Chapter

Hematopoiesis

2 Amber M. D’Souza, Pedro A. de Alarcón, and Mervin C. Yoder

Hematopoiesis refers to the continuous produc- Overview of Hematopoiesis

tion and release of blood cells into the circula-
tion. As blood cells become old or injured, self- Early Blood Cell Investigators
renewing hematopoietic stem cells (HSC)
With the invention of the microscope came the
proliferate and differentiate to replenish multi-
identification of the first blood cell in the late
ple hematopoietic lineages. This process
1600s: erythrocytes. Dutch scientist Antonie van
produces nearly 200 billion red blood cells,
Leeukwenhoek described red blood cells as “san-
10 billion white blood cells, and 400 billion pla-
guinous globules” that were “flexible and pliant”
telets every day. In addition to the requirement
in order to pass through small vessels [2].
for high cell production, the concentration of
Colorless white blood cells were not identified
individual blood cell lineages is precisely regu-
for nearly 200 more years, when a medical student
lated in the peripheral blood and tissues. The
named Paul Ehrlich devised a triacid stain that
production and use of circulating blood cells
allowed for the identification of different white
increase during periods of altered homeostasis
blood cell lineages based on size, nuclear shape,
such as defense against infection or replenish-
and cytoplasmic elements. Platelets were first
ment of circulating red cells after hemorrhage.
described in the mid 1800s as small globules
When the tightly regulated production of blood
derived from plasma [3], and later established as
cells fails, the host may encounter life-
the third element of blood, distinct from white
threatening anemia or other cytopenias or suf-
and red cells, by Giulio Bizzozero [4]. With addi-
fer from excessive neoplastic growth of blood
tional improvements in microscopes and triacid
cells manifesting as leukemia.
stains, the field of hematology began to expand,
During embryogenesis, hematopoiesis
and changes in blood cell number, morphology,
occurs in tightly regulated, tissue specific sites,
and function were correlated with specific human
including the extraembryonic yolk sac, para-
diseases. Further progress in studying hematopoi-
aortic mesonephric area, fetal liver, and the pre-
esis was largely limited to conjecture and opinion
term marrow. A harmonious relationship
derived from morphologic studies of blood
between hematopoietic stem cells, neighboring
cell-containing tissues until the middle of the
tissue stromal components, and hematopoietic
twentieth century, when hematologists began to
growth factors promotes the evolution of red
examine the hematopoietic consequences of ani-
cell production throughout human develop-
mal exposure to ionizing radiation [2].
ment. This chapter will briefly review the cellu-
lar model of hematopoiesis starting in the
human embryo. Although human hematopoi- Methods of Identifying Hematopoietic
esis is the focus of this chapter, certain concepts
gathered from murine studies will be presented Stem Cells and Progenitor Cells
to demonstrate some basic hematopoietic prin- Assays of hematopoietic stem and progenitor cell
ciples [1]. (HSPC) potential were first developed in the
1950s–1960s. After discovering that whole-body

Neonatal Hematology, Pathogenesis, Diagnosis, and Management of Hematologic Problems, 3rd edition, ed. Pedro
A. de Alarcón, Eric J. Werner, Robert D. Christensen, and Martha C. Sola-Visner. Published by Cambridge
University Press. © Cambridge University Press 2021.

