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Critical Reviews in Oncology/Hematology xxx (2010) xxx–xxx

Neoplastic stem cells: Current concepts and clinical perspectives

Axel Schulenburg a,d,∗ , Kira Brämswig b , Harald Herrmann c,d , Heidrun Karlic d ,
Irina Mirkina c,d , Rainer Hubmann c,d , Sylvia Laffer d , Brigitte Marian e , Medhat Shehata c,d ,
Clemens Krepler d,f , Hubert Pehamberger d,f , Thomas Grunt b,d , Ulrich Jäger c,d ,
Christoph C. Zielinski b,d , Peter Valent c,d
aBone Marrow Transplantation Unit, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria
b Division of Clinical Oncology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria
c Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria
d Ludwig Boltzmann Cluster Oncology, Vienna, Austria
e Department of Internal Medicine I, Institute for Cancer Research, Medical University of Vienna, Vienna, Austria
f Department of Dermatology, Medical University of Vienna, Vienna, Austria

Accepted 6 January 2010

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.1. Identification of putative CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Definition of CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Limitations of in vivo CSC assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. In vitro assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Antigens commonly expressed on CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. General problems with the so-called ‘stem cell markers’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Myeloid neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8. Lymphoid neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.1. Acute lymphoblastic leukemia (ALL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.2. Multiple myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9. Solid tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.1. Head and neck squamous cell cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.2. Colon CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.3. Liver CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.4. Pancreas CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.5. Breast CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.6. Brain tumor stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.7. Prostate CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.8. Sarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9.9. Other solid tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
10. Melanoma stem cells (MSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
11. CSC plasticity—the Hydra Model of CSC development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
12. Strategies for the successful elimination of CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
13. Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author at: Department of Medicine I, Stem Cell Transplantation Unit, Medical University of Vienna, Währinger Gürtel 18-20,

A-1090 Wien, Austria. Tel.: +43 1 404006085; fax: +43 1 404005701.

E-mail address: axel.schulenburg@meduniwien.ac.at (A. Schulenburg).

1040-8428/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2010.01.001

Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abstract
Neoplastic stem cells have initially been characterized in myeloid leukemias where NOD/SCID mouse-repopulating progenitors supposedly
reside within a CD34+/Lin− subset of the malignant clone. These progenitors are considered to be self-renewing cells responsible for the in
vivo long-term growth of neoplastic cells in leukemic patients. Therefore, these cells represent an attractive target of therapy. In some lymphoid
leukemias, NOD/SCID mouse-repopulating cells were also reported to reside within the CD34+/Lin− subfraction of the clone. More recently,
several attempts have been made to transfer the cancer stem cell concept to solid tumors and other non-hematopoietic neoplasms. In several of
these tumors, the cell surface antigens AC133 (CD133) and CD44 are considered to indicate the potential of a cell to initiate permanent tumor
formation in vivo. However, several questions concerning the phenotype, self-renewal capacity, stroma-dependence, and other properties of
cancer- or leukemia-initiating cells remain to be solved. The current article provides a summary of our current knowledge on neoplastic
(cancer) stem cells, with special emphasis on clinical implications and therapeutic options as well as a discussion about conceptual and
technical limitations.
© 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cancer stem cells; Targeted therapy; Drug resistance

