Article in press - uncorrected proof
Biol. Chem., Vol. 389, pp. 813–824, July 2008 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.092
Review
Lineage development of hematopoietic stem and
progenitor cells
Bernd Giebel* and Michael Punzel*
Institute for Transplantation Diagnostics and Cellular
Therapeutics, Heinrich Heine University, D-40225
Düsseldorf, Germany
* Corresponding authors
e-mail: giebel@itz.uni-duesseldorf.de;
punzel@itz.uni-duesseldorf.de
Abstract
Hematopoietic stem cells have the potential to develop
into multipotent and different lineage-restricted progeni-
tor cells that subsequently generate all mature blood cell
types. The classical model of hematopoietic lineage
commitment proposes a first restriction point at which all
multipotent hematopoietic progenitor cells become com-
mitted either to the lymphoid or to the myeloid devel-
opment, respectively. Recently, this model has been
challenged by the identification of murine as well as
human hematopoietic progenitor cells with lymphoid dif-
ferentiation capabilities that give rise to a restricted sub-
set of the myeloid lineages. As the classical model does
not include cells with such capacities, these findings
suggest the existence of alternative developmental path-
ways that demand the existence of additional branches
in the classical hematopoietic tree. Together with some
phenotypic criteria that characterize different subsets of
multipotent and lineage-restricted progenitor cells, we
summarize these recent findings here.
Keywords: hematopoietic progenitor cell (HPC);
hematopoietic stem cell (HSC); lineage specification.
Introduction
Hematopoietic stem cells (HSCs) are defined as clono-
genic cells that possess the dual abilities to self-renew
and to differentiate into mature blood cells of all lineages,
and that contain the capacity to regenerate the whole
blood system for a lifetime of an organism. These fea-
tures became evident after transplantation of HSC-con-
taining cell fractions either obtained from adult bone
marrow (BM), from peripheral blood of granulocyte-col-
ony stimulating factor (G-CSF) treated individuals or from
umbilical cord blood (CB) of newborns into immunocom-
promised hosts; apart from experiments in animals, clin-
ical applications demonstrate that the blood system of
treated patients can fully be restored by donor HSC for
the rest of their normal lasting life time (Bensinger et al.,
1997; Korbling and Anderlini, 2001; Ballen, 2005; Ring-
den and Le Blanc, 2005). Both exhaustion as well as
enforced expansion of HSC might result in clinical rele-
vant cytopenia or leukemia, respectively. Therefore, the
size of the HSC-pool and thus the decision whether
HSCs self-renew or become committed to differentiate
needs to be biased and tightly regulated. Additionally, to
establish and maintain an appropriate composition of dif-
ferentiated blood cells, processes controlling lineage
specification and differentiation need to be highly con-
trolled as well. Thus, it is a central issue of modern stem
cell research to uncover the mechanisms governing the
development of HSCs and their committed progeny
which are hierarchically organized.
One fundamental requirement to uncover such mech-
anisms is a reliable model which predicts the underlying
hierarchical organization as well as the relationship of the
individual cell lineages to each other. For several years,
the classical hematopoietic commitment model was the
prevailing model according to which multipotent HSCs
give rise to multipotent hematopoietic progenitor cells
(HPCs), which then create progenitor cells either being
restricted to the myeloid or lymphoid development (Reya
et al., 2001). As this classical hematopoietic model has
been challenged by several new data, we summarize
recent findings that imply the existence of additional
developmental branches within both the murine and the
human primitive hematopoietic system that are not
included in the classical model.
Murine hematopoietic differentiation
As already mentioned, all adult hematopoietic cells derive
from multipotent HSCs, which are capable to replenish
the peripheral blood system of lethally irradiated recipi-
ents after transplantation. In the murine system, the pool
of HSCs has been initially characterized by the Weissman
group as cells that do not express any lineage specific
antigens (Lin-), that are positive for the stem cell asso-
ciated antigen (Sca-1) and that express low amounts of
the T-cell antigen Thy1 (Lin-Sca-1qThy-1low cells). Later,
mouse HSCs were grouped in the Lin-Sca-1qc-kitq (LSK)
cell compartment (Spangrude et al., 1988; Ikuta and
Weissman, 1992; Morrison and Weissman, 1994; Osawa
et al., 1996). As shown in Figure 1 and summarized in
Table 1, the LSK compartment contains different hier-
archical classes of stem cells, which can be distin-
guished in clonogenic long-term self-renewing HSCs
(LT-HSCs), transiently self-renewing HSCs (short-term
HSCs), and non-self-renewing multipotent progenitors
(MPPs) (Smith et al., 1991; Morrison and Weissman,
1994; Osawa et al., 1996; Randall et al., 1996; Akashi et
Brought to you by | BUPMC 2008/271
MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
814 B. Giebel and M. Punzel
Figure 1 Developmental route(s) in adult murine hematopoiesis.
Within the murine hematopoietic system, HSCs are discriminat-
ed in long-term self-renewing HSCs (LT-HSCs) and transiently
self-renewing HSCs (short-term HSCs) that give rise to non-self-
renewing multipotent progenitors (MPPs). In contrast to the clas-
sical model which predicts that MPPs become specified either
as common myeloid progenitors (CMPs) or as common lym-
phoid progenitors (CLPs), recent findings suggest the existence
of additional developmental routes. The pool of MPPs can be
subdivided into cells containing the full CMP potential (FLT3lowV-
CAM1q cells) and in cells containing some residual CMP activ-
ities that are primed to develop along the lymphoid routes
(FLT3qV-CAM1q cells). In addition to the classical model in
which granulocyte/macrophage progenitors (GMPs) and mega-
karyocyte/erythrocyte progenitors (MEPs) are derivatives of the
CMP, cells have been found that contain the CLP and GMP
potential but lack the MEP capacity. These cells are termed as
lymphoid primed multipotent progenitor cells (LMPPs) and are
characterized as FLT3qV-CAM-1- cells. Within the group of the
FLT3qV-CAM-1- cells, IL-7Rq cells seem to contain CLPs and
no myeloid activities, and CCR9q cells still contain CLP capac-
ities but are primed to develop along the T-cell lineage (ETP).
al., 2000). The LT-HSCs are highly enriched within the
LSK-Thy1.1low cell fraction, and a subset of them can
nearly be purified as CD34- LSK cells that are located in
the tip of the side population (‘Tip’-SP CD34- KSL) (Mor-
rison and Weissman, 1994; Morrison et al., 1997; Mat-
suzaki et al., 2004). However, no sharp definition for
ST-HSCs or for MPPs has been described so far. More
recently, a set of new markers, cell surface receptors of
the signaling lymphocyte activation molecule (SLAM)
family, were found that also help to discriminate subpo-
pulations. In this context, HSCs are described as
CD150qCD48-CD244- cells, MPP as CD244qCD48-
CD150- cells and the most restricted progenitors as
CD48qCD244qCD150- cells (Kiel et al., 2005).
Downstream of the LSK compartment, cells lose their
multipotent capacities, and at a single cell level progen-
itors have been identified whose developmental capaci-
ties are either restricted to the myeloid or to the lymphoid
lineage, respectively (Kondo et al., 1997; Akashi et al.,
2000). Therefore, it was postulated that MPPs further dif-
ferentiate into either the common myeloid progenitors
(CMPs) or the common lymphoid progenitors (CLPs),
which sustain either granulocyte/macrophage progeni-
tors (GMPs), as well as megakaryocyte/erythrocyte pro-
genitors (MEPs) or T-cell, B-cell and Natural Killer (NK)
cell differentiation potentials, respectively (Kondo et al.,
1997; Akashi et al., 2000; Reya et al., 2001). Kondo et al.
(1997) described CLPs as a subpopulation of Lin-Sca-
1lowc-kitlowThy-1- cells that express the interleukin-7
receptor a chain (IL-7Ra) and are unable to differentiate
into myeloid cell types. In agreement with these findings,
the lymphopoiesis in IL-7Ra-deficient mice is severely
impaired (Peschon et al., 1994; He and Malek, 1996;
Maki et al., 1996; Kondo et al., 1997). The CMPs con-
taining fraction of Lin-Sca-1-c-kitq cells lacks lymphoid
gene expression and does not express IL-7Ra (Akashi et
al., 2000). Furthermore, and according to the expression
of the Fcg receptor (FcgR) and of CD34 the CMPs con-
taining fraction can be subdivided in three populations:
that of the (i) FcgRlowCD34q cells, (ii) the FcgRlowCD34-
cells, and (iii) the FcgRhighCD34q cells. In contrast to the
FcgRlowCD34q cell fraction which contains CMP activity,
the developmental potential of the FcgRlowCD34- is
restricted to the megakaryocyte/erythrocyte lineages and
defines MEPs, while that of the FcgRhighCD34q cells is
restricted to the granulocyte/macrophage development
and defines GMPs (Figure 1) (Akashi et al., 2000).
According to the discovery of CMPs and CLPs, it was
widely accepted that at this developmental stage all
hematopoietic cells become either restricted to the mye-
loid or the lymphoid development, as it has been for-
mulated in the ‘classical’ hematopoietic model (Reya et
al., 2001).
However, by using an additional set of markers, such
as FMS-related tyrosine kinase 3 (Flt-3) and vascular cell
adhesion molecule 1 (VCAM-1) (Adolfsson et al., 2001;
Christensen and Weissman, 2001), Flt-3qV-CAM-1- cells
have been described which still contain lymphoid devel-
opmental capacities and give rise to granulocytes and
macrophages but not to megakaryocytes or erythro-
cytes. Cells with these features seem to define a new
group of primitive hematopoietic cells termed as lym-
phoid primed multipotent progenitor cells (LMPPs) and
do not fit into the classical model of hematopoiesis in
which the first step of lineage commitment simply selects
between the lymphoid and the myeloid lineages (Adolfs-
son et al., 2005; Lai and Kondo, 2006). In this context,
comparison of gene expression profiles of LMPPs with
that of HSCs revealed a significant down-regulation of
genes required for granulocyte/macrophage differentia-
tion, and an up-regulation of genes known to be impor-
tantly involved in lymphoid differentiation, such as Ikaros
(Adolfsson et al., 2005; Lai and Kondo, 2006; Yoshida et
al., 2006; Mansson et al., 2007). Therefore, these obser-
vations led to the hypothesis that in addition to the clas-
sically described development of the GMPs from CMPs,
GMPs can arise from LMPPs that are also predicted to
generate CLPs (Figure 1) (Adolfsson et al., 2005; Buza-
Vidas et al., 2007).
Very recently, it was observed further that a subset of
the MPPs expresses the chemokine receptor 9 (CCR9)
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Table 1 Murine hematopoietic stem and progenitor cell populations.