10
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irradiation of animals led to significant pancyto-
penia (i.e. reduction in blood cell counts), scientist
Lo Jacobson discovered that mice were protected
from hematopoietic effects of radiation if their
spleens were shielded or if irradiated mice were
subsequently infused with blood cells from
a healthy mouse [5]. Confirmation that hemato-
poietic cells, not plasma, conferred radiation pro-
tection was reported by Ford and colleagues in
1956 [6]. In 1961, Till and McCulloch provided
evidence that single, multipotent hematopoietic
precursors (i.e. cells giving rise to more than one
lineage of blood cells) could be identified in vivo
by injecting the donor marrow cells into the
spleen of a lethally irradiated recipient animal
and examining the recipient spleen for hemato-
poietic colonies 8 to 12 days later ([7]; Fig. 2.1).
Each colony of hematopoietic cells in the spleen
arose from a single precursor cell, the colony-
forming unit in the spleen cell (CFU-S) [7].
Although this method advanced the study of
hematopoietic stem cell biology, little was known
in the early 1960s of the mechanisms that caused
or permitted stem cells to differentiate into
mature cells.
Fig. 2.1 Image of the various erythroid colonies. Examples of
colonies derive from murine definitive and primitive erythroid
progenitors. Immature BFU-E derived colonies, counted at day
7 of culture, typically consist of one or more large colonies
surrounded by smaller colonies, which constitute a “burst.”
Late-stage BFU-E derived colonies, counted at day 3 of culture,
consist of smaller single colonies. CFU-E derived colonies,
counted at day 2 of culture, consist of a single small colony
containing 16–64 hemoglobinizing cells. Primitive erythroid
progenitors (EryP-CFC) derived from the mouse embryo or
from differentiating mouse embryonic stem cells generate
single compact grape-like colonies of large primitive
erythroblasts at 5 days of in vitro culture. Abbreviations: d, day.
(From Joyce A. Lloyd (ed): Erythropoiesis: Methods and
Protocols, Methods in Molecular Biology, vol 1698, Springer,
2018, p. 119.)
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Hematopoiesis
In the mid 1960s, several groups demon-
strated that murine hematopoietic cells could
be cultured in vitro [8, 9]. Additionally, these
single precursor cells, called colony-forming
unit in culture (CFU-C), could differentiate into
myeloid colonies when grown in the presence of
soluble fluid from murine organs (i.e. urine or
pregnant uterine extracts), which became known
as colony-stimulating activity (CSA). Later work
revealed that these colonies were composed of
granulocytes, macrophages, or both (CFU-GM),
and that the specific cell type formed was based
on the type of CSA added. Later, using anemic
mouse serum as a growth factor, Axelrad and
coworkers demonstrated that red blood cell colo-
nies were clonally derived in vitro, and they
called the most primitive red cell precursors the
erythroid burst-forming unit (BFU-E) and the
most committed erythroid precursors colony-
forming unit-erythroid (CFU-E) cells ([10]; see
Fig. 2.1). Multipotent progenitor cells were also
identified and called colony-forming unit-mix
(CFU-Mix). These assays, developed for studying
murine hematopoiesis, were rapidly adapted for
human studies and continue to be sensitive
assays for detecting human hematopoietic pro-
genitor cells [11].
Because the frequency of human and murine
hematopoietic stem cells is estimated to be 1 of
104 to 105 marrow cells [12, 13], the primary
obstacle in studying human hematopoietic stem
cell biology is that few methods can identify these
rare cells. As such, results of studies of murine
hematopoietic stem cells were essential to advan-
cing our understanding of human hematopoi-
esis. In fact, it was murine bone marrow
transplantation experiments that provided the
evidence to reliably identify hematopoietic stem
cells, which were defined as cells that self-renew
in vivo, proliferate, and differentiate into all
lineages of circulating peripheral blood cells for
more than 4 months after transplantation into
recipient animals [14]. Because of ethical con-
straints, similar in vivo reconstitution assays in
human patients are not possible, and several
alternative in vitro and in vivo estimates of
hematopoietic precursors have been developed.
In vitro studies have identified two types of
hematopoietic progenitor cells. Lineage-committed
hematopoietic progenitor cells form colonies
in vitro in response to one or two recombinant
growth factors. In contrast, more primitive
11
 Hematopoiesis
multipotent hematopoietic cells form in vitro colo-
nies only when cultured with multiple recombinant
growth factors. These results have been interpreted
as evidence that the more primitive hematopoietic
stem and progenitor cells (HSPC) require complex
growth factor combinations to fuel or support line-
age commitment and progenitor cell differentiation.
Plating hematopoietic stem cells in special double-
layer agar cultures with multiple recombinant cyto-
kines permits identification of blood progenitors
that are highly proliferative (i.e., high proliferative
potential colony-forming cells (HPP-CFC)). High
proliferative potential colony-forming cells produce
colonies containing more than 50,000 cells that are
visible without magnification (most CFU-Cs are less
than 50,000 and microscopic) and are considered to
be the most primitive progenitor cell grown in semi-
solid medium without added stromal cells.
Additionally, complex in vitro cultures of
hematopoietic stem cells with a monolayer of
bone marrow or fetal liver stromal cells (includes
macrophages, endothelial cells, fibroblasts, and
preadipocytes) results in sustained hematopoiesis
for several months [15]. The most primitive
hematopoietic cells (i.e. those giving rise to hema-
topoietic progenitors) reside beneath the stromal
monolayers, whereas the mature blood cells pro-
duced are released into the tissue culture medium
as nonadherent cells [16]. Using limiting dilution
analysis, the number of such murine or human
long-term culture-initiating cells (LTC-IC) that
give rise to multipotent and committed progeni-
tors for 3 to 4 weeks in vitro can be calculated [17].
As such, LTC-IC are considered to be the most
primitive hematopoietic precursor detectable by
means of in vitro assays.
In attempts to develop an in vivo system
for detection of human hematopoietic stem cells,
several groups developed xenotransplantation
models. Enriched populations of human hemato-
poietic stem cells from human bone marrow, cord
blood, or fetal liver have been observed to engraft
in the pre-immune sheep fetus (<60 days of gesta-
tion) with long-term evidence of multilineage per-
ipheral human blood cell chimerism in the
transplanted animals [18]. Nonobese diabetic
(NOD) mice bred with severe combined immu-
nodeficient (SCID) mice produce NOD/SCID
mice that accept human hematopoietic grafts
[19]. This NOD/SCID model permits calculation
of the frequency of human repopulating cells in
a donor sample, but it remains unclear whether
12
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this assay is robust and can accurately identify the
same hematopoietic stem cells that repopulate the
blood system of a larger animal such as
a nonhuman primate [20]. More recent immuno-
deficient murine models apparently permit higher
levels of human cell engraftment and even full
lymphoid reconstitution under certain circum-
stances, but these models have not yet been com-
pared to the nonhuman primate models for
detection of engrafting long-term repopulating
human stem cells [21–24].
Stem Cell Model of Hematopoiesis
The stem cell theory of hematopoiesis posits that
the hematopoietic system comprises a continuum
of functionally distinct hematopoietic cell com-
partments. Stem cells are rare, self-renewing, rela-
tively quiescent cells (Fig. 2.2). In the mouse,
hematopoietic stem cells have a half-life (i.e.,
time to 50% of stem cells cycled) of approximately
19 days [25], though slower rates may be identi-
fied in a subset [26]. Stem cells can divide to give
rise to a daughter cell that retains all of the plur-
ipotentiality of the parent (i.e., self-renewal) or
can proliferate into daughter cells that have lost
some multipotentiality and have become com-
mitted to producing progenitor cells [27]. After
the commitment decision is made, the progenitor
cell gives rise to progressively more lineage-
committed hematopoietic progenitor cells and
eventually to mature blood cells (see Fig. 2.2). It
remains unclear how hematopoietic stem cells
become committed to follow a certain lineage,
but thought to be regulated by multiple environ-
mental factors including tissue specific growth
factors, cytokines, metabolic products, oxygen
levels, extracellular matrix molecules, or cell-cell
interaction and feedback loops [28–32].
With the development of flow cytometry,
monoclonal antibodies to cell-surface antigens,
and in vivo and in vitro assays for primitive
hematopoietic cells, investigators succeeded in
identifying enriched populations of human
hematopoietic stem cells and provided evidence
that a single murine hematopoietic stem cell is
sufficient to restore multilineage hematopoiesis
in an irradiated host [33, 34]. Purification of
hematopoietic stem cells permitted investigators
to pursue basic investigations of stem cell biology
that translated into new methods for human
hematopoietic transplantations and improved
 Hematopoiesis