1. Introduction which subpopulations with variable capacity of long-term

survival can be detected and separated using monoclonal anti-
The principle concept of cancer stem cells (CSC) has bodies. Such antibodies are directed against organ-specific
gained acceptance in recent years [1–4]. The CSC concept or/and lineage-specific antigens or so-called ‘stem cell mark-
postulates the existence of subfractions of “tumor stem cells” ers’, and can be employed to enrich (separate) stem cells
within each neoplasm. These CSC exhibit the capacity for by fluorescence-activated cell sorting (FACS) from primary
self-renewal and unlimited growth, and in this regard dif- cell samples [1–4,7]. The different subsets of cancer cells
fer from more mature neoplastic cells (progeny) that have (putative stem cells and more mature cells) are then investi-
only a limited capacity to divide and to survive. The con- gated for their capacity to repopulate immunodeficient mice
cept of tumor stem cells may provide explanations for the with the tumor/leukemia (stem cell function). In fact, CSC
failure of certain treatments to induce long-term remission. should be able to reproducibly establish the original cancer or
In fact, in many instances, conventional chemotherapy may leukemia (all or most components of the disease) in a xeno-
act only on more mature cells, whereas immature neoplastic transplant model (also in secondary recipient mice). In most
stem cells exhibit resistance, so that these drugs fail to target studies, non-obese diabetic severe combined immunodefi-
and eliminate CSC [1–4]. Thereby, the CSC concept points to cient (NOD/SCID) mice have been used [1–4,8,9]. However,
the need to develop new treatment strategies through which depending on the type of tumor, other mouse systems may
CSC can be eliminated. A prerequisite for the evaluation of provide an even better engraftment [10,11]. Despite limita-
CSC as “target-cell” in oncology is their identification and tions (non-human microenvironment, slowly growing tumors
knowledge about target expression profiles. Therefore, sub- may not establish during the lifetime of mice), immun-
stantial efforts have recently been made to identify CSC in odeficient mouse models remain a widely used approach
various types of cancer and to identify molecular targets and and are considered the best available standard-model for
expression profiles in these cells. the identification of cancer- and leukemia-initiating cells
A number of different monoclonal antibodies directed in primary tissue samples. Depending on the type of neo-
against various cell surface antigens have been used to iden- plasm, primary neoplastic cells are injected intravenously
tify CSC-enriched cell populations in various neoplasms, and (leukemias, metastatic carcinomas), subcutaneously (skin
to purify these cells for molecular and functional studies [5,6]. tumors, solid tumors), or directly into solid organs [12–17].
These experiments focus on the identification and character- In case of leukemias, NOD/SCID mice are usually irradi-
ization of molecular targets, and effects of natural ligands, ated sublethally in order to provide proper engraftment of
response modifiers, and targeted drugs on these cells. leukemic cells in the bone marrow cavities of mice [18,19].
The current article provides a summary of our current An unresolved question is whether and what cytokines and
knowledge about cancer- and leukemia-initiating cells, with what human microenvironmental cells are required to facil-
special focus on clinical implications and perspectives. itate optimal engraftment and growth/survival of neoplastic
cells in various disease models.
1.1. Identification of putative CSC After injection, tumor/leukemia cell growth is monitored
over several weeks. When a tumor or leukemia has devel-
The identification of CSC is usually based on differen- oped, the mouse is sacrificed and the neoplasm examined
tial expression of cell surface antigens (markers) through for histologic and molecular features [1–4]. Key questions in

Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
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each experiment are whether the xenotransplant tumor indeed
resembles the original neoplasm and whether indeed most or
all elements (subclones) of the original neoplasm are found in
the xenotransplant tumor [2]. To further document long-term
engraftment and thus to confirm the stem cell function of CSC
in xenotransplant tumors, these tumors can be recovered from
mouse tissues and can be transplanted into secondary recipi-
ent mice, where self-renewing CSC should again form tumor
lesions and all components of the primary tumor/leukemia as
well as a new CSC pool [1,2]. It is important in each project to
demonstrate that the more mature cells are unable to repop-
ulate leukemias/tumors in the same mice [20] which may be
difficult to demonstrate in slowly growing/developing neo-
plasms, as the time of development of the human neoplasm
may exceed the lifetime of the mouse. Moreover, in such neo-
plasms, it may be difficult to delineate between engraftment
of “real” stem cells (CSC) and the persistence of more mature
progenitors that have only a limited capacity to divide. There-
fore, in these neoplasms (e.g. chronic leukemias), it may be
of particular importance to confirm engraftment and growth
of neoplastic cells in secondary recipient mice in order to
learn whether these cells exhibit or lack stem cell function.
Another important question is whether engrafted cells indeed
are derived from neoplastic stem cells (CSC) or derive from
normal stem cells.
2. Definition of CSC
Cancer/leukemia stem cells (CSC) are undifferentiated
cells and are defined by three key features [1,21]: first, these
cells can differentiate into most or all types of cells that are
produced by the original tumor. Second, CSC have the ability
to self-renew. Finally, CSC maintain the stem cell pool and
most (or even all) mature elements of the tumor/leukemia for
unlimited time periods by balancing between self-renewal
(proliferation without maturation) and proliferation plus dif-
ferentiation and maturation by asymmetrical cell division(s)
[1,4]. The process(es) of cell division, of self-renewal, and
of differentiation of CSC are considered to be regulated by
a network of cytokines and by the microenvironment, simi-
lar to normal stem cells [1,4]. In many instances, the same
cytokine that regulates growth of normal (stem) cells in a cer-
tain organ will also promote the growth and self-renewal of
neoplastic stem cells [1,4].
3. Limitations of in vivo CSC assays
Despite the obvious value of an in vivo model that is
sufficient to demonstrate the tumor-initiating potential of
distinct subpopulations of neoplastic cells, a number of lim-
itations of the xenotransplant assay have to be considered.
First, neoplasms with a low growth rate (e.g. indolent neo-
plasms, low-grade malignancies, preneoplasm) are difficult
to analyze in a mouse xenotransplant model as in most
instances, the development phase of the neoplasm exceeds
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
3
the lifetime of the mouse. Second, the microenvironment is
usually species-specific and often tumor-specific. In fact, the
microenvironment of the normal mouse may differ in several
aspects from the tumor microenvironment that supports the
growth of neoplastic (stem) cells in the natural (human) host.
Likewise, microenvironment receptors and cytokines in the
mouse may not cross-interact in all cases with the respec-
tive receptors expressed on human tumor/leukemia (stem)
cells. To overcome this problem, NOD/SCID mice have been
treated with human cytokines or are cotransplanted with
microenvironmental cells in order to facilitate better growth
of neoplastic (stem) cells [9]. Another important limitation
is that most mouse models that have been used in the past,
including NOD/SCID mice, harbour a residual immune sys-
tem through which these mice can eliminate subfractions of
injected cancer/leukemia cells, especially when these cells
display numerous immunogenic antigens (more mature cells)
or when cells are antibody-laden (antibody-stained) cells.
The same cells may, however, grow and form tumors in
mice with a more severely impaired immune system [22,23].
Finally, most neoplasms may grow in permanently estab-
lished subclones with subclone-specific stem cells. In other
words, the stem cell pool in most neoplasms is composed of
several (many) different subsets of stem cells with varying
biological properties and diverse growth characteristics, and
it remains unknown whether all (relevant) subclones can be
grown in one xenotransplant model.
4. In vitro assays
As stem cell research using NOD/SCID mice is expen-
sive and time-consuming and may have several limitations,
in vitro long-term growth assays are often used in order to
screen for stem cell fractions or CSC-regulating compounds.
Such in vitro long-term growth assays have been established
for myeloid and lymphoid neoplasms as well as various solid
tumors [24–28]. Interestingly, in most instances, a stroma
cell layer supports the long-term growth of immature neo-
plastic cells in these assays, which is in line with the concept
that the (tumor) microenvironment essentially contributes to
growth and survival of CSC and thus the development and
biology of these neoplasms (stem cell niche). In myeloid neo-
plasms, the Dexter type long-term culture system, originally
established for normal pluripotent hematopoietic progenitor
cells [29] has been employed and found to support the long-
term growth of leukemias [30]. However, interestingly, some
of these leukemias may even have a “growth-disadvantage”
compared to normal stem cells (long-term culture initiating
cells) in these cultures [31]. In lymphoid neoplasms, includ-
ing CLL and ALL, it is well known that stromal cells can