Defined mouse Phenotype Determination Frequency References

population function source
HSC Lin-Sca1qc-kitq (LSK) In vivo Tx 0.05% of mouse BM Spangrude et al., 1988; Morrison and Weissman, 1994;
Osawa et al., 1996
LT-HSC LSK Thy1.1low In vivo Tx BM Smith et al., 1991; Morrison and Weissman, 1994
Flt3-VCAM-1q Adolfsson et al., 2001; Christensen and Weissman, 2001
CD34-CD38q 1 LT-HSC in 5 LSK34- Osawa et al., 1996; Randall et al., 1996
‘Tip’-side-population 21% HSCs (0.008% of Matsuzaki et al., 2004
CD150qCD48-CD244- BM) Kiel et al., 2005
ST-HSC LSK Thy1.1int In vivo Tx BM Reya et al., 2001
Flt3lowVCAM-1q Adolfsson et al., 2001; Christensen and Weissman, 2001
CD34qCD38- Osawa et al., 1996; Randall et al., 1996
CD150qCD48-CD244- Kiel et al., 2005
MPP LSK Thy1.1- In vivo Tx BM
Flt3lowVCAM-1q Adolfsson et al., 2001; Christensen and Weissman, 2001
CD34qCD38- Osawa et al., 1996; Randall et al., 1996
CD150-CD48-CD244q Kiel et al., 2005
Flt3lowVCAM-1q MPP LSK Thy1.1-CD34q In vivo Tx BM Lai and Kondo, 2006
Flt3lowVCAM-1q Primitive CMP potential
Flt3qVCAM-1q MPP LSK Thy1.1-CD34q In vivo Tx 15% of CD34q Lai and Kondo, 2006
Flt3qVCAM-1q CLP 15% of Thy1.1-
(marginal CMP) LSK
BM
Subfraction of LMPP
LMPP LSK Flt3qVCAM-1- In vivo Tx 30% of CD34q Adolfsson et al., 2005; Lai and Kondo, 2006
IL-7Raq CLPqGMP (qMegE) 30% of Thy1.1- Forsberg et al., 2006; Mansson et al., 2007
LSK
Article in press - uncorrected proof

BM
ELP LSK Flt3q RAG1qCD34q In vivo Tx 5% of CD34q Igarashi et al., 2002
CLPqGMP 5% of Thy1.1-
LSK
BM
subfraction of LMPP
ETP precursor LSK Flt3qVCAM-1- CLPq(GMP) BM Lai and Kondo, 2007
CCR9q
CLP Lin-Sca1lowc-KitlowThy1.1- In vivo Tx BM Kondo et al., 1997
IL-7Raq
CMP Lin-Sca1-c-KitqIL-7Ra- In vivo Tx BM Akashi et al., 2000
FcgRlowCD34q Akashi et al., 2000
CMP/MEP FcgRlowCD34- Akashi et al., 2000
CMP/GMP FcgRhighCD34q Akashi et al., 2000