LT- LT- HSC HSC

a HSC
b HSC pool c pool
IT-
HSC
ST-
HSC
ST-
HSC
MPP MPP
2 MPP 4
3

CMP CMP LMPP

MEP GMP CLP MEP EoBP/GMP CLP

Erythrocytes Monocytes B cells Erythrocytes Monocytes Neutrophils B cells Erythrocytes MegakaryocytesB cells Monocytes DCs
Megakaryocytes Eosinophils T cells Megakaryocytes Eosinophils DCs T cells T cells Eosinophils
Neutrophlils NK/ILCs NK/ILCs NK/ILCs Neutrophils
DCs

Fig. 2.2 Timeline of hierarchical models of hematopoiesis. (a) Visualization based on cutting-edge research around the year 2000:
HSC are represented as a homogeneous population, downstream of which the first lineage bifurcation separates the myeloid and
lymphoid branches via the common myeloid progenitor (CMP) and common lymphoid progenitor (CLP) populations. (b) During the
years 2005–2015, this visualization incorporates new findings: The HSC pool is now accepted to be more heterogeneous both in
terms of self-renewal (vertical axis) and differentiation properties (horizontal axis), the myeloid and lymphoid branches remain
associated further down in the hierarchy via the lymphoid-primed multipotential progenitor (LMPP) population, the GMP
compartment is shown to be fairly heterogeneous. (c) From 2016 onwards, single-cell transcriptomic snapshots indicate
a continuum of differentiation. Each red dot represents a single cell and its localization along a differentiation trajectory.
Abbreviations: DCs, dendritic cells; EoBP, eosinophil–basophil progenitor; GMP, granulocyte–monocyte progenitors; LT, long-term;
ILCs, innate lymphoid cells; MEP, megakaryocyte–erythrocyte progenitors; NK, natural killer cells; ST, short-term. (From Laurenti E,
Göttgens B. From haematopoietic stem cells to complex differentiation landscapes. Nature 2018; 553(7689):418–26.) (See plate
section for color version.)

methods of somatic gene transfer. Further stu- Embryonic Hematopoiesis

dies to examine hematopoietic stem cell function
in patients with bone marrow failure syndromes
and hematologic malignancies will also probably
translate into clinical therapies.
A significant challenge to the in vitro study
of hematopoietic stem cells is the realization
that many of the growth factors required to
stimulate the in vitro proliferation and differen-
tiation of hematopoietic progenitor cells are
associated with measurable declines in the con-
centrations of functional hematopoietic stem
cells. Although certain stromal cell lines and
primary human or murine stromal cells can
maintain hematopoietic stem cell function
in vitro, only in the last several decades have
tissue culture conditions been developed that
permit expansion of murine hematopoietic cell
numbers with retention of individual hemato-
poietic stem cell self-renewal and reconstituting
ability [35–37]. The conditions to support
in vitro expansion of adult human and murine
bone marrow hematopoietic progenitor cells
indicate that the proliferative potential and
growth factor requirements for survival in vitro
differ among hematopoietic cells during onto-
geny [36, 38].
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Studies of Murine Embryonic
Hematopoiesis
The murine visceral yolk sac is composed of visc-
eral endoderm and mesoderm cells that proliferate,
differentiate, migrate, and co-localize during gas-
trulation. The endoderm cells face the yolk sac
cavity and are highly active in the absorption of
material from the maternal circulation and in the
biosynthesis and secretion of numerous nutrients
[39]. In the mouse, yolk sac endoderm cells are also
the primary route for transporting maternal
immunoglobulins into the embryo. The mesoderm
cells of the yolk sac give rise to blood islands con-
taining blood cells and endothelial cells [40–42].
The simultaneous appearance of endothelial and
hematopoietic cells in the blood islands, the appar-
ent intimate cell-to-cell contact of the blood island
endothelial and hematopoietic cells, and the shared
requirements for certain growth factors and
growth factor receptors support the idea that
blood island endothelial cells and hematopoietic
cells must be derived from a common precursor,
called the hemangioblast [41–43]. Study of the
hemangioblast remains elusive, although some
13
 Hematopoiesis