inhibit apoptosis and support the growth of leukemic cells in
vitro [32,33]. In various solid tumors and melanomas, iso-
lated cells are found to form three-dimensional spheres that
presumably are composed of immature neoplastic cells and
supporting/nutritive stromal cells, and thus may resemble an
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in vitro model of the so-called stem cell niche [34]. Such
sphere formation has been described for neural cancer (stem)
cells, especially glioblastomas, colon cancer, breast cancer,
and melanoma cells [24–28]. The culture system is based on
the ability of cancer cells to form three dimensional spheres
in vitro, and the ability of sphere-derived cells to induce long-
term growth of tumors [24–28]. In fact, at least some of
the tumor cells within these spheres are considered to have
self-renewal capacity and tumor-initiating (CSC) potential.
5. Antigens commonly expressed on CSC
Neoplastic stem cells are considered to express a simi-
lar antigen pattern, to display similar functional properties,
and to be regulated by similar receptor ligands when com-
pared to normal stem cells (derived from the same organ
system). Therefore, many stem cell/progenitor cell markers
are also markers of neoplastic stem cells. These antigens
include cytokine receptors, homing receptors, and various
drug transporters (Table 1).
To identify potential surface markers of CSC, it is
helpful to look for interactions of these cells with the sur-
rounding microenvironment. Based on the behaviour of
normal stem cells, CSC should interact with the supporting
microenvironment via several biologically relevant surface
receptors mediating specific functions. One specific stem cell
function related to the microenvironment is “stem cell hom-
ing” [35–38]. Major homing receptors discussed as being
expressed on CSC are integrins, selectin-ligands, chemokine
receptors, other cytoadhesion molecules (CAMs), and lig-
ands of matrix molecules such as L1 or the hyaluronic
acid receptor CD44 [39,40]. L1 is also involved in the
epithelial–mesenchymal transition (EMT) and is found on
the edge of invasive colon cancer and its metastases [41,42].
Together with L1, CD133 appears to be necessary for tumor
growth of gliomas [43]. CD133 and CD44 show overlapping
expression in various tumors and CSC [26,44,45]. CD133, a
glycoprotein also known as Prominin 1 (PROM1), is a mem-
ber of the pentaspan family of transmembrane glycoproteins
(5-transmembrane, 5-TM) which specifically bind to cellular
protrusions [46–48]. CD133 is expressed in hematopoietic
stem cells, endothelial progenitors, as well as on glioblas-
toma, colon CSC, and other solid tumor CSC [46–48].
The CD44 protein is a cell-surface glycoprotein involved
in cell–cell interactions, adhesion, migration, and homing
[49]. It is a receptor for hyaluronic acid and can also inter-
act with other ligands, such as osteopontin, collagen species,
and matrix metalloproteinases (MMPs) [49]. A specialized
sialofucosylated glycoform of CD44, called HCELL, is usu-
ally expressed on human hematopoietic stem cells, and is a
highly potent E-selectin and L-selectin ligand [50]. HCELL
functions as a “bone marrow homing receptor” directing
migration of human hematopoietic stem cells and mesenchy-
mal stem cells to the bone marrow [50]. HCELL is expressed
on breast-, prostate-, pancreatic-, and colon CSC, and is reg-
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
ulated by the WNT pathway [13,16,45,51]. The frequently
observed expression of CD44 and CD133 on CSC suggests
a functional role for these receptors in CSC biology. How-
ever, it remains unknown whether these receptors are indeed
necessary for stem cell functions or are just expressed on
CSC without a specific function. Whatever the answer to this
question is, these markers are commonly found on immature
neoplastic cells (CSC) and may thereby help in the identifi-
cation of CSC in various organs and tumors. More recently,
it has been reported that in a colon adenoma strain, LT97,
the CD44+ subpopulation exhibits a faster growth rate and
expresses higher levels of other stem cell antigens (such
as Musashi-1 and EphB2) compared to the CD44-negative
fraction of adenoma cells [52]. Similarly, the CD133+ frac-
tion of the HCT116 colon cancer cell line has recently been
described to display higher levels of stem cell antigens and
a faster growth rate in vitro and in vivo compared to the
CD133− fraction [53].
Another basic requirement for CSC is to protect them-
selves against various external toxic stimuli and drugs, which
makes them often drug-resistant. One example for such a
drug transporter protein is ABCG2 which is able to pump
out not only cell-specific substances but also exogenous tox-
ins and cytotoxic drugs, and thus is responsible (in part) for
resistance of CSC against various drugs. Other drug trans-
porters that have been discussed as indicating the long-term
repopulation-potential of malignant cells include MDR-1
and ABCB5 [54,55]. Various specific dyes like the Hoechst
33342 dye, are also transported from the cell into the extra-
cellular space via specific transporters, which allows the
definition of the so-called “side population” which seems
closely related to the stem cell fraction in many tumors
[56]. A similar distribution and association with stem cells
has been described for the enzyme aldehyde dehydrogenase
(aldolase) [57–61]. This enzyme has been described to be
involved in the metabolization of several cytostatic drugs
including cyclophosphamide and thereby may be associated
with chemoresistance [60,62,63].
Lastly, CSC are considered to respond to various exter-
nal physiologic (sometimes paracrine or autocrine) stimuli
including various cytokines. In line with this assumption,
CSC express certain cytokine and chemokine receptors
on their cell surface. For example the IL-3 receptor