Download Date | 1/30/18 11:39 AM

Authenticated
Brought to you by | BUPMC MIR (I894)
Hematopoietic stem cell lineage development 815
 Article in press - uncorrected proof
816 B. Giebel and M. Punzel
and preferentially home to the thymus. This together with
the concept that these cells are phenotypically related to
the most immature early T-lineage progenitors (ETPs) led
to the suggestion that although these CCR9q MPPs pos-
sess limited GMP potential and can give rise to T-, B-
and dendritic cells, they are already primed for T-cell
development (Lai and Kondo, 2007). This implies that
even cells that seem to be committed to a certain lineage
are not necessarily determined and might retain a certain
degree of developmental flexibility, sometimes called
plasticity. In addition and compatible to this hypothesis,
it was found that pax5 mutant cells that already started
to rearrange their immunoglobulin genes cannot proceed
with the B-cell development and possess long-term and
multipotent reconstitution capacities (Schaniel et al.,
2002a,b). In this context, over-expression experiments
revealed that Pax5 inhibits T-cell development, but does
not block early myeloid-lineage development (Cotta et
al., 2003). It rather promotes maintenance of bipheno-
typic myeloid progenitors which co-express myeloid and
B-cell lineage-associated genes (Montecino-Rodriguez
et al., 2001; Anderson et al., 2007).
Taken together, all these findings strongly suggest the
existence of murine progenitor cells that follow alterna-
tive developmental pathways which do not correspond
to the classical model of hematopoiesis, and thus define
new roadmaps during early hematopoiesis.
Unresolved controversies
Although the latter data seem to be contradictory to the
classical hematopoietic model, the Weissman group
demonstrated that transplantation of approximately 500
green fluorescent protein (GFP)-expressing Flt-3qLSK
cells that were designated to contain LMPP capacities
(Adolfsson et al., 2005; Lai and Kondo, 2006) result in a
robust reconstitution of GFPq platelets and GFPq ery-
throid progenitor cells (Forsberg et al., 2006). Conse-
quently, the hypothesis of alternative hematopoietic road
maps that might bypass the CMPs during GMP commit-
ment became questionable. According to Forsberg et al.
(2006), this hypothesis might mistakenly be based on
experimental artifacts probably related to the usage of
too little cell amounts and to the readouts that have been
performed. They suppose that the LMPPs are rather late
MPPs with reduced self-renewal capacities, but still con-
tain the full multi-lineage potentials (Forsberg et al.,
2006). The Jacobsen group, however, argues against that
and takes into account that the Flt-3qLSK-cell population
like any stem and progenitor cell population is hetero-
geneous and that since the Weissman group used
approximately 500 cells for transplantation into single
mice, they might have included some infrequent progen-
itor cells residing within this fraction that contained MEP
potentials (Mansson et al., 2007). To shed some more
light into this controversial discussion, they suggest sub-
fractionation of the cell population thought to contain
LMPP capacities and perform further analyses.
Hematopoietic lineage specification
The observations that hematopoietic cells can divide
asymmetrically (Beckmann et al., 2007; Chang et al.,
2007) and that their development depends on the sur-
rounding environment, the hematopoietic niches (Calvi et
al., 2003; Zhang et al., 2003; Kiel et al., 2005) demon-
strate that the development of hematopoietic cells is
orchestrated by a combination of intrinsic and extrinsic
signals. These signals become integrated to finally alter
the genetic program of corresponding cells. Thus, it
becomes evident that transcription factors as key regu-
lators of genetic programs play important roles in spec-
ifying the cell fate of hematopoietic cells during
hematopoietic lineage development. Certain transcription
factors, such as members of the GATA and SCL/Tal-
1gene families, act at different levels during hemato-
poietic development and seem to function as
‘hematopoietic master regulators’. For example, during
embryonal stages SCL/Tal-1 acts together with members
of the E-protein family (E2A) to induce mesodermal pro-
genitor cells to acquire the HSC fate. Mice lacking SCL/
Tal-1 activities are embryonic lethal and do not develop
a hematopoietic system (Robb et al., 1995, 1996; Shiv-
dasani et al., 1995; Porcher et al., 1996), whereas gained
SCL/Tal-1 activity increases the content of embryonic
hematopoiesis (Gering et al., 1998; Mead et al., 2001).
During adult hematopoiesis, SCL/Tal-1 is specifically
expressed in primitive hematopoietic cells as well as in
cells of the megakaryocyte/erythrocyte lineage and has
been found to control the differentiation of the latter cells
(Elefanty et al., 1998; Akashi et al., 2000; Ramalho-San-
tos et al., 2002; Mikkola et al., 2003; Zhang et al., 2005).
In contrast to SCL/Tal-1, E2A expression now is required
for governing the lymphoid differentiation (Bain et al.,
1994, 1997, 1999).
A typical paradigm of how lineage decisions can be
controlled is the antagonism between PU.1 and Gata-1.
PU.1 directs the differentiation of hematopoietic progen-
itors into macrophages, neutrophils and B-lymphocytes
(DeKoter et al., 1998; DeKoter and Singh, 2000), while
Gata-1 promotes development along the MEP lineage
(Pevny et al., 1991; Fujiwara et al., 1996; Shivdasani et
al., 1997). It has been shown that PU.1 interacts with
Gata proteins, i.e., PU.1 and Gata proteins antagonize
activities of each other by direct protein-protein interac-
tions. Additionally, PU.1 can inhibit the expression of
Gata factor encoding genes (Nerlov and Graf, 1998;
Rekhtman et al., 1999; Zhang et al., 1999, 2000; Nerlov
et al., 2000; Walsh et al., 2002). As it has been shown
that the amount of PU.1 decides upon whether hema-
topoietic progenitors follow the lymphoid development or
whether they differentiate into macrophages, it becomes
evident why enforced PU.1 or Gata expression can alter
the development of hematopoietic progenitor cells
(DeKoter and Singh, 2000; Iwasaki et al., 2003).
Remarkably, over-expression experiments revealed
that enforced expression of PU.1 and Gata proteins as
well as that of other myeloid transcription factors fre-
quently can override the lymphoid program and subse-
quently reprogram these progenitors towards certain
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
myeloid differentiation pathways (Iwasaki et al., 2003,
2006; Hsu et al., 2006). Transcription factors, whose
enforced expression is sufficient to enable committed
CMPs to give rise to lymphoid cells, have to our knowl-
edge not been defined yet. This together with the obser-
vation that HSCs express large numbers of myeloid but
not lymphoid genes and that during lymphoid develop-
ment myeloid gene expression progressively decreases
and lymphoid gene expression subsequently increases
suggests that myelopoiesis and most likely the granulo-
cyte macrophage development defines a default com-
mitment pathway for multipotent hematopoietic pro-
genitors that actively need to be maintained (Akashi et
al., 2003; Iwasaki and Akashi, 2007; Mansson et al.,
2007). In this context, lymphopoiesis seems to become
initiated when activities of pro-myelopoietic factors are
inhibited or become modulated, e.g., by the activity of
the Notch signaling pathway (Franco et al., 2006; Laiosa
et al., 2006; Rothenberg, 2007). Depending on the inte-
grated intrinsic and extrinsic signals, the default myeloid
commitment can theoretically differently be modulated
and thus might explain the existence of classical as well
as alternative lineage developmental pathways, i.e., that
of CMPs, LMPPs and GMPs.
This concept further helps to explain the observation
of Muller-Sieburg and colleagues, who found that indi-
vidual HSCs differ in their abilities to give rise to myeloid
or lymphoid daughter cells. According to these obser-
vations, HSCs can differently be biased, either towards
the lymphoid or to the myeloid lineage (Muller-Sieburg
et al., 2002, 2004).
Human hematopoiesis
It is evident that progress in the discovery of lineage
development and hematopoietic differentiation in the
mouse model has mainly been achieved by two experi-
mental approaches: (i) by analyses of genetic alterations
that result in changes of the lineage composition and/or
in alterations in the phenotype of distinct hematopoietic
cells, and (ii) by the isolation of distinct subpopulations
containing cells with HSC/HPC potential and their sub-
sequent functional analyses, either performed in vivo
using transplantation models or in vitro at clonal levels.
Although, knowledge about biological processes
obtained in mice can often be transferred to humans and
other mammals, some processes are not transferable
between species. In this context, essential differences
are found between the hematopoiesis in mouse and in
man. Before reviewing these differences, we would like
to add some comments about human HSCs.
Caveats in human HSC research
The fact that stem cell grafts can fully restore the blood
system of immunocompromised patients is proof for
human HSCs (Bensinger et al., 1997; Korbling and
Anderlini, 2001; Ballen, 2005; Ringden and Le Blanc,
2005). However, the functional readout of a hematopoi-
Hematopoietic stem cell lineage development 817
etic stem cell requires transplantation of donor stem cells
into an isogeneic host. For ethical reasons, human cell
fractions cannot be tested experimentally whether or not
they contain HSCs or more mature progenitor cells that
are unable to mediate long-term engraftment. Thus, it is
difficult to purify defined cell populations containing true
human HSC activity.
Over the last two decades, surrogate xenogeneic in
vivo-transplantation models have been established to cir-
cumvent these problems. Nowadays, xenografts into
immunodeficient NOD-SCID mice are the gold standard
readout for human long-term repopulating hematopoietic
cells (Kamel-Reid and Dick, 1988; Lapidot et al., 1992;
Larochelle et al., 1996), but also the fetal sheep model
(Zanjani et al., 1992).
Although, the usage of these animal surrogate models
has rigorously increased the knowledge about human
primitive hematopoietic cells, it should be kept in mind
that the quality and frequency of human HSCs cannot
directly be compared to that obtained in syngeneic mice
transplants. There are several limitations, such as the for-
eign homing environment that skews lineage readout
together with the usual short period (less than 7 weeks)
between transplantation and engraftment analyses.
Indeed, it has been reported that most of the primitive
hematopoietic cells obtained from non-human primates,
i.e., from baboons, that are able to engraft NOD-SCID
mice, only contain short-term but not long-term recon-
stituting activities and therefore should not be designated
as true HSCs (Ramirez et al., 1998; Horn et al., 2003;
Glimm et al., 2005; Horn and Blasczyk, 2007).
In addition to the xenotransplantation experiments, a
number of different in vitro readout systems had been
established that have recently been extensively reviewed
(Coulombel, 2004). These assays also revealed, and
surely will reveal, comprehensive knowledge about
human primitive hematopoietic cells, which has been the
basis for improvements of applications using human
HSC sources in clinical trials. Furthermore, this knowl-
edge is important to increase our understanding of the
etiology of certain diseases, e.g., that of leukemia.
Phenotypic description of human primitive
hematopoietic cells
As already pointed out, murine LT-HSCs are highly
enriched within the LSK cell fraction of cells which do
not express the cell surface antigen CD34. However,
most human cells with repopulating cell activities have
been identified as CD34q cells. Hence, in contrast to
mice, CD34 has become a surrogate marker of human
HSCs (Krause et al., 1996). Apart from CD34, there are
other substantial differences in the immunophenotype
between HSCs in man and mouse. Primitive hematopoi-
etic cells that have lost their repopulating capacities and
became committed to differentiate show higher expres-
sion levels of the CD38 antigen. Therefore, the majority
of the most primitive human hematopoietic cells is found
within the CD34qCD38- cell fraction (Terstappen et al.,
1991; Hao et al., 1996; Larochelle et al., 1996). In the
murine system, LT-HSCs have been characterized as
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
818 B. Giebel and M. Punzel
CD34-CD38q cells and more committed MPPs as
CD34qCD38- cells (Osawa et al., 1996; Randall et al.,
1996). Therefore, CD38 is a primitive marker in mice and
a differentiation marker in humans.
In addition to CD34, the AC133 antigen, prominin-1/
CD133, which is co-expressed on most of the CD34q
cells, was characterized as a stem cell surrogate marker
in humans (Yin et al., 1997; de Wynter et al., 1998) and
becomes localized on the uropod of cultivated CD34q
cells (Giebel et al., 2004). As a very small fraction of
immature cells with repopulating activities has been iden-
tified to reside within the Lin-CD34-CD38- cell fraction
which expresses CD133 (Bhatia et al., 1998; Zanjani et
al., 1998; Gallacher et al., 2000), CD133 seems to
describe the most primitive human hematopoietic cells
more precisely than CD34. In agreement with this, we
observed that upon cultivation CD34qCD133q cells gen-
erate a secondary CD34qCD133low/- population, which
has lost the primitive characteristics of CD34qCD133q
cells and increases over time (Giebel et al., 2006; von
Levetzow et al., 2006; Beckmann et al., 2007). Within the
murine hematopoietic system, CD133 (Prominin) was
found to be expressed on 36% of the BM-derived CD34q
cells (Corbeil et al., 2000), but to our knowledge its
expression has not been systematically analyzed within
the HSC-containing LSK fraction, yet. However, in con-
trast to primitive human hematopoietic cells, expanded
murine HSCs neither express CD133 nor CD62L (Zhang
and Lodish, 2005). According to our analyses, CD62L is
specifically expressed on cultivated human CD34q
CD133q cells (Beckmann et al., 2007).
In addition to absence of lineage-specific antigens
(Lin-) and low-level expression of CD38 as one of the
most applied phenotypical criteria to describe human
HSC candidates, other antigens have been used to dis-
criminate more primitive from more mature CD34q cells
(Table 2). Some of the combinations used are Lin-
CD34qHLA-DR- (Verfaillie et al., 1990), CD34qc-kitqCD38-
CD33-Rho123- (Liu and Verfaillie, 2002), Lin-CD133q
ALDHhi (Hess et al., 2006) or Lin-CD34qCD38-Rho123low
(McKenzie et al., 2007). Recently, together with CD71
and CD62L, which have been used to discriminate prim-
itive hematopoietic cell fractions before (Lansdorp and
Dragowska, 1992; Mayani et al., 1993; Mohle et al., 1995;
Koenig et al., 1999), we qualified the tetraspanins CD53
and CD63 as new markers, that together with CD133
help to discriminate more primitive cultured
CD34qCD133qwCD53qCD62LqCD63-CD71lowx cells from
more mature cultivated CD34qCD133low/-wCD53-CD62L-
CD63qCD71qx cells (Beckmann et al., 2007). It may be
that this new combination of antigens will help to solve
the dilemma that upon cultivation certain cell surface
antigens, such as c-kit or CD38, lose most of their pre-
dictive value for the identification of distinct functional
subpopulations of very primitive hematopoietic cells
(Dorrell et al., 2000; Danet et al., 2001).
Pros and cons for the classical model of
hematopoiesis in humans
Comparable to the murine system and in agreement with
the classical model of hematopoiesis criteria that dis-
criminate human multipotent HPCs from lineage-restrict-
ed CMPs or CLPs have been described as well. In this
context, BM-derived Lin-CD34qCD38qCD45RAqCD10-
wHLA-DRq c-kit-Thy1-x cells and CB-derived CD34qCD38-
CD7q wCD45RAqHLA-DRqc-kit-Thy1-x cells contain CLP
activities (Galy et al., 1995; Hao et al., 2001; Hoebeke et
al., 2007). Cells with CMP activities have been identified
as BM-derived Lin-CD34qCD38qIL-3RalowCD45RA- cells
that generate both Lin-CD34qCD38qIL-3RalowCD45RAq
cells containing the clonogenic potential for granulocyte
and macrophage (GMPs) development, and Lin-
CD34qCD38qIL-3Ra-CD45RA- cells containing that for
megakaryocyte and erythrocyte (MEPs) development
(Manz et al., 2002).
In controversy to the classical hematopoietic model,
Haddad and colleagues described two human CB-
derived cell populations (Lin-CD34qCD45RAhighCD10q
and Lin-CD34qCD45RAhighCD7q) that although they were
either primed to the T-cell and NK cell development or to
the B-cell lineage, respectively, still sustained robust gra-
nulomonocytic differentiation potentials (Haddad et al.,
2004). Pointing toward the same direction, progeny of
CD34qCD10-CD19- cells can follow the course of early
B-cell development and perform B-cell specific DJH rear-
rangements without losing their capacity to differentiate
into NK-cells, T-cells and macrophages (Reynaud et al.,
2003). Additionally, CD34qCD19q cells that belong to a
small CXCR4- B-cell progenitor subpopulation have been
found to contain capacities for granulocyte, macrophage
and red blood cell development (Hou et al., 2005).
Similarly, in our own single cell analyses in which we
deposited individual CB-derived Lin-CD34qCD38- cells
and studied the primitive hematopoietic re-initiation
potential of their arising daughter cells in clonogenic
myeloid (long-term culture initiating cell; LTC-IC), as well
as in clonogenic lymphoid assays (NK cell initiating cell;
NK-IC), we retrospectively identified cells which gave rise
to NK cells and macrophages but not to granulocytes
(Giebel et al., 2006). According to their colony formation
potential, we termed such corresponding cells as
macrophage NK-ICs (M-NK-ICs). Although we do not
know yet whether these cells retain other lymphoid
developmental capacities, cells that contain develop-
mental potentials for NK cells and macrophages while
simultaneously lacking granulocyte development should
not exist according to the classical model of
hematopoiesis.
In addition, cells that gave rise to NK cells and also
generated granulocytes should – at least according to
the classical model – contain both the full myeloid as well
as the full lymphoid developmental potentials. Accord-
ingly, and even though it remains to be determined
whether such cells contain megakaryocytic and erythroid
potentials, these primitive progenitors have been named
myeloid lymphoid initiating cells or ML-ICs (Punzel et al.,
1999). Due to the fact that these cells are able to reinitiate
multiple myeloid as well as lymphoid long-term hema-
topoiesis in vitro, they are considered as one of the most
primitive in vitro measurable stem cell equivalent of
human hematopoietic cells (Punzel et al., 1999). Follow-
ing the classical model, ML-ICs should give rise to
daughter cells retaining the multipotent ML-IC capacity
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Table 2 Human hematopoietic stem and progenitor cell populations.