reports have suggested that isolating the cell is
feasible in the murine or chick embryo [43–46].
The precise temporal and spatial origins of the
first hematopoietic stem cells in the murine
embryo remain unknown. Studies using chicks
and frogs indicate that the mesoderm cells that
become committed to the formation of blood cells
migrate from the posterior primitive streak and
into the embryo and the extraembryonic yolk sac
[40]. The mesoderm cells contributing to the for-
mation of murine blood cells are also restricted to
the posterior primitive streak at 6.75 days post-
conception (DPC). Some mesoderm cells fated to
become blood cells migrate along the extraem-
bryonic endoderm cells and participate in form-
ing the extraembryonic yolk, sac blood islands
[40]. Presumably, certain mesoderm cells are
acted on by local factors to undergo differentia-
tion into endothelial and hematopoietic stem and
progenitor cells. Although little is known about
the factors that induce murine mesodermal cell
commitment to blood and endothelial differentia-
tion, some evidence suggests that certain tran-
scription factors and growth factor receptors are
required for this process [28, 47]. Because blood
cells arise from different sites at different times
during murine embryogenesis, the site of origin of
hematopoietic stem cells remains a vital question
in experimental hematology. Hematopoietic stem
cells may arise de novo in each hematopoietic site
during ontogeny [48, 49]. Alternatively, all hema-
topoietic stem cells may arise in the embryonic
yolk sac and/or the para-aortic splanchnopleura
(PS) and migrate to all other sites during devel-
opment [50–53]. Several groups of investigators
have identified hematopoietic stem cell activity in
the mouse that appears to originate from the
ventral wall of the embryonic paired dorsal aorta
and/or from the subendothelial mesenchyme of
the PS [54–58]. The most recent data suggest that
all murine hematopoietic stem and progenitor
cells that reside in the bone marrow compartment
are derived from hemogenic endothelium present
in embryonic and extraembryonic vessels [57, 59,
60]. It is also apparent that biomechanical forces
generated by flowing blood promote the emer-
gence of the first blood cells [61–63].
Differentiated blood cells are first recogniz-
able in the murine yolk sac at 7 DPC. The pri-
mary cells observed are nucleated red blood cells
expressing embryonic forms of hemoglobin [64].
Hematopoiesis peaks in the murine yolk sac at 11
DPC, and the liver becomes the predominant site
of hematopoiesis at 12 DPC. The murine fetal

Fig. 2.3 The composition of the HSPC compartment changes in space and time. HSPCs are found in many organs in the body
across a lifetime. Cells of different colors represent distinct HSPC subsets. It is unclear whether all HSPC subsets and differentiation
trajectories are present in the same proportion in each of the organs. Current evidence suggests that age-related changes result from
a combination of shifts in the composition of the HSPC pool, as well as phenotypic changes in particular cell types driven by intrinsic
genetic or epigenetic changes and systemic alterations of the microenvironment. Abbreviation: AGM, aorta gonad mesonephros.
(From Laurenti E, Göttgens B. From haematopoietic stem cells to complex differentiation landscapes. Nature 2018;553
(7689):418–26.) (See plate section for color version.)