(CD123/CD131), SCF receptor (KIT) and the G-CSF-
receptor are usually expressed on leukemic CSC in AML
[64,65]. It has also been described that EGF receptor fam-
ily members including HER2 are expressed on epithelial
CSC including mammary CSC [66,67]. There is also evi-
dence that IGF- and FGF-receptors play an important role
in solid tumors and may be expressed on solid tumor CSC
[68,69]. More recently, it has been described that pancreatic
CSC express CXCR4 [12]. A clinically important question
is whether solid tumor CSC or leukemic CSC express recep-
tors for erythropoietin (EPO), G-CSF, or GM-CSF, as these
cytokines are often applied in cancer/leukemia patients [70].
At least for AML and some tumor cell types it has been
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Table 1
Cell surface molecules detectable on stem and progenitor cells.
Marker/antigen Background and function of marker/antigen
CD133 CD133 antigen is a transmembrane molecule expressed on hematopoietic stem and progenitor cells, on circulating
endothelial progenitor cells, neural stem cells, renal and prostate stem cells [215].
CD34 CD34 is an adhesion molecule expressed on human hematopoietic stem and progenitor cells, endothelial progenitor
cells and vascular endothelial cells [142].
CD44 CD44 is a cell adhesion receptor, and its ligands are hyaluronate and the cytokine osteopontin. CD44 is expressed in
lymphoid, mieloid and erythroid cells, mesenchymal stem cells and may be useful predictor of lymph node metastasis
[1].
CD29 CD29 is a beta1 integrin involved in cell adhesion embryogenesis, tissue repair, immune response and metatastatic
diffusion of tumor cells reacting with thrombocytes, monocytes and a T and B lymphocytes and is also expressed on
mesenchymal stem cells [216].
CD24 CD24 antigen is a glycosylphosphatidylinositol-linked membrane sialoglycoprotein. CD24 is present on B cells, from
the stage pre-B to the mature B cell stage, but not on plasma cells. It is expressed on mature granulocytes and on a
variety of epithelial cell types [1].
CD166 CD166 molecule, a mesenchymal stem cell marker that displayed heterogeneous expression patterns in CRC epithelial
cells (15, 16) and whose increased expression levels were previously associated with poor clinical outcome in CRC
patients [45].
CD326 EpCAM (CD326) is a pan-epithelial differentiation antigen expressed on the basolateral surface of various carcinomas
to varying degrees. As a homotypic cell adhesion molecule, it is intimately integrated within the Cadherin–Catenin and
WNT pathways. It has recently been shown to modulate the expression of proto-oncogenes such as c-myc [217].
CD90 Thy-1 (CD90) is expressed on many cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts.
Activation of Thy-1 can promote T cell activation, and this role of Thy-1 is reviewed elsewhere. Thy-1 also affects
numerous nonimmunologic biological processes, including cellular adhesion, neurite outgrowth, tumor growth,
migration, and cell death [218].
CD123 The specific [alpha] subunit of the interleukin-3 receptor (IL-3R-alpha, CD123) is expressed on hematopoietic cells,
including monocytes, neutrophils, basophils, and megakaryocytes but not on peripheral T cells, natural killer cells,
platelets, and red blood cells [219].
CD9 CD9 belongs to a tetraspanin superfamily and is expressed in a variety of blood cells including pre-B lymphocytes but
not in HSCs. It is also expressed in many types of solid tumors, and is involved in a various kinds of cell processes, such
as cell adhesion, motility, and signalling events through an association with integrin family proteins [220].
CD20 CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35), is a hydrophobic transmembrane protein with a
molecular weight of approximately 35 kDa located on pre-B and mature B lymphocytes. The antigen is expressed on
most B-cell non-Hodgkin’s lymphomas but is not found on stem cells, pro-B cells, normal plasma cells or other normal
tissues. Plasma blasts and stimulated plasma cells may express CD20. CD20 regulates an early step(s) in the activation
process for cell cycle initiation and differentiation, and possibly functions as a calcium ion channel. CD20 is not shed
from the cell surface and does not internalize upon antibody binding. Free CD20 antigen is not found in the circulation;
thus a drug that reacts with CD20, such as an antibody, would not be neutralized before binding to its target cell [221].
ABCB1 (MDR1) and ABCG2 ABCB1 and ABCG2 are recognized as belonging to a family of at least 48 human ABC transporters involved in a
variety of essential cellular transport processes. They are the products of MDR genes and confer multidrug resistance by
pumping out chemotherapy. They also pump out Hoechst dye and rhodamine. Reversal of MDR in vitro was easily
attained with a variety of inhibitors like verapamil [222].
ABCB5 ABCB5 [subfamily B (MDR/TAP)] is a novel human ABC transporter encoded on chromosome 7p15.3 [223]. ABCB5,
like ABCB1, acts as an energy-dependent drug efflux transporter for the fluorescent probe rhodamine-123 [224].
CLL-1 Human CLL-1 (also known as MICL or CLEC12A), is a type II transmembrane glycoprotein and member of the large
family of C-type lectin-like receptors involved in immune regulation. The intracellular domain of CLL-1 contains both
an immunotyrosine-based inhibition motif as well as a YXXM motif, suggesting a role for CLL-1 as a signalling
receptor [86,225].
Erythropoietin receptor The erythropoietin receptor (EpoR) consists of two peptide chains and is a member of the cytokine receptor family. The
interaction of erythropoietin with its cell surface receptor induces a conformational change of receptor homodimers
leading to the activation of intracellular signal transduction that mediates the ability of erythropoietin to support the
proliferation, terminal differentiation and survival [226].