Defined human Phenotype Determination Frequency References

population function source

HSC CD34qCD38-c-kitqLin- Terstappen et al., 1991; Hao et al., 1996;

Larochelle et al., 1996
Additional marker CD133q(CD34-/q) Preimmune fetal sheep, Fetal liver, UCB, BM, PB Bhatia et al., 1998; de Wynter et al., 1998;
NOD/SCID mice; Zanjani et al., 1998; Yin et al., 1997
in vitro differentiation, Gallacher et al., 2000; Giebel et al., 2006;
flow cytometry Beckmann et al., 2007

ALDHhigh(CD133q) NOD/SCID/b2m-/- mice; UCB Hess et al., 2004, 2006

HLA-DRlow(CD34q)
CD33-Rho123-(CD34q)
CD71-CD62Lq(CD34q)
CD53qCD63-(CD133q)
in vitro differentiation,
flow cytometry
In vitro differentiation, BM Verfaillie et al., 1990
flow cytometry
NOD/SCID, UCB Liu and Verfaillie, 2002
In vitro differentiation, McKenzie et al., 2007
flow cytometry
In vitro differentiation, UCB, BM, PB, cultured Lansdorp and Dragowska, 1992; Mayani et al.,
flow cytometry UCB 1993; Mohle et al., 1995; Koenig et al., 1999;
Beckmann et al., 2007
In vitro differentiation, Cultured UCB Beckmann et al., 2007
flow cytometry

CLP CD34qCD38qLin-CD10q In vitro differentiation, BM Galy et al., 1995

HLA-DRqc-kit-Thy1.1- flow cytometry,
Gene expression
CD34qCD38-CD7q In vitro differentiation, UCB Hao et al., 2001; Hoebeke et al., 2007
HLA-DRqc-kit-Thy1.1- flow cytometry,
Gene expression
Article in press - uncorrected proof

CLP (B-cells)q CD34qLin-CD38qIL-3Ralow In vitro differentiation, UCB Haddad et al., 2004

GMP-potential CD45RAhighCD10q flow cytometry,
gene expression
CLP (T/NK-cells)q CD34qLin-CD38qIL-3Ralow UCB Haddad et al., 2004
GMP-potential CD45RAhighCD7q
CLPqmacrophages CD34qCD10-CD19- In vitro differentiation, UCB Reynaud et al., 2003
CD79aqIL-7Raq flow cytometry,
gene expression
BMP(B-cells)q CD34qCD19qCXCR4- In vitro differentiation, BM Hou et al., 2005
GMPqerythroid flow cytometry ;0.3% mononuclear cell fraction (MNC) BM
M-NK-IC CD34qCD38- In vitro differentiation UCB Giebel et al., 2004
CMP CD34qLin-CD38qIL-3Ralow NOD/SCID/b2m-/- mice, ;0.28% MNC BM; ;0.4% MNC UCB Manz et al., 2002
CD45RA-CD16-CD32- in vitro differentiation
GMP IL-3RalowCD45RAq ;0.35% MNC BM; ;0.3% MNC UCB Manz et al., 2002