14
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liver remains the primary site of hematopoiesis
until the time of birth, when the bone marrow
and to some extent the spleen provide lifelong
provision of circulating blood cells (Fig. 2.3).
Although red blood cell production predomi-
nates in the liver and spleen, white blood cell
production predominates in the bone marrow
compartment [64].
The pattern of hematopoietic cell differentia-
tion also varies greatly in the yolk sac and PS.
Primitive red blood cells, macrophages, and
megakaryocytes are produced in the early yolk
sac. The red blood cells are called primitive
because they are large, nucleated cells that contain
forms of hemoglobin expressed only during the
embryonic phase of development. Other hemato-
poietic progenitors emerging in the yolk sac
include BFU-E (expressing primitive and adult-
type hemoglobins), CFU-GM, and CFU-Mix that
vary with the age of the donor embryo [65].
Culture of hematopoietic progenitors from the
PS gives rise to BFU-E and CFU-GM that are
composed of cells with definitive features; red
blood cells are enucleated and synthesize adult-
type hemoglobins, and granulocytes predominate
over macrophages in the CFU-GM. Of interest,
more recent data suggest that all of the circulating
definitive progenitor cells that seed the fetal liver
at 10 DPC and differentiate into mature elements
by 12 DPC are derived from the yolk sac and not
the embryo proper [66].
Studies of Human Embryonic
Hematopoiesis
The inability to use transplantation methods for
identification of hematopoietic stem cells in
human patients has limited the analysis of stem
cell function during human embryonic hematopoi-
esis. However, much of the ontogeny of blood cell
production in the human embryo has been
described using morphologic approaches and
some in vitro hematopoietic progenitor cell assays.
Human hematopoiesis occurs in a sequential pro-
cess throughout the embryonic and fetal period:
the yolk sac, the aorta–gonad–mesonephros region
(AGM), the fetal liver, and finally the bone marrow
(see Fig. 2.3). This section reviews the formation
and function of the human yolk sac, determination
of the site and kinetics of blood cell appearance,
and determination of the number and behavior of
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Hematopoiesis
cultured human embryonic hematopoietic pro-
genitor cells.
The human yolk sac develops in three stages:
a formative period, a period of yolk sac function,
and a period of yolk sac regression [67]. The first
phase of yolk sac formation can be further sub-
divided into formation of the primary and sec-
ondary yolk sacs. The primary yolk sac is formed
after implantation (7 to 8 DPC) by proliferation
and differentiation of primitive endoderm cells
into visceral endoderm adjacent to the developing
embryonic disk and parietal endoderm projecting
into the uterine lumen. This simple bilaminar
structure has no identifiable function, but the
endoderm cells lining the primary yolk sac give
rise to mesodermal precursors (i.e. intermediate
cells) that colonize the space between the yolk sac
cavity and the surrounding trophoblast cells. The
primary yolk sac then appears to collapse, with
heterogeneous foci of re-expansion breaking up
the primary yolk sac into smaller vesicles. The
secondary yolk sac is formed (12 to 15 DPC)
from the remnants of the primary yolk sac (i.e.
parietal endoderm and mesoderm) that are con-
tiguous with the visceral endoderm underlying
the embryonic disk.
The secondary yolk sac is an active site of pro-
tein biosynthesis, nutrient transport, and hemato-
poiesis. A variety of proteins are synthesized
including albumin, alpha–fetoprotein, prealbumin,
apolipoproteins, insulin-like growth factor, trans-
ferrin, vascular endothelial growth factor, and col-
ony-stimulating factor 1 (i.e. macrophage colony-
stimulating factor) [67]. The endodermal compo-
nent of the secondary yolk sac is a transporter of
proteins and vitamins [68]. Another primary func-
tion of the secondary yolk sac is to produce hema-
topoietic cells. The first primitive red blood cells in
the yolk sac adhere to surrounding endothelial
cells, and these hematopoietic-endothelial masses
are called blood islands [69]. Commensurate with
blood island formation, the mesenchymal layer in
the secondary yolk sac thickens, but the endoderm
layer is not affected.
Macroscopically, the secondary yolk sac begins
as a small structure (<0.4 mm) but steadily
increases in size to 2 mm by day 19, to 4–5 mm
by 7 weeks, and 6–6.5 mm by the end of week 10.
As the yolk sac matures, an extensive network of
superficial capillaries is formed as the blood island
endothelial cells migrate toward one another and
15
 Hematopoiesis
form vascular channels. The secondary yolk sac
circulation is fed and drained by the vitelline
arteries and veins, which connect the embryonic
to the extraembryonic circulation. Initially, the
vitelline vessels empty into the sinus venosus of
the liver, establishing a preportal-type circulation.
Later in gestation, the yolk sac regresses, new capil-
laries arising from the developing gut contribute to
the vitelline circulation, and as the liver cords pro-
liferate, the portal circulation is established [70,
71]. Although evident until week 12 of develop-
ment, the yolk sac begins regressing around week
10, with progressive evidence of tissue degenera-
tion and with loss of blood flow through the yolk
sac vascular network [67, 72].
Many twentieth century investigators using
microscopic analysis of tissue sections of human
embryonic tissues described the sequence of hema-
topoietic development that occurs in the human
embryo and fetus [73–75]. During human develop-
ment in utero, blood cells first arise in the yolk sac
between days 16 and 19, followed sequentially by
fetal liver hematopoiesis by weeks 5 to 6 and engraft-
ment and expansion of hematopoietic cells in devel-
oping long bones by weeks 8 to 19 (e.g., most bones
become hematopoietic between weeks 11 and 12)
[76, 77]. The liver functions as the primary site of
hematopoiesis from weeks 6 through 22 of gestation
until the bone marrow becomes the lifelong predo-
minant site of blood cell production. Distinct pat-
terns of blood cell differentiation and proliferation
occur in each specific site of hematopoiesis during
ontogeny [78, 79].
The most extensive morphologic analysis of
hematopoiesis in the developing human was
reported in a 1979 monograph by Kelemen and
colleagues [76]. These investigators studied tissue
sections from 190 human embryos of between 3 and
28 weeks of gestation. Hematopoietic cells were first
observed in the human embryo at day 18 in the
mesenchymal layer of the secondary yolk sac. Most
blood islands were composed of hematopoietic cells
completely surrounded by endothelial cells, but
some extravascular free aggregates of blood cells
were present in the mesenchyme close to the visc-
eral endoderm. The 3- to 4-week embryo had more
defined vessels, many of which were empty of hema-
topoietic cells. Frequently, small clusters of undiffer-
entiated cells called hemocytoblasts (equivalent to
the hemangioblast in the mouse) associated with the
endothelium protruded into the vessel lumen.
Concomitantly, clusters of primitive erythroblasts
16
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(>20 μm cells with central to eccentric nucleus and
orthochromatic cytoplasmic staining) began to fill
capillaries and larger vessels (e.g., vitelline, umbili-
cal, chorionic, and amniotic). Wherever the number
of primitive erythroblasts predominated in vessels,
few hemocytoblasts were detectable. This was
a surprising finding, because the erythroblasts had
previously been thought to originate from the
hemocytoblasts [48]. At the sixth week of develop-
ment, intraembryonic and extraembryonic blood
vessels became macroscopically visible, and defini-
tive erythroblasts (<20 μm cells with condensed
nuclei and abundant hemoglobin cytoplasmic stain-
ing) and primitive erythroblasts could be identified
in the yolk sac vessels. The only other mature blood
cell in the yolk sac was the macrophage. In contrast
to normal fetal liver and bone marrow patterns of
differentiation, macrophages in the yolk sac arose
without evidence of a monocytic precursor. An
overall decline in hematopoiesis was observed in
the human yolk sac after the eighth week of
gestation.
Hematopoiesis occurred in the liver by the fifth
to sixth week of gestation. The establishment of the
systemic circulation (weeks 4 to 5) had just com-
menced before liver hematopoiesis at week 5. The
predominant blood cell observed in the large vessels
and in developing liver sinusoids was the primitive
erythroblast. Over the next 4 weeks, there was a shift
away from the primitive to the definitive erythro-
blast. Mature enucleated red blood cell production
in the fetus was associated with a 40-fold increase in
the total liver mass, with hematopoietic cells com-
prising 60% of the total cell number (weeks 11 to
12). Macrophages, megakaryocytes, granulocytes,
and lymphocytes also were identifiable during
weeks 5 through 22. Macrophages were the predo-
minant mature blood cell type identified early in
liver hematopoiesis (weeks 5 to 6), but the concen-
tration of macrophages diminished thereafter.
Megakaryocytes were present throughout the liver
phase of hematopoiesis, but a notable shift from
megakaryocytes with one to two nuclei to megakar-
yocytes with four or more nuclei occurred as liver
hematopoiesis diminished (weeks 18 to 21) and
bone marrow hematopoiesis began to flourish. The
few granulocytes were restricted to the connective
tissue of the hepatic portal area from weeks 5
through 15. A significant increase in granulocytes
was observed beyond week 21 of development, coin-
ciding with enhanced hematopoiesis in the bone
marrow. Lymphocytes were sparse until week 7 of