described that CSC indeed express receptors for SCF, G-CSF,
and sometimes also for GM-CSF [65].
6. General problems with the so-called ‘stem cell
markers’
One general problem is that the so-called stem cell mark-
ers are by far not specific for stem cells or progenitor cells.
Rather, most of these antigens are broadly expressed on var-
ious mesenchymal cells. Likewise, CD44 is expressed not
only on hematopoietic and non-hematopoietic stem cells
but also on most mature cells, including monocytes, lym-
phocytes, granulocytes, epithelial cells, and melanocytes
[50,71]. Similarly, in most leukemias and solid neoplasms,
CD44 is expressed on mature cells. Prominin (CD133) is
expressed on myeloid progenitor cells and on various (imma-
ture and mature) mesenchymal cells, including endothelial

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cells [46–48]. Even the CD34 antigen (hematopoietic pre-
cursor cell antigen-1, HPCA1) is not only expressed on
hematopoietic stem cells but also on more mature progen-
itors and also on endothelial cells [72]. All in all, no stem
cell specific antigen has been identified so far. Therefore, it
is essential to apply combinations of markers and antibodies
and to define stem cell-enriched fractions on the basis of typ-
ical antigen combinations. Usually, one or two markers are
employed by investigators to gate for or to exclude a germ
layer or an organ system (e.g. CD45 as pan-hematopoietic cell
marker). In certain instances a cocktail of antibodies is used
to exclude more mature cells in a certain organ system, e.g.
more mature hematopoietic cells by lineage-specific mark-
ers (“Lin-cocktail” defining Lin-negative progenitor cells)
[19,73]. Here, one problem may be that in certain leukemias,
CSC may aberrantly express lineage-related marker antigens.
In these leukemias, application of the “Lin-cocktail” would
lead to a loss of CSC (sub)fractions.
Finally, markers that are typically expressed on immature
cells of a given neoplasm or organ, are applied, e.g. CD34 for
lymphohematopoietic cells and most myeloid leukemias. In
these defined progenitor fractions, further subpopulations in
which CSC reside, are defined. This is performed by antibod-
ies that positively or negatively identify these subfractions.
Based on antibody-binding patterns and identification of sub-
fractions enriched in CSC, these cells can be isolated by
multi-color flow cytometry and cell sorting. The read out that
is then used to confirm the presence of CSC is either an in
vitro long-term culture system or – preferably – the immun-
odeficient mouse model. In most instances, the NOD/SCID
mouse has been employed in these assays. However, there are
a number of caveats that have to be considered when using
sorted cells in these mouse models and bioassays. The most
important caveat may be that the antibody itself may stimu-
late or may interfere with in vitro growth, engraftment, or/and
long-term repopulation of progenitor cells in NOD/SCID
mice [22]. Therefore, it has to be excluded by appropriate
control experiments, that the antibodies used to positively
or negatively define CSC would per se induce or inhibit
engraftment of CSC in NOD/SCID mice, or would interfere
with in vitro CSC growth in the bioassay applied or with
in vivo engraftment [22]. Recently, Taussig et al. revealed a
procedure-related problem in the description of AML CSC
defined by expression of CD34 and lack of CD38 [22]. In their
paper, they were able to show that the original description
of leukemic stem cells as CD38-negative cells in the mouse
model may be due to the clearance of leukemic CSC together
with the CD38 antibody by the residual immune system of
NOD/SCID mice. Clearance was not observed when mice
were further immunosuppressed by drugs or when the anti-
body was degraded into fab fragments which allowed CD38+
leukemic cells to repopulate these mice [22]. Moreover,
CD34+/CD38+ grew well in more severely immunodeficient
IL2rgamma(null) mice (NSG mice) [22]. All in all, it appears
that NOD/SCID mice may not be the most suitable strain to
study CSC growth in solid tumors and leukemias. Rather,
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
more severely immunodeficient mice (NOG or NSG mice)
may be required for optimal engraftment of CSC [10,11]. It
can therefore be expected that stem cell research will employ
NOG or NSG mice in various CSC models in the future.
Recently, Kim et al. found that a polyclonal anti-human
CD24 rabbit antibody initiates cross-linking of CD24 on
tumor cells, and that this cross-link induces apoptosis in
breast cancer cells in a cell culture system using MCF-7
cells [74]. This may have implications for the characteriza-
tion of breast cancer stem cells, since these cells supposedly
reside within the CD44+CD24low (CD24-negative) fraction
of tumor cells. If CD24+ tumor cells would just be elimi-
nated (faster than CD24low cells) by the antibody-induced
cross-linking of CD24, the CD24-negativity could no longer
be regarded as a stem cell-related feature. Whether indeed
CD24-induced stem cell depletion occurs after antibody-
binding remains to be shown.
Another major limitation may be that CSC receptors and
their ligands and homing receptors in the tissue environment
are sometimes species-specific. Therefore, CSC are often
injected directly into a certain target organ (e.g. bone marrow,
pancreas, brain, others) in these mice [12–17,44,75,76]. Nev-
ertheless, it is difficult to know whether some of the CSC will
not (cannot) grow in a xenotransplant model simply because
essential mouse-derived ligands are not cross-reacting with
human receptors expressed on CSC. A possible solution to
this problem may be to co-transplant human stroma cells,
to humanize mice (organ-specific microenvironment), or to
inject mice with all important (human) ligands in order to
guarantee proper engraftment of CSC.
Probably the most important problem with CSC markers
and CSC-reactive antibodies is stem cell plasticity. In fact,
it has been described that leukemic and solid tumor CSC
fractions are usually composed of several different subclones
defined by varying profiles of molecular markers, point muta-
tions in critical target genes (often causing drug resistance in
subclones), and expression of cell surface receptors including
stem cell markers. This means, that CSC in a given tumor may
display varying combinations of cell surface antigens which
makes it difficult to define all CSC subfractions (subclones)
by monoclonal antibodies. In some instances, the phenotypic
heterogeneity may also be associated with functional hetero-
geneity. However, so far very little is known about cell surface
antigens defining subfractions of CSC in various solid tumors
and leukemias. Likewise, in AML, leukemic CD34+/Lin−
(stem) cells are often composed of a CD133+ and a CD133−
subfraction [77].
In the following paragraphs, we review human neoplasms
where CSC have recently been identified.
7. Myeloid neoplasms
It is generally assumed that most if not all myeloid
neoplasms derive from a clonal immature hematopoietic
progenitor (stem) cell. Therefore, myeloid neoplasms are
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optimal models to study the CSC hypothesis. Normal
hematopoietic stem cells are considered to reside within
the Lin-negative and CD38-negative portion of CD34-
positive progenitor cells. Leukemic stem cells have first
been described in AML, and later in CML [19,73,78]. In
other myeloid neoplasms, neoplastic stem cells are less well
defined. The phenotype of leukemic stem cells is considered
to be similar to that of normal stem cells (Lin−, CD34+,
CD38− and in part CD38+ ) [8,19,79] (Table 2). Several
attempts have been made to identify markers that distin-
guish between normal and neoplastic meyloid stem cells.
This would enable therapeutic approaches that might be
able to spare normal stem cells [80]. Indeed, several surface
molecules may be expressed abundantly on CD34+/Lin−
AML stem cells, whereas normal hematopoietic stem cells
lack or express only low levels of these antigens. Exam-
ples for antigens preferentially expressed on leukemic CSC
in AML are the alpha chain of the IL-3 receptor (CD123),
the Mylotarg-receptor Siglec-3 (CD33), CD96, CXCR4, and
CLL-1 [64,81–86]. CD33 is of special interest as CD33-
targeting drugs are available and are used to treat patients
with (refractory) AML. Fig. 1 shows the effects of Mylotarg
on survival of CD34+/CD38+ and CD34+/CD38− AML
cells. Neoplastic stem cells have been defined in several
but not all variants of AML [8,19]. Likewise, in a group
of AML patients, leukemic blast cells and immature pro-
genitors are CD34-negative. In these patients, it is very
difficult to define stem cell compartments. Other examples
are promyelocytic leukemia, NPM-mutated AML variants,
and monoblastic leukemias where most of the neoplastic cells
may be CD34-negative cells [87]. In many of these leukemias
it even remains to be demonstrated that AML stem cells reside
within the (small) CD34+ subpopulation of clonal leukemic
cells. It is also important to note that AML types in which the
stem cell origin of the leukemia can be demonstrated using a
NOD/SCID mouse xenotransplant model are those variants
where blast cells exhibit a high proliferative potential and
resistance against chemotherapy [88].
In CML, most clonal cells are CD34-negative cells. In
these patients, a complex multi-step enrichment technique
for the isolation of putative CD34+/Lin− stem cells has been
proposed [73]. Indeed, when these cells were injected into
NOD/SCID or NOD/SCID-beta2microglobulin−/− mice,
they were found to repopulate these mice with a CML-like
disease. By contrast, more mature CD34+ CML cells only
produced an early transient leukemic repopulation that was
no longer detectable after 6 weeks [89]. Similar to the AML
stem cell, the CML stem cell was found to express the IL-
3 receptor [90]. Other target antigens like CD33, CD44, or
CD117 were also found to be expressed on CD34+/CD38−
stem cells in CML patients [64]. CML stem cells also
express ABCB1 and ABCG2, whereas the levels of OCT-
1 expressed on CML stem cells are rather low [55,91]. Both
ABCG molecules are known to mediate the efflux of ima-
tinib and other drugs and thus confer resistance. OCT-1,
on the other hand, mediates the uptake of imatinib, and
low expression of these transporter molecules is associ-
ated with low drug-uptake and thus low intracellular levels
of imatinib. All these mechanisms may contribute to the
intrinsic resistance of CML CSC against imatinib [55,92].