Download Date | 1/30/18 11:39 AM

Authenticated
Brought to you by | BUPMC MIR (I894)
MeP IL-3Ra-CD45RA- ;0.13% MNC BM; ;0.05% MNC UCB Manz et al., 2002
Hematopoietic stem cell lineage development 819
 Article in press - uncorrected proof
820 B. Giebel and M. Punzel
or to daughter cells, being committed to either the lym-
phoid (CLPs) or the myeloid (CMPs) lineage develop-
ment. Thus, in the ML-IC assay, CLPs should give rise
to NK cells but not to myeloid colonies, CMPs should
generate myeloid colonies but not NK cells. However, in
our studies in which we analyzed the cell fate of individ-
ual daughter cells, we never observed that CD34qCD38-
cells originally containing the ML-IC capacity gave rise
to one daughter cell fulfilling the CMP criteria and another
one fulfilling that of a CLP (Giebel et al., 2006). In all
cases studied, at least one daughter cell retained the full
ML-IC potential of its mother cell, and in more than 80%
of the cases, one daughter cell acquired a restricted cell
Figure 2 Developmental route(s) in neonatal and adult human
hematopoiesis.
Clinical trials revealed that the heterogeneous pool of human
CD34q cells contains HSC activities that can restore the hema-
topoietic system of immunocompromised patients. However, for
ethical reasons subfractions should not be transplanted in
humans, their developmental potential can only be estimated in
in vitro assays or in xenogenic models, e.g., in NOD/SCID mice
or in fetal sheep. According to these surrogate HSC analyses,
the most primitive human hematopoietic cells seem to belong
to the Lin-CD133qc-kitq cell fraction. Although a small propor-
tion of such cells lack CD34 expression – maybe the most prim-
itive human hematopoietic cells detected so far –, the vast
majority of these cells express CD34. These cells can be dis-
criminated in more primitive CD34qCD38low/- cells and more
mature CD34qCD38q cells. Similar to the mouse system, recent
findings have suggested that multipotent progenitors, enriched
within the CD34qCD38low/- cell fraction, not only give rise to
CMPs and CLPs, as predicted by the classical model of hema-
topoiesis, but also to cells that display reduced developmental
capabilities of both the myeloid and the lymphoid developmental
routes. In this context, T/NK-cell-primed and B-cell-primed cells
have been identified that still possess GMP abilities (T/NK GMP,
B GMP), as well as cells with CLP and macrophage potentials
(M CLP). Additionally, some putatively lineage restricted lym-
phocytes still contain capabilities to produce macrophages
(M-NK, M-B, M-T). Abbreviations: ML-IC, myeloid-lymphoid ini-
tiating cell; E-LTC-IC, extended long-term culture initiating cell;
CAFC, cobblestone forming cell; LTC-IC, long-term culture ini-
tiating cell; M-NK-IC, macrophage-NK cell initiating cell; NK-IC,
NK cell initiating cell; CFU, colony-forming unit.
fate. Remarkably, in all cases in which such daughter
cells generated NK cells, we also observed the devel-
opment of macrophages, suggesting that under the con-
ditions used ML-ICs do not give rise to cells fulfilling the
CLP criteria, but give rise to cells with M-NK-IC activities
(Giebel et al., 2006). As cells with improved M-NK-IC
activities frequently generated daughter cells, one of
which retained the M-NK-IC potential and one which only
generated NK cells and thus could in principal reflect
CLP activities (Giebel et al., 2006), we propose that under
the conditions used in our experiments, CLPs arise from
multipotent HPCs via progenitor cells containing M-NK-
IC activities. These findings within the early human
hematopoietic system strongly support the data obtained
from the groups of Jacobsen and Kondo which have
been described previously (Adolfsson et al., 2005; Lai
and Kondo, 2006). Taking the current literature into
account, a proposed model of human hematopoietic
lineage development is shown in Figure 2.
Conclusion
In summary, recent findings in mouse and in man predict
additional or alternative roadmaps within the model of
the hematopoietic development (Figures 1 and 2). There-
fore, the hypothesis that myeloid and lymphoid progen-
itors are strictly separated at a single branching point, as
proposed in the classical model of hematopoiesis, can-
not be kept in this form anymore. It seems more likely
that there are different developmental routes a multi-
potent hematopoietic cell can follow. As extrinsic factors
play important roles in governing the cell fate of hema-
topoietic progenitor cells and because primitive hema-
topoietic cells are motile and sometimes leave their
niches enter the blood or lymph stream and return to
functional niches (Goodman and Hodgson, 1962; Wright
et al., 2001; Abkowitz et al., 2003; Massberg et al., 2007),
progenitors will be influenced by a bundle of different
places and different signals. In this context, the order
how progenitor cells receive certain signals should
already influence their developmental potential. With an
example of the developing external sensory organs of
Drosophila melanogaster, it has been shown that cells
which transduce the Notch-signaling in a first decision
step but not in a second acquire a different cell fate than
those cells that only transduce the Notch signal in the
second decision step (Campos-Ortega, 1996). Taking
into account how primitive hematopoietic cells receive
certain extrinsic signals, there should be many different
ways how the cell fate of developing hematopoietic cells
can be primed. Thus, the discovery of progenitor cells
that are specified by different routes may be the tip of an
iceberg, which is not compatible with the classical model
of hematopoiesis.
In this context, the old discussion whether hemato-
poietic cell fates are specified in a deterministic or sto-
chastic manner can be repeated (McCulloch, 1983).
According to our data in which most of the primitive
hematopoietic cells which had ML-IC capacities gave
rise to differently specified daughter cells (Giebel et al.,
2006), and due to the identification of asymmetrically
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
segregating proteins (Beckmann et al., 2007), we suggest
that very primitive hematopoietic cells are intrinsically
determined to acquire different cell fates, which is sup-
ported by the finding that within the murine system clon-
ally expanded HSCs maintain their tendency to give rise
to either myeloid-biased or lymphoid-biased HSC clones
(Muller-Sieburg et al., 2002, 2004). This concept would
support the deterministic model of hematopoiesis (Mul-
ler-Sieburg and Sieburg, 2006). We further hypothesize
that as long as cells with identical developmental poten-
tial receive cell fate controlling signals in the same order,
they should also obey the deterministic model and follow
the same developmental routes. As soon as the order
and combination of signals is altered, cells will finally
adopt different developmental probabilities. A different
combination of extrinsic signals might therefore account
for observed variabilities among different functional read-
out systems (Ramirez et al., 1998; Horn et al., 2003;
Glimm et al., 2005; Horn and Blasczyk, 2007). Due to the
multitude of different cell fate influencing factors, we
assume that there is a high degree of randomness within
the order how cells receive such signals. Therefore, we
suggest that the process of hematopoietic cell fate spec-
ification finally remains at least partially stochastic.
Future work and more detailed analyses with defined
combinations of extrinsic signals will be required to finally
improve the hierarchical concept of the hematopoietic
tree and should define the basis for new functional anal-
yses that help to dissect the mechanisms involved in
governing self-renewing and differentiation processes
within the primitive hematopoietic systems.
Acknowledgments
We thank Julia Beckmann, Verdon Taylor and Johannes Fischer
for discussion and critics on the manuscript. Our studies were
supported by grants from the Deutsche Forschungsgemein-
schaft (SPP1109 GI 336/1-4), the Forschungskommission of the
HHU-Düsseldorf und the Leukämieliga e.V.
References
Abkowitz, J.L., Robinson, A.E., Kale, S., Long, M.W., and Chen,
J. (2003). Mobilization of hematopoietic stem cells during
homeostasis and after cytokine exposure. Blood 102,
1249–1253.
Adolfsson, J., Borge, O.J., Bryder, D., Theilgaard-Monch, K.,
Astrand-Grundstrom, I., Sitnicka, E., Sasaki, Y., and Jacob-
sen, S.E. (2001). Upregulation of Flt3 expression within the
bone marrow Lin-Sca1qc-kitq stem cell compartment is
