appropriately for the series, i.e., pink for
eosinophilic, blue for basophilic, neutral for neutrophilic
Metamyelocyte: cell is about the size of a mature granulocyte; nucleus with
slight indentation; granules present that stain appropriately for the series, i.e., pink
for eosinophilic, blue for basophilic, neutral for neutrophilic
Band cell: cell is about the size of a mature granulocyte; nucleus with definite
indentation - looks like a horseshoe; prominent granules that stain appropriately
for the series
Mature (segmented) granulocyte: cell is mature and looks like normal, mature
granulocytes in the blood with lobed nucleus and prominent granules that stain
appropriately for the series.

VII. Thrombopoiesis

Platelets, also called thrombocytes, play an important role in hemostasis by.

plugging holes in blood vessels to prevent bleeding
promoting formation of clots to further prevent bleeding
helping to repair damaged blood vessels
Platelet granules contain the secretory material that platelets produce to help
repair damaged blood vessels, growth factor and many other proteins. Some of these are:
platelet factor 4 - regulates vascular permeability, calcium mobilization from
bone, chemotaxis of monocytes and neutrophils
beta thromboglobulin - function unknown; used to monitor activation of platelets
in some diseases
coagulation factors - fibrinogen, factor V, factor VIII
fibronectin, thrombospondin, platelet-derived growth factor - all may be involved
in repair of damaged blood vessels
serotonin (taken up from plasma and stored in granules)
lysosomal enzymes such as hydrolases
Platelets are formed in the bone marrow from megakaryocytes (30-100 m
diameter), very large cells with a polyploid, multilobed nucleus. Platelets are released
from fragmenting megakaryocytes in at least two ways:
extension of pseudopodia through the wall of the sinuses; pseudopodia contain
"strings" of platelets that are pinched off and released into the circulation
passage of mature megakaryocyte into circulation and fragmenation in the
pulmonary vascular bed

VIII. Lymphoiesis
The lymphoid pathway subdivides into the B cell pathway; the T cell pathway; and the
"null" cell, or "non-B, non-T' cell pathway.
B cell development to the stage of the mature B lymphocyte is completed within
the bone marrow. Further differentiation into Plasma Cells or memory B cells
does not occur until the mature (but nave) B lymphocyte encounters specific
antigen.
T cell development to the stage of the precursor T lymphocyte occurs within the
bone marrow. The precursor T lymphocytes (otherwise known as pre-Ts) then
must go to the thymus to complete maturation. When mature T lymphocytes leave
the thymus, they leave as mature (but nave ) Tc (T cytotoxic lymphocytes) or Th
(T helper lymphocytes). Further differentiation does not occur until the mature T
cells encounter antigen (presented to the T cell in association with MHC proteins).
The complex orchestration of hematopoiesis through which the elaborate array of blood
cells described above is produced requires three physiologic components, each of which
is essential. These are:
the stem cell pool itself (described above);
the hematopoietic inductive microenvironment, which is made up of the
bone marrow stroma and vasculature; and
hematopoietic cytokines, which are the hormones that regulate
hematopoiesis through both endocrine and paracrine mechanisms