development, and even through week 13, they
remained a minor population (1% of blood cells)
in the liver. It was not evident whether these lym-
phocytes were of the B or T cell lineage, but the
thymus did not have lymphocytes until week 9. The
percentage of hemocytoblasts (presumed stem cells)
was high (10% of blood cells) in the 5- to 6-week
embryo liver but declined for the remainder of the
liver phase of hematopoiesis.
The earliest bone marrow sample in which
hematopoietic cells were present was taken from
an embryo at week 8 of development. The predomi-
nant cell type at 8 to 9 weeks was the primitive
erythroblast. Definitive erythroblast production
increased greatly from weeks 11 through 14, and
by 14 weeks, nearly all erythroblasts were definitive
types. It was speculated that primitive erythroblasts
were presented to the marrow by means of the
systemic circulation and that only definitive erythro-
blasts were produced in the marrow. Erythrocytic
cells constituted about 35% of total marrow cells
by week 12 and 20% to 30% thereafter. At weeks 8
to 9 of embryonic development, granulocytes were
observed in the marrow of long bones. Neutrophilic
granulocytes composed 30% to 40% of total marrow
cells between weeks 10 and 13. By 21 weeks, neu-
trophilic granulocytes were the major blood cell
produced, representing nearly 60% of hematopoietic
cells in the marrow. Eosinophilic granulocytes (1%)
were present in the marrow of embryos at week 10
and gradually increased to nearly 5% of total marrow
cells by week 21. Basophilic granulocytes were few in
the 10-week embryo and remained low (0.3%) even
in the 28-week embryo. Macrophages were most
numerous in the embryonic marrow at weeks 10
through 16, whereas monocytes peaked between
weeks 12 and 16. Lymphocytes increased from 12%
of total marrow cells at week 12 to 20% to 30% in
older embryos. Megakaryocytes were present in all
samples examined. The percentage of immature
“blast cells,” which probably represented hemato-
poietic stem and progenitor cells, varied from 15%
at weeks 12 through 13 to 1% to 4% at weeks 21
through 23. Whereas erythropoiesis exceeded gran-
ulocytopoiesis in the 10- to 11-week-old embryo
(erythropoiesis/granulopoiesis ratio of 0.8), granu-
lopoiesis predominated by week 12 and reached
a nearly adult ratio of 3 (1.8 to 3.3) by the twenty-
first week of human development.
Splenic hematopoiesis in the human embryo
has been difficult to prove. At 8 weeks of gestation,
the spleen is composed primarily of a meshwork
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Hematopoiesis
of mesenchymal cells with some primitive ery-
throblasts contained in the blood vessels [76].
Few macrophage and neutrophil precursors were
present in the spleen at 8 weeks. The hematopoie-
tic cells in the spleen were dispersed throughout,
but most erythroblasts were confined to the vas-
cular system. This distribution of hematopoietic
cells in the spleen contrasted with the same-stage
embryo liver in which the developing blood cells
were confined to focal sites of hematopoiesis.
Similar observations were made by other investi-
gators [80], and these results raised the question
of whether the hematopoietic cells observed in the
embryonic spleen were circulating blood cells or
were cells produced in the spleen [80].
Calhoun and coworkers used several methods
to determine whether the human mid-gestation
spleen (13 to 22 weeks’ gestation) was an active
site of hematopoiesis [81]. Liver, spleen, and
bone marrow samples were examined by histol-
ogy, and cell suspensions from each site were
prepared for enumeration of mature blood cells
and their immediate precursors. Lymphocytes,
macrophages, and erythroid cells predominated
at 13 weeks’ gestation. Neither mature nor
immature neutrophils were identified in the
spleen until 16 weeks’ gestation. Throughout
the study period, active foci of hematopoiesis
were present in the liver and bone marrow but
never in the spleen. Because the distribution of
blood cells observed for the splenic suspensions
was similar to the blood cell concentrations pre-
viously reported after fetal blood sampling [82],
these results in total suggest that the mid-
gestation human spleen is not normally an active
hematopoietic organ [81].
Numerous investigators extended these mor-
phologic observations by examining the onset of
human hematopoiesis by means of functional
assays of hematopoietic CFU-C formation and
by using cell-surface antigen detection to identify
specific hematopoietic lineages (i.e. monoclonal
antibodies with immunohistochemical or FACS
analysis). Although many studies have focused
on human fetal liver and marrow hematopoiesis,
little is known about the biology of hematopoietic
stem and progenitor cells that arise in the human
yolk sac. The data reviewed indicate that the tran-
sient population of yolk sac hematopoietic cells
differs in many aspects from hematopoietic cells
produced within the embryo proper or in the liver