accompanied by loss of self-renewal capacity. Immunity 15,
659–669.
Adolfsson, J., Mansson, R., Buza-Vidas, N., Hultquist, A., Liuba,
K., Jensen, C.T., Bryder, D., Yang, L., Borge, O.J., Thoren,
L.A., et al. (2005). Identification of Flt3q lympho-myeloid
stem cells lacking erythro-megakaryocytic potential a revised
road map for adult blood lineage commitment. Cell 121,
295–306.
Akashi, K., Traver, D., Miyamoto, T., and Weissman, I.L. (2000).
A clonogenic common myeloid progenitor that gives rise to
all myeloid lineages. Nature 404, 193–197.
Akashi, K., He, X., Chen, J., Iwasaki, H., Niu, C., Steenhard, B.,
Zhang, J., Haug, J., and Li, L. (2003). Transcriptional acces-
sibility for genes of multiple tissues and hematopoietic line-
Hematopoietic stem cell lineage development 821
ages is hierarchically controlled during early hematopoiesis.
Blood 101, 383–389.
Anderson, K., Rusterholz, C., Mansson, R., Jensen, C.T., Bacos,
K., Zandi, S., Sasaki, Y., Nerlov, C., Sigvardsson, M., and
Jacobsen, S.E. (2007). Ectopic expression of PAX5 promotes
maintenance of biphenotypic myeloid progenitors coexpress-
ing myeloid and B-cell lineage-associated genes. Blood 109,
3697–3705.
Bain, G., Maandag, E.C., Izon, D.J., Amsen, D., Kruisbeek, A.M.,
Weintraub, B.C., Krop, I., Schlissel, M.S., Feeney, A.J., van
Roon, M., et al. (1994). E2A proteins are required for proper
B cell development and initiation of immunoglobulin gene
rearrangements. Cell 79, 885–892.
Bain, G., Engel, I., Robanus Maandag, E.C., te Riele, H.P.,
Voland, J.R., Sharp, L.L., Chun, J., Huey, B., Pinkel, D., and
Murre, C. (1997). E2A deficiency leads to abnormalities in
alphabeta T-cell development and to rapid development of
T-cell lymphomas. Mol. Cell. Biol. 17, 4782–4791.
Bain, G., Quong, M.W., Soloff, R.S., Hedrick, S.M., and Murre,
C. (1999). Thymocyte maturation is regulated by the activity
of the helix-loop-helix protein, E47. J. Exp. Med. 190,
1605–1616.
Ballen, K.K. (2005). New trends in umbilical cord blood trans-
plantation. Blood 105, 3786–3792.
Beckmann, J., Scheitza, S., Wernet, P., Fischer, J.C., and Giebel,
B. (2007). Asymmetric cell division within the human hema-
topoietic stem and progenitor cell compartment: identifica-
tion of asymmetrically segregating proteins. Blood 109,
5494–5501.
Bensinger, W.I., Buckner, D., and Gahrton, G. (1997). Allogeneic
stem cell transplantation for multiple myeloma. Hematol.
Oncol. Clin. North Am. 11, 147–157.
Bhatia, M., Bonnet, D., Murdoch, B., Gan, O.I., and Dick, J.E.
(1998). A newly discovered class of human hematopoietic
cells with SCID-repopulating activity. Nat. Med. 4, 1038–
1045.
Buza-Vidas, N., Luc, S., and Jacobsen, S.E. (2007). Delineation
of the earliest lineage commitment steps of haematopoietic
stem cells: new developments, controversies and major
challenges. Curr. Opin. Hematol. 14, 315–321.
Calvi, L.M., Adams, G.B., Weibrecht, K.W., Weber, J.M., Olson,
D.P., Knight, M.C., Martin, R.P., Schipani, E., Divieti, P., Bring-
hurst, F.R., et al. (2003). Osteoblastic cells regulate the
haematopoietic stem cell niche. Nature 425, 841–846.
Campos-Ortega, J.A. (1996). Numb diverts notch pathway off
the tramtrack. Neuron 17, 1–4.
Chang, J.T., Palanivel, V.R., Kinjyo, I., Schambach, F., Intlekofer,
A.M., Banerjee, A., Longworth, S.A., Vinup, K.E., Mrass, P.,
Oliaro, J., et al. (2007). Asymmetric T lymphocyte division in
the initiation of adaptive immune responses. Science 315,
1687–1691.
Christensen, J.L. and Weissman, I.L. (2001). Flk-2 is a marker in
hematopoietic stem cell differentiation: a simple method to
isolate long-term stem cells. Proc. Natl. Acad. Sci. USA 98,
14541–14546.
Corbeil, D., Roper, K., Hellwig, A., Tavian, M., Miraglia, S., Watt,
S.M., Simmons, P.J., Peault, B., Buck, D.W., and Huttner,
W.B. (2000). The human AC133 hematopoietic stem cell anti-
gen is also expressed in epithelial cells and targeted to plas-
ma membrane protrusions. J. Biol. Chem. 275, 5512–5520.
Cotta, C.V., Zhang, Z., Kim, H.G., and Klug, C.A. (2003). Pax5
determines B- versus T-cell fate and does not block early
myeloid-lineage development. Blood 101, 4342–4346.
Coulombel, L. (2004). Identification of hematopoietic stem/pro-
genitor cells: strength and drawbacks of functional assays.
Oncogene 23, 7210–7222.
Danet, G.H., Lee, H.W., Luongo, J.L., Simon, M.C., and Bonnet,
D.A. (2001). Dissociation between stem cell phenotype and
NOD/SCID repopulating activity in human peripheral blood
CD34q cells after ex vivo expansion. Exp. Hematol. 29,
1465–1473.
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
822 B. Giebel and M. Punzel
de Wynter, E.A., Buck, D., Hart, C., Heywood, R., Coutinho, L.H.,
Clayton, A., Rafferty, J.A., Burt, D., Guenechea, G., Bueren,
J.A., et al. (1998). CD34qAC133q cells isolated from cord
blood are highly enriched in long-term culture-initiating cells,
NOD/SCID-repopulating cells and dendritic cell progenitors.
Stem Cells 16, 387–396.
DeKoter, R.P. and Singh, H. (2000). Regulation of B lymphocyte
and macrophage development by graded expression of
PU.1. Science 288, 1439–1441.
DeKoter, R.P., Walsh, J.C., and Singh, H. (1998). PU.1 regulates
both cytokine-dependent proliferation and differentiation of
granulocyte/macrophage progenitors. EMBO J. 17, 4456–
4468.
Dorrell, C., Gan, O.I., Pereira, D.S., Hawley, R.G., and Dick, J.E.
(2000). Expansion of human cord blood CD34qCD38- cells
in ex vivo culture during retroviral transduction without a cor-
responding increase in SCID repopulating cell (SRC) fre-
quency: dissociation of SRC phenotype and function. Blood
95, 102–110.
Elefanty, A.G., Begley, C.G., Metcalf, D., Barnett, L., Kontgen, F.,
and Robb, L. (1998). Characterization of hematopoietic pro-
genitor cells that express the transcription factor SCL, using
a lacZ ‘knock-in’ strategy. Proc. Natl. Acad. Sci. USA 95,
11897–11902.
Forsberg, E.C., Serwold, T., Kogan, S., Weissman, I.L., and Pas-
segue, E. (2006). New evidence supporting megakaryocyte-
erythrocyte potential of flk2/flt3q multipotent hematopoietic
progenitors. Cell 126, 415–426.
Franco, C.B., Scripture-Adams, D.D., Proekt, I., Taghon, T.,
Weiss, A.H., Yui, M.A., Adams, S.L., Diamond, R.A., and
Rothenberg, E.V. (2006). Notch/Delta signaling constrains
reengineering of pro-T cells by PU.1. Proc. Natl. Acad. Sci.
USA 103, 11993–11998.
Fujiwara, Y., Browne, C.P., Cunniff, K., Goff, S.C., and Orkin, S.H.
(1996). Arrested development of embryonic red cell precur-
sors in mouse embryos lacking transcription factor GATA-1.
Proc. Natl. Acad. Sci. USA 93, 12355–12358.
Gallacher, L., Murdoch, B., Wu, D.M., Karanu, F.N., Keeney, M.,
and Bhatia, M. (2000). Isolation and characterization of
human CD34-Lin- and CD34qLin- hematopoietic stem cells
using cell surface markers AC133 and CD7. Blood 95,
2813–2820.
Galy, A., Travis, M., Cen, D., and Chen, B. (1995). Human T, B,
natural killer, and dendritic cells arise from a common bone
marrow progenitor cell subset. Immunity 3, 459–473.
Gering, M., Rodaway, A.R., Gottgens, B., Patient, R.K., and
Green, A.R. (1998). The SCL gene specifies haemangioblast
development from early mesoderm. EMBO J. 17,
4029–4045.
Giebel, B., Corbeil, D., Beckmann, J., Hohn, J., Freund, D., Gie-
sen, K., Fischer, J., Kogler, G., and Wernet, P. (2004). Seg-
regation of lipid raft markers including CD133 in polarized
human hematopoietic stem and progenitor cells. Blood 104,
2332–2338.
Giebel, B., Zhang, T., Beckmann, J., Spanholtz, J., Wernet, P.,
Ho, A.D., and Punzel, M. (2006). Primitive human hemato-
poietic cells give rise to differentially specified daughter cells
upon their initial cell division. Blood 107, 2146–2152.
Glimm, H., Schmidt, M., Fischer, M., Schwarzwaelder, K., Wiss-
ler, M., Klingenberg, S., Prinz, C., Waller, C.F., Lange, W.,
Eaves, C.J., and von Kalle, C. (2005). Efficient marking of
human cells with rapid but transient repopulating activity in
autografted recipients. Blood 106, 893–898.
Goodman, J.W. and Hodgson, G.S. (1962). Evidence for stem
cells in the peripheral blood of mice. Blood 19, 702–714.
Haddad, R., Guardiola, P., Izac, B., Thibault, C., Radich, J., Dele-
zoide, A.L., Baillou, C., Lemoine, F.M., Gluckman, J.C., Pflu-
mio, F., and Canque, B. (2004). Molecular characterization of
early human T/NK and B-lymphoid progenitor cells in umbil-
ical cord blood. Blood 104, 3918–3926.
Hao, Q.L., Thiemann, F.T., Petersen, D., Smogorzewska, E.M.,
and Crooks, G.M. (1996). Extended long-term culture reveals
a highly quiescent and primitive human hematopoietic pro-
genitor population. Blood 88, 3306–3313.