Table 2
Published cell surface phenotype of neoplastic stem cells in various malignancies.
Disease Surface marker Refs.
AML CD34+, CD38−, CD44, CD123+ Bonnet and Dick [19]
ALL Ph+ CD34+, CD38− Cobaleda et al. [105]
TEL-AML1–positive CD34+/CD38−/low/CD19+ Hong et al. [106]
c-ALL CD34+/CD19+ Kong et al. [107]
Childhood B-ALL CD133(+)/CD19(−) and CD38(−) Cox et al. [108]
B lineage ALL CD34+/CD9+ Nishida et al. [109]
CML CD34+, CD38−, CD123+ Holyoake et al. [73]
Breast CD44+, CD24−/low, ESA+ Al-Hajj et al. [13]
CD44+ Li et al. [16]
Pancreas
CD133+ Hermann et al. [12]
CD133+ Ma et al. [131]
Liver
CD90+ Yang et al. [17]
CD133 Ricci-Vitian et al. [26], O’Brien et al. [44]
Colon
CD44 Dalerba et al. [45]
CD133+/alpha 2 beta 1 integrin/CD44+ Collins et al. [142]
Prostate
CD44+/CD24− Hurt et al. [51]
Sarcoma CD133+ Suva et al. [151]
CD20+ Fang et al. [24]
Melanoma
CD133+ Monzani et al. [162]
CD133+ Singh et al. [15]
CNS
Hemm et al. [27]