or bone marrow.
17
 Hematopoiesis
Migliaccio and colleagues performed a careful
examination of the kinetics of yolk sac CFU-C in
human embryos from 4.5 to 10 weeks’ gestation
[83]. This stage encompassed the period of yolk
sac hematopoietic decline and initiation of liver
hematopoiesis. CFU-E and BFU-E were abundant
in the yolk sac of the 4.5-week embryos. CFU-
GMs were also present in high concentration. At
5 weeks, the liver contained abundant primitive
erythroblasts and some macrophages in the hepa-
tic sinusoids. The concentrations of CFU-E,
BFU-E, and CFU-GM in the liver rose rapidly
over the next 3 weeks and reached a plateau by
8 to 9 weeks of gestation. Although definitive
erythropoiesis (i.e. CFU-E and BFU-E) predomi-
nated for the remainder of the fetal liver phase of
hematopoiesis, CFU-GMs increased from week 8
through 12 and remained stable until almost 20
weeks, when the number of myeloid progenitor
cells declined [84]. Concomitant with the changes
in progenitor cell concentrations, the percentage
of circulating and tissue-restricted mature blood
cells of the myeloerythroid lineages changed as
hematopoiesis shifted from the yolk sac to the
liver. A gradual shift from primarily embryonic
to primarily fetal and adult hemoglobin molecules
also occurred in yolk sac and liver BFU-E-derived
red blood cells respectively, during the fifth
through eighth weeks of gestation [85].
Investigators have also attempted to isolate
and enrich populations of hematopoietic stem
and progenitor cells from the developing human
embryo. Through the use of monoclonal antibo-
dies and FACS, a stem cell-surface “phenotype”
has emerged that is useful in isolating hemato-
poietic progenitor and putative hematopoietic
stem cells [86]. Because numerous monoclonal
antibodies may recognize the same cell-surface
antigen, an international classification is assigned
to each monoclonal antibody; all monoclonal
antibodies reacting with a single blood cell-
surface antigen are given a cluster of differentia-
tion (CD) number designation [87].
The CD34 antigen is one that has been used
extensively in the positive selection of human
adult hematopoietic stem and progenitor cells
[88]. CD34, a cell-surface sialomucin, is also
expressed on human fetal liver hematopoietic
cells; more CD34 is expressed on the fetal hema-
topoietic progenitor cells than on adult hemato-
poietic progenitor cells [89]. The cell-surface
phenotype that permits the highest enrichment
18
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of committed and primitive hematopoietic pro-
genitor cells is found in embryonic and fetal
hematopoietic cells that do not express any
mature blood cell lineage antigens (lin−), CD38
(a transmembrane glycoprotein), or CD71 (trans-
ferrin receptor) but express CD4, CD34, CD117
(KIT tyrosine kinase receptor), and CD90 (Thy-1
antigen) [89–91]. Other antigens that are more
controversial with regard to enriching for hema-
topoietic stem and progenitor cell activity in the
embryo and fetus include expression of CD15
(3-fucosyl-N-acetyl-lactosamine), and human
major histocompatibility complex class II antigen
(HAD-DR)[90].
The CD34+ hematopoietic progenitor cells
have been identified as early as day 23 of human
gestation in the yolk sac and within the embryo
proper [92]. The intraembryonic CD34+ cells
were localized to the ventral aspect of the dorsal
aorta around the peri-umbilical region, or the
AGM. The hematopoietic clusters were in the
luminal side in tight association with the endothe-
lial cells [92]. When tissue explants of the peri-
umbilical aortic region, liver, heart, limbs, blood,
and umbilical cord of embryos at days 30 through
40 of gestation were co-cultured in vitro with
a murine bone marrow stromal cell line, fivefold
more hematopoietic progenitors were derived
from the peri-umbilical aortic region than from
all other sites combined [92]. Additionally, long-
term hematopoietic engraftment was achieved by
transplanting cells from the AGM into immuno-
deficient mice [93]. These results confirmed the
presence of hematopoietic progenitor cells in the
human embryo at sites distinct from the yolk sac
and liver and more recently have defined vascular
endothelium as the cells giving rise to the hema-
topoietic clusters [94, 95]. Huyhn and coworkers
isolated yolk sac, liver, and embryonic tissues
from 25 to 40-day-gestation embryos and plated
the enzyme-disaggregated samples in methylcel-
lulose cultures for detection of hematopoietic pro-
genitor cells [96]. Although the concentration of
BFU-E in the yolk sacs and embryo proper were
similar, 7–12-fold higher numbers of CFU-GM
were present in the embryo proper than in the
yolk sac. These results were interpreted as evi-
dence that intraembryonic hematopoietic precur-
sors occur in high concentrations when the liver is
initiating hematopoiesis.
In addition to differences in progenitor cell
concentration in the 25- to 50-day yolk sac, liver,