Hao, Q.L., Zhu, J., Price, M.A., Payne, K.J., Barsky, L.W., and
Crooks, G.M. (2001). Identification of a novel, human multi-
lymphoid progenitor in cord blood. Blood 97, 3683–3690.
He, Y.W. and Malek, T.R. (1996). Interleukin-7 receptor a is
essential for the development of gdq T cells, but not natural
killer cells. J. Exp. Med. 184, 289–293.
Hess, D.A., Meyerrose, T.E., Wirthlin, L., Craft, T.P., Herrbrich,
P.E., Creer, M.H., and Nolta, J.A. (2004). Functional charac-
terization of highly purified human hematopoietic repopula-
ting cells isolated according to aldehyde dehydrogenase
activity. Blood 104, 1648–1655.
Hess, D.A., Wirthlin, L., Craft, T.P., Herrbrich, P.E., Hohm, S.A.,
Lahey, R., Eades, W.C., Creer, M.H., and Nolta, J.A. (2006).
Selection based on CD133 and high aldehyde dehydrogen-
ase activity isolates long-term reconstituting human hema-
topoietic stem cells. Blood 107, 2162–2169.
Hoebeke, I., De Smedt, M., Stolz, F., Pike-Overzet, K., Staal, F.J.,
Plum, J., and Leclercq, G. (2007). T-, B- and NK-lymphoid,
but not myeloid cells arise from human CD34qCD38-CD7q
common lymphoid progenitors expressing lymphoid-specific
genes. Leukemia 21, 311–319.
Horn, P.A. and Blasczyk, R. (2007). Severe combined immuno-
deficiency-repopulating cell assay may overestimate long-
term repopulation ability. Stem Cells 25, 3271–3272.
Horn, P.A., Thomasson, B.M., Wood, B.L., Andrews, R.G., Mor-
ris, J.C., and Kiem, H.P. (2003). Distinct hematopoietic stem/
progenitor cell populations are responsible for repopulating
NOD/SCID mice compared with nonhuman primates. Blood
102, 4329–4335.
Hou, Y.H., Srour, E.F., Ramsey, H., Dahl, R., Broxmeyer, H.E.,
and Hromas, R. (2005). Identification of a human B-cell/mye-
loid common progenitor by the absence of CXCR4. Blood
105, 3488–3492.
Hsu, C.L., King-Fleischman, A.G., Lai, A.Y., Matsumoto, Y.,
Weissman, I.L., and Kondo, M. (2006). Antagonistic effect of
CCAAT enhancer-binding protein-a and Pax5 in myeloid or
lymphoid lineage choice in common lymphoid progenitors.
Proc. Natl. Acad. Sci. USA 103, 672–677.
Igarashi, H., Gregory, S.C., Yokota, T., Sakaguchi, N., and Kin-
cade, P.W. (2002). Transcription from the RAG1 locus marks
the earliest lymphocyte progenitors in bone marrow. Immu-
nity 17, 117–130.
Ikuta, K. and Weissman, I.L. (1992). Evidence that hematopoietic
stem cells express mouse c-kit but do not depend on steel
factor for their generation. Proc. Natl. Acad. Sci. USA 89,
1502–1506.
Iwasaki, H. and Akashi, K. (2007). Myeloid lineage commitment
from the hematopoietic stem cell. Immunity 26, 726–740.
Iwasaki, H., Mizuno, S., Wells, R.A., Cantor, A.B., Watanabe, S.,
and Akashi, K. (2003). GATA-1 converts lymphoid and mye-
lomonocytic progenitors into the megakaryocyte/erythrocyte
lineages. Immunity 19, 451–462.
Iwasaki, H., Mizuno, S., Arinobu, Y., Ozawa, H., Mori, Y., Shi-
gematsu, H., Takatsu, K., Tenen, D.G., and Akashi, K. (2006).
The order of expression of transcription factors directs hier-
archical specification of hematopoietic lineages. Genes Dev.
20, 3010–3021.
Kamel-Reid, S. and Dick, J.E. (1988). Engraftment of immune-
deficient mice with human hematopoietic stem cells. Science
242, 1706–1709.
Kiel, M.J., Yilmaz, O.H., Iwashita, T., Terhorst, C., and Morrison,
S.J. (2005). SLAM family receptors distinguish hematopoietic
stem and progenitor cells and reveal endothelial niches for
stem cells. Cell 121, 1109–1121.
Koenig, J.M., Baron, S., Luo, D., Benson, N.A., and Deisseroth,
A.B. (1999). L-selectin expression enhances clonogenesis of
CD34q cord blood progenitors. Pediatr. Res. 45, 867–870.
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
Kondo, M., Weissman, I.L., and Akashi, K. (1997). Identification
of clonogenic common lymphoid progenitors in mouse bone
marrow. Cell 91, 661–672.
Korbling, M. and Anderlini, P. (2001). Peripheral blood stem cell
versus bone marrow allotransplantation: does the source of
hematopoietic stem cells matter? Blood 98, 2900–2908.
Krause, D.S., Fackler, M.J., Civin, C.I., and May, W.S. (1996).
CD34: structure, biology, and clinical utility. Blood 87, 1–13.
Lai, A.Y. and Kondo, M. (2006). Asymmetrical lymphoid and
myeloid lineage commitment in multipotent hematopoietic
progenitors. J. Exp. Med. 203, 1867–1873.
Lai, A.Y. and Kondo, M. (2007). Identification of a bone marrow
precursor of the earliest thymocytes in adult mouse. Proc.
Natl. Acad. Sci. USA 104, 6311–6316.
Laiosa, C.V., Stadtfeld, M., Xie, H., de Andres-Aguayo, L., and
Graf, T. (2006). Reprogramming of committed T cell progen-
itors to macrophages and dendritic cells by C/EBP a and
PU.1 transcription factors. Immunity 25, 731–744.
Lansdorp, P.M. and Dragowska, W. (1992). Long-term erythro-
poiesis from constant numbers of CD34q cells in serum-free
cultures initiated with highly purified progenitor cells from
human bone marrow. J. Exp. Med. 175, 1501–1509.
Lapidot, T., Pflumio, F., Doedens, M., Murdoch, B., Williams,
D.E., and Dick, J.E. (1992). Cytokine stimulation of multi-
lineage hematopoiesis from immature human cells engrafted
in SCID mice. Science 255, 1137–1141.
Larochelle, A., Vormoor, J., Hanenberg, H., Wang, J.C., Bhatia,
M., Lapidot, T., Moritz, T., Murdoch, B., Xiao, X.L., Kato, I.,
et al. (1996). Identification of primitive human hematopoietic
cells capable of repopulating NOD/SCID mouse bone mar-
row: implications for gene therapy. Nat. Med. 2, 1329–1337.
Liu, H. and Verfaillie, C.M. (2002). Myeloid-lymphoid initiating
cells (ML-IC) are highly enriched in the rhodamine-c-
kitqCD33-CD38- fraction of umbilical cord CD34q cells. Exp.
Hematol. 30, 582–589.
Maki, K., Sunaga, S., Komagata, Y., Kodaira, Y., Mabuchi, A.,
Karasuyama, H., Yokomuro, K., Miyazaki, J.I., and Ikuta, K.
(1996). Interleukin 7 receptor-deficient mice lack gammadelta
T cells. Proc. Natl. Acad. Sci. USA 93, 7172–7177.
Mansson, R., Hultquist, A., Luc, S., Yang, L., Anderson, K., Kha-
razi, S., Al-Hashmi, S., Liuba, K., Thoren, L., Adolfsson, J.,
et al. (2007). Molecular evidence for hierarchical transcrip-
tional lineage priming in fetal and adult stem cells and
multipotent progenitors. Immunity 26, 407–419.
Manz, M.G., Miyamoto, T., Akashi, K., and Weissman, I.L. (2002).
Prospective isolation of human clonogenic common myeloid
progenitors. Proc. Natl. Acad. Sci. USA 99, 11872–11877.
Massberg, S., Schaerli, P., Knezevic-Maramica, I., Köllnberger,
M., Tubo, N., Moseman, E.A., Huff, I.V., Junt, T., Wagers, A.J.,
Mazo, I.B., and von Andrian, U.H. (2007). Immunosurveil-
lance by hematopoietic progenitor cells trafficking through
blood, lymph, and peripheral tissues. Cell 131, 994–1008.
Matsuzaki, Y., Kinjo, K., Mulligan, R.C., and Okano, H. (2004).
Unexpectedly efficient homing capacity of purified murine
hematopoietic stem cells. Immunity 20, 87–93.
Mayani, H., Dragowska, W., and Lansdorp, P.M. (1993). Cyto-
kine-induced selective expansion and maturation of erythroid
versus myeloid progenitors from purified cord blood precur-
sor cells. Blood 81, 3252–3258.
McCulloch, E.A. (1983). Stem cells in normal and leukemic
hemopoiesis (Henry Stratton Lecture, 1982). Blood 62, 1–13.
McKenzie, J.L., Takenaka, K., Gan, O.I., Doedens, M., and Dick,
J.E. (2007). Low rhodamine 123 retention identifies long-term
human hematopoietic stem cells within the Lin-CD34qCD38-
population. Blood 109, 543–545.
Mead, P.E., Deconinck, A.E., Huber, T.L., Orkin, S.H., and Zon,
L.I. (2001). Primitive erythropoiesis in the Xenopus embryo:
the synergistic role of LMO-2, SCL and GATA-binding pro-
teins. Development 128, 2301–2308.
Mikkola, H.K., Klintman, J., Yang, H., Hock, H., Schlaeger, T.M.,
Fujiwara, Y., and Orkin, S.H. (2003). Haematopoietic stem
cells retain long-term repopulating activity and multipotency
Hematopoietic stem cell lineage development 823
in the absence of stem-cell leukaemia SCL/tal-1 gene.
Nature 421, 547–551.
Mohle, R., Murea, S., Kirsch, M., and Haas, R. (1995). Differential
expression of L-selectin, VLA-4, and LFA-1 on CD34q pro-
genitor cells from bone marrow and peripheral blood during
G-CSF-enhanced recovery. Exp. Hematol. 23, 1535–1542.
Montecino-Rodriguez, E., Leathers, H., and Dorshkind, K.
(2001). Bipotential B-macrophage progenitors are present in
adult bone marrow. Nat. Immunol. 2, 83–88.
Morrison, S.J. and Weissman, I.L. (1994). The long-term repo-
pulating subset of hematopoietic stem cells is deterministic
and isolatable by phenotype. Immunity 1, 661–673.
Morrison, S.J., Wandycz, A.M., Hemmati, H.D., Wright, D.E., and
Weissman, I.L. (1997). Identification of a lineage of multi-
potent hematopoietic progenitors. Development 124, 1929–