IX. Medullary (Hematopoietic) Microenvironment

The medullary microenvironment or the bone marrow stroma contains cells and the
extracellular matrix.
There are many different cell types, including macrophages, fibroblasts, endothelial
cells, smooth muscle cells, T-lymphocytes, monocytes etc. These cells, in
combination with components of the extracellular matrix and basement membranes,
form the so-called Hematopoietic inductive microenvironment which maintains
the functional integrity of this complex system of resident and circulating cells. Cells
of the hematopoietic microenvironment show low or no detectable cell growth and
are believed to be in the G0 phase of the cell cycle .
The extracellular matrix occupies the space between cells. Many of the components
of the extracellular matrix are connected to proteins of the cytoskeleton by
transmembrane proteins.The matrix is a complex network of different combinations
of collagens, proteoglycans (PG), hyaluronic acid, laminin, fibronectin , and many
other glycoproteins including proteolytic enzymes involved in degradation and
remodeling of the extracellular matrix. The matrix plays an important structural and
functional role in multicellular organisms and is more than a scaffold that fills
extracellular spaces. Many of its components are engaged in processes mediating
cell-to-cell. Some of the matrix components, in particular the proteoglycans ,
function as modulators of the biological activities of growth factors and in
angiogenesis
The microenvironment of the bone marrow is an essential ingredient of
hematopoiesis. Several important aspects of blood cell production are managed by the
stroma of the marrow.
First, a number of the classes of cells that comprise the stroma (e.g. endothelial cells,
fibroblasts, macrophages) are rich sources of hematopoietic cytokines that act on
stem cells and early committed progenitor cells. In this setting, hematopoietic
cytokines are able to function as paracrine hormones since they are produced by
cells that are in close proximity to the cells upon which they act.
Secondly, the marrow provides an important sorting mechanism that permits only the
most immature cells (stem cells, committed progenitor cells) and the most mature
cells (e.g. red blood cells, neutrophils, platelets) to exit the marrow (HOMING). In
order to enter the blood stream, cells must penetrate pores in the endothelium that are
much smaller than a red blood cell. Only cells with no nucleus (red blood cells,
platelets) or with a very distensible nucleus (neutrophils, lymphocytes, stem cells)
can ordinarily leave the marrow. All of the maturing blood elements have large, rigid
nuclei that restrict them to the marrow.
Finally, the bone marrow serves as an important reservoir for mature neutrophils. In
a healthy individual, over 95% of the mature neutrophils in the body are in the bone
marrow, ready to be released on a moment's notice should a bacterial infection occur.
These neutrophils are said to be in the bone marrow's neutrophil storage pool.

X. Hematopoietic Cytokines

The hormones that regulate blood cell production are called hematopoietic cytokines.
Most of them were discovered starting in the mid 1960's. These growth factors are
present at extremely low concentrations and biological activity at concentrations as low
as 10-12 M. Some of the natural hormones were laboriously biochemically purified, and
starting in the mid 1980's, various laboratories began to clone the genes that encoded
many of these glycoproteins. As a consequence, it became possible to express synthetic
hematopoietic cytokines as recombinant proteins. Now, more than 20 of these hormones
have been cloned and sequenced, and more are emerging every day.
CSFs- act in a stepwise manner inducing proper maturation.
IL-3 [multi-CSF] acts early, possibly even at the level of the pluripotent stem cell,
to induce formation of the nonlymphoid cells (erythrocytes, monocytes,
granulocytes[neutrophils, eosinophils, basophils], and megakaryocytes).
GM-CSF acts at a slightly later stage, but it also induces formation of all the
nonlymphoid blood cells.
M-CSF and G-CSF act still later to promote the formation of monocytes and
granulocytic cells, respectively.
Erythropoietin (Epo) - is mainly a differentiation factor for late determined and
differentiated progenitor cells of erythropoiesis. It determines their differentiation
and maturation into erythrocytes. In addition Epo also regulates the proliferation
of erythropoietic progenitor cells . The Epo sensitivity progressively increases
with differentiation of immature progenitor cells (see: BFU-E , CFU-E . Epo has
not been shown to act on pluripotent hematopoietic stem cells
IL-4 - stimulates B progenitors, mast progenitors, and basophil progenitors
IL-5 - stimulates eosinophil progenitor
IL-6 - stimulates the myeloid stem cell
IL-7 - induces the differentiation of lymphoid progenitor into B progenitor and T
progenitor
IL-8 - stimulates the neutrophil progenitor
IL-9 - stimulates mast cell growth
Committment of a progenitor cell is associated with the expression on the cell
membrane of membrane receptors that are specific for particular cytokines.
Hematopoiesis is a continuous process throughout adulthood. Production of mature
blood cells equals their loss. Estimated that the average human must produce 3.7X1011
blood cells per day. This process is regulated by complex mechanisms.
Cell division and differentiation during hematopoiesis are balanced by apoptosis - there
must by maintenance of a steady state.
During apoptosis you see:
a decrease in cell volume
modification of the cytoskeleton with pronounced membrane blebbing
condensation of chromatin
degradation of DNA into oligonulceosomal fragments
shedding of apoptotic bodies

quick phagocytosis to prevent inflammation

If apoptosis fails, a leukemic state can occur.