and embryo samples, distinct differences in the
biology of the hematopoietic progenitors were
also observed. Yolk sac BFU-Es were distinct
from liver and embryo proper-derived BFU-Es by
being larger and containing dispersed myeloid cells
on microscopic examination. It appeared that the
yolk sac BFU-Es were more primitive (i.e. contain-
ing erythroid and myeloid progenitor activity) than
typical adult marrow-derived BFU-Es [12]. Dose-
response curves analyzing the in vitro growth fac-
tor responsiveness of the progenitor cells did not
indicate any difference in the growth factor sensi-
tivity of progenitors from these sites [96]. The
CFU-E concentrations were low in the yolk sac
and embryo proper but were abundant in the
liver. These CFU-E results contrast with the find-
ings of Migliaccio and coworkers, who observed
high numbers of yolk sac CFU-Es in the 4.5- to
5-week human embryonic yolk sac [83]. The dis-
crepant results have not been resolved.
The yolk sac and embryo proper are known to
be composed of hematopoietic cells demonstrat-
ing HPP-CFC activity. The CD34+ hematopoietic
cells recovered from the yolk sac and embryo
yielded 45 HPP-CFC and 20 HPP-CFC, respec-
tively, with 10,000 CD34+ cells were plated with
specific combinations of growth factors [96]. All
CD34+ progenitors and HPP-CFC in the yolk sac
and embryo proper failed to express CD38 or
CD33, consistent with the pattern of expression
observed for adult marrow hematopoietic stem
cells.
The LTC-IC concentration is reported to be
higher in fetal liver CD34+CD38− cells than in
phenotypically similar adult marrow cells, and
the number of secondary colonies derived
from cultured LTC-IC was sevenfold higher
in fetal liver compared with adult marrow
CD34+CD38− cells [97]. Hematopoietic stem
cells from the livers of 12- to 15-week gestation
human fetuses can engraft and produce circu-
lating blood cell progeny for more than 2 years
after in utero transplantation into preimmune
fetal sheep [18, 98]. Human fetal liver cells can
also engraft and repopulate blood cell lineages
in human patients with a variety of hemato-
poietic disorders [99, 100].
No published reports have characterized the
biology of hematopoietic stem and progenitor
cells in the human late embryonic and early fetal
bone marrow. Several groups have isolated
enriched populations of hematopoietic cells from
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Hematopoiesis
the bone marrow (e.g. tibias and femurs) of
human fetuses at 12 to 24 weeks’ gestation.
Turner and colleagues compared in vivo repopu-
lating ability of CD34+ cells isolated from fetal
bone marrow, adult bone marrow, and mobilized
peripheral blood cells (e.g. patients treated for 5 to
7 days with recombinant growth factors and blood
cells collected by apheresis)[101]. The fetal mar-
row cells expanded to constitute 23% of the total
number of marrow and spleen cells in 15% of the
recipient mice, whereas adult marrow cells only
engrafted in 2% of the recipients. Human periph-
eral blood cells engrafted in 3% of the mice but
only in the T lymphocyte lineage. The investiga-
tors concluded that fetal marrow appears superior
to adult marrow and mobilized peripheral blood
in engrafting in this xenograft model and that
human hematopoiesis can occur in this model
without the administration of human cytokines
to recipient mice. One factor that may have con-
tributed to the high level of fetal marrow engraft-
ment in the mice was that 18- and 9-fold more
CD34+ cells were recovered in the fetal marrow
than in the mobilized peripheral blood and adult
bone marrow, respectively.
While the use of human embryonic hema-
topoietic stem cells has not been widely applied
for therapeutic transplantation, human umbili-
cal cord blood has emerged as an effective alter-
native to adult bone marrow as a source of
transplantable stem cells [102–106]. Human
embryonic stem (hES) cell-derived hematopoie-
tic stem cells has been proposed as an emerging
hopeful inexhaustible alternative to all current
sources of stem cells, though progress in defin-
ing full multilineage engraftment from a hES
cell has been elusive [107–110]. Most encoura-
ging are the increasing amounts of literature on
reprogramming human somatic cells to pluri-
potent stem cells [111–113]. Indeed, recent
work has demonstrated that human pluripotent
stem cells can be differentiated into human
hematopoietic stem and progenitor cells and
that adult murine endothelial cells may be
directly programmed into hematopoietic stem
cells [114, 115]. These exciting developments
may provide novel approaches to treating
human disease, but more work is needed to
understand the molecular regulation of hema-
topoietic stem cell development, and as such,
both great challenges and life-altering opportu-
nities lie ahead.
19
 Hematopoiesis
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