1939.
Muller-Sieburg, C.E. and Sieburg, H.B. (2006). The GOD of
hematopoietic stem cells: a clonal diversity model of the
stem cell compartment. Cell Cycle 5, 394–398.
Muller-Sieburg, C.E., Cho, R.H., Thoman, M., Adkins, B., and
Sieburg, H.B. (2002). Deterministic regulation of hemato-
poietic stem cell self-renewal and differentiation. Blood 100,
1302–1309.
Muller-Sieburg, C.E., Cho, R.H., Karlsson, L., Huang, J.F., and
Sieburg, H.B. (2004). Myeloid-biased hematopoietic stem
cells have extensive self-renewal capacity but generate
diminished lymphoid progeny with impaired IL-7 responsive-
ness. Blood 103, 4111–4118.
Nerlov, C. and Graf, T. (1998). PU.1 induces myeloid lineage
commitment in multipotent hematopoietic progenitors.
Genes Dev. 12, 2403–2412.
Nerlov, C., Querfurth, E., Kulessa, H., and Graf, T. (2000). GATA-
1 interacts with the myeloid PU.1 transcription factor and
represses PU.1-dependent transcription. Blood 95,
2543–2551.
Osawa, M., Hanada, K., Hamada, H., and Nakauchi, H. (1996).
Long-term lymphohematopoietic reconstitution by a single
CD34-low/negative hematopoietic stem cell. Science 273,
242–245.
Peschon, J.J., Morrissey, P.J., Grabstein, K.H., Ramsdell, F.J.,
Maraskovsky, E., Gliniak, B.C., Park, L.S., Ziegler, S.F., Wil-
liams, D.E., Ware, C.B., et al. (1994). Early lymphocyte
expansion is severely impaired in interleukin 7 receptor-defi-
cient mice. J. Exp. Med. 180, 1955–1960.
Pevny, L., Simon, M.C., Robertson, E., Klein, W.H., Tsai, S.F.,
D’Agati, V., Orkin, S.H., and Costantini, F. (1991). Erythroid
differentiation in chimaeric mice blocked by a targeted muta-
tion in the gene for transcription factor GATA-1. Nature 349,
257–260.
Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F.W., and
Orkin, S.H. (1996). The T cell leukemia oncoprotein SCL/
tal-1 is essential for development of all hematopoietic line-
ages. Cell 86, 47–57.
Punzel, M., Wissink, S.D., Miller, J.S., Moore, K.A., Lemischka,
I.R., and Verfaillie, C.M. (1999). The myeloid-lymphoid initi-
ating cell (ML-IC) assay assesses the fate of multipotent
human progenitors in vitro. Blood 93, 3750–3756.
Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C.,
and Melton, D.A. (2002). ‘‘Stemness’’: transcriptional profiling
of embryonic and adult stem cells. Science 298, 597–600.
Ramirez, M., Rottman, G.A., Shultz, L.D., and Civin, C.I. (1998).
Mature human hematopoietic cells in donor bone marrow
complicate interpretation of stem/progenitor cell assays in
xenogeneic hematopoietic chimeras. Exp. Hematol. 26,
332–344.
Randall, T.D., Lund, F.E., Howard, M.C., and Weissman, I.L.
(1996). Expression of murine CD38 defines a population of
long-term reconstituting hematopoietic stem cells. Blood 87,
4057–4067.
Rekhtman, N., Radparvar, F., Evans, T., and Skoultchi, A.I.
(1999). Direct interaction of hematopoietic transcription fac-
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM
 Article in press - uncorrected proof
824 B. Giebel and M. Punzel
tors PU.1 and GATA-1: functional antagonism in erythroid
cells. Genes Dev. 13, 1398–1411.
Reya, T., Morrison, S.J., Clarke, M.F., and Weissman, I.L. (2001).
Stem cells, cancer, and cancer stem cells. Nature 414,
105–111.
Reynaud, D., Lefort, N., Manie, E., Coulombel, L., and Levy, Y.
(2003). In vitro identification of human pro-B cells that give
rise to macrophages, natural killer cells, and T cells. Blood
101, 4313–4321.
Ringden, O. and Le Blanc, K. (2005). Allogeneic hematopoietic
stem cell transplantation: state of the art and new perspec-
tives. APMIS 113, 813–830.
Robb, L., Rasko, J.E., Bath, M.L., Strasser, A., and Begley, C.G.
(1995). scl, a gene frequently activated in human T cell leu-
kaemia, does not induce lymphomas in transgenic mice.
Oncogene 10, 205–209.
Robb, L., Elwood, N.J., Elefanty, A.G., Kontgen, F., Li, R., Bar-
nett, L.D., and Begley, C.G. (1996). The scl gene product is
required for the generation of all hematopoietic lineages in
the adult mouse. EMBO J. 15, 4123–4129.
Rothenberg, E.V. (2007). Negotiation of the T lineage fate deci-
sion by transcription-factor interplay and microenvironmental
signals. Immunity 26, 690–702.
Schaniel, C., Bruno, L., Melchers, F., and Rolink, A.G. (2002a).
Multiple hematopoietic cell lineages develop in vivo from
transplanted Pax5-deficient pre-B I-cell clones. Blood 99,
472–478.
Schaniel, C., Gottar, M., Roosnek, E., Melchers, F., and Rolink,
A.G. (2002b). Extensive in vivo self-renewal, long-term
reconstitution capacity, and hematopoietic multipotency of
Pax5-deficient precursor B-cell clones. Blood 99,
2760–2766.
Shivdasani, R.A., Mayer, E.L., and Orkin, S.H. (1995). Absence
of blood formation in mice lacking the T-cell leukaemia onco-
protein tal-1/SCL. Nature 373, 432–434.
Shivdasani, R.A., Fujiwara, Y., McDevitt, M.A., and Orkin, S.H.
(1997). A lineage-selective knockout establishes the critical
role of transcription factor GATA-1 in megakaryocyte growth
and platelet development. EMBO J. 16, 3965–3973.
Smith, L.G., Weissman, I.L., and Heimfeld, S. (1991). Clonal
analysis of hematopoietic stem-cell differentiation in vivo.
Proc. Natl. Acad. Sci. USA 88, 2788–2792.
Spangrude, G.J., Heimfeld, S., and Weissman, I.L. (1988). Puri-
fication and characterization of mouse hematopoietic stem
cells. Science 241, 58–62.
Terstappen, L.W., Huang, S., Safford, M., Lansdorp, P.M., and
Loken, M.R. (1991). Sequential generations of hematopoietic
colonies derived from single nonlineage-committed
CD34qCD38- progenitor cells. Blood 77, 1218–1227.
Verfaillie, C., Blakolmer, K., and McGlave, P. (1990). Purified
primitive human hematopoietic progenitor cells with long-
term in vitro repopulating capacity adhere selectively to irra-
diated bone marrow stroma. J. Exp. Med. 172, 502–509.
von Levetzow, G., Spanholtz, J., Beckmann, J., Fischer, J.,
Kogler, G., Wernet, P., Punzel, M., and Giebel, B. (2006).
Nucleofection, an efficient nonviral method to transfer genes
into human hematopoietic stem and progenitor cells. Stem
Cells Dev. 15, 278–285.
Walsh, J.C., DeKoter, R.P., Lee, H.J., Smith, E.D., Lancki, D.W.,
Gurish, M.F., Friend, D.S., Stevens, R.L., Anastasi, J., and
Singh, H. (2002). Cooperative and antagonistic interplay
between PU.1 and GATA-2 in the specification of myeloid cell
fates. Immunity 17, 665–676.
Wright, D.E., Wagers, A.J., Gulati, A.P., Johnson, F.L., and
Weissman, I.L. (2001). Physiological migration of hemato-
poietic stem and progenitor cells. Science 294, 1933–1936.
Yin, A.H., Miraglia, S., Zanjani, E.D., Almeida-Porada, G., Oga-
wa, M., Leary, A.G., Olweus, J., Kearney, J., and Buck, D.W.
(1997). AC133, a novel marker for human hematopoietic stem
and progenitor cells. Blood 90, 5002–5012.
Yoshida, T., Ng, S.Y., Zuniga-Pflucker, J.C., and Georgopoulos,
K. (2006). Early hematopoietic lineage restrictions directed by
Ikaros. Nat. Immunol. 7, 382–391.
Zanjani, E.D., Pallavicini, M.G., Ascensao, J.L., Flake, A.W., Lan-
glois, R.G., Reitsma, M., MacKintosh, F.R., Stutes, D., Har-
rison, M.R., and Tavassoli, M. (1992). Engraftment and
long-term expression of human fetal hemopoietic stem cells
in sheep following transplantation in utero. J. Clin. Invest. 89,
1178–1188.
Zanjani, E.D., Almeida-Porada, G., Livingston, A.G., Flake, A.W.,
and Ogawa, M. (1998). Human bone marrow CD34- cells
engraft in vivo and undergo multilineage expression that
includes giving rise to CD34q cells. Exp. Hematol. 26,
353–360.
Zhang, C.C. and Lodish, H.F. (2005). Murine hematopoietic stem
cells change their surface phenotype during ex vivo expan-
sion. Blood 105, 4314–4320.
Zhang, P., Behre, G., Pan, J., Iwama, A., Wara-Aswapati, N.,
Radomska, H.S., Auron, P.E., Tenen, D.G., and Sun, Z.
(1999). Negative cross-talk between hematopoietic regula-
tors: GATA proteins repress PU.1. Proc. Natl. Acad. Sci. USA
96, 8705–8710.
Zhang, P., Zhang, X., Iwama, A., Yu, C., Smith, K.A., Mueller,
B.U., Narravula, S., Torbett, B.E., Orkin, S.H., and Tenen,
D.G. (2000). PU.1 inhibits GATA-1 function and erythroid dif-
ferentiation by blocking GATA-1 DNA binding. Blood 96,
2641–2648.
Zhang, J., Niu, C., Ye, L., Huang, H., He, X., Tong, W.G., Ross,
J., Haug, J., Johnson, T., Feng, J.Q., et al. (2003). Identifi-
cation of the haematopoietic stem cell niche and control of
the niche size. Nature 425, 836–841.
Zhang, W.J., Park, C., Arentson, E., and Choi, K. (2005). Mod-
ulation of hematopoietic and endothelial cell differentiation
from mouse embryonic stem cells by different culture con-
ditions. Blood 105, 111–114.
Brought to you by | BUPMC MIR (I894)
Authenticated
Download Date | 1/30/18 11:39 AM