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Fig. 1. Apoptosis in AML stem cells induced by Mylotarg (GO). Blast cells obtained from a patient with AML (FAB M0) were incubated with control medium
(upper graphs) or with gemtuzumab/ozogamicin (GO = Mylotarg), 1 ␮g/ml, at 37 ◦ C for 48 h (lower graphs). Then, apoptosis was analyzed by combined flow
cytometry using antibodies against CD34 and CD38, and AnnexinV-FITC. The percentage of apoptotic cells was analyzed in CD34+/CD38+ cells (right panel)
and in the CD34+/CD38− fraction of the clone (left panel) after gating for vital (7-AAD negative) cells.

More recently, imatinib-resistance of CML stem cells has
been employed to screen for stem cell-related markers in
BCR/ABL transformed (imatinib-exposed) murine cells [93].
In these studies, several antigens potentially involved in
leukemic stem cell growth and survival have been identi-
fied. One of these CML stem cell-related genes is the Alox5
gene that encodes the 5-lipoxygenase (5-LO) [87]. In the
absence of Alox5/5-LO, BCR/ABL failed to induce a CML-
like disease in mice, whereas Alox5-deficiency showed no
effects on growth of normal stem cells [93]. These data
suggest that growth and survival of leukemic stem cells
in CML are regulated by specific gene products, and the
same may hold true for AML. Also, recent data suggest
that neoplastic stem cells in various myeloid neoplasms
may use similar if not identical signalling pathways for
growth and survival in vivo. These pathways include the PI3
kinase-mTOR pathway, WNT-␤-catenin pathway, and Notch
signalling-pathway [94]. However, all these pathways may
be shared by normal and leukemic stem cells, and therefore
may not be optimal targets, at least when treatment should
spare normal (stem) cells. Also, Hedgehog signalling seems
to be important for the development of myeloid leukemias
and may be a promising target pathway [95]. Other path-
ways found in leukemic stem cells are the JAK2-STAT5
pathway and NF-kappa-B pathway. In most instances, sig-
nalling is initiated by specific oncoproteins, like BCR-ABL in
CML, or PML-RAR-alpha in acute promyelocytic leukemia
[96–100].
In other myeloid neoplasms, very little is known about
the biology and phenotype of neoplastic stem cells. Florian
et al. examined the phenotype of CD34+/CD38− cells in
various myeloid neoplasms including myelodysplastic syn-
dromes, CML, and systemic mastocytosis [64]. In all these
myeloid neoplasms, the phenotype, determined by antibody-
staining, appeared to be very similar, and included major
surface targets such as CD123 and CD33 [64]. In classi-
cal JAK2 V617F-mutated myeloproliferative disorders, the
JAK2 mutant is usually detectable in the CD34+/CD38−
fraction of clonal cells [101,102]. However, JAK2 V617F
may not be detectable in all neoplastic stem cell subclones
in these patients [103], and leukemic progression (sec-
ondary AML) is often accompanied by a loss of the JAK2
mutant, which may be explained by expansion of a more
immature JAK2 V617F-negative subclone during disease
progression [103]. Otherwise, very little is known about the
biology, target expression profiles, and phenotypes of neo-
plastic stem cells in JAK2+ MPN. Recently, a defective stem
cell niche has been discussed as an important factor con-
tributing to the pathogenesis of JAK2-mutated neoplasms
[104].

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8. Lymphoid neoplasms
8.1. Acute lymphoblastic leukemia (ALL)
Little is known about subpopulations of CD34+ ALL
cells that display stem cell function. In 2000, Cobaleda et
al. showed that in Ph+ (BCR/ABL+) ALL, the NOD/SCID
mouse-repopulating ALL stem cell resides within the
CD34+/CD38− fraction of the clone, similar to AML
stem cells [105]. When ≥100 CD34-positive/CD38-negative
cells were injected intravenously, leukemias developed after
4–6 weeks in these mice [105]. Hong et al. recently
described that the CD34+/CD38−/low/CD19+ cells in TEL-
AML1–positive c-ALL (0.002% of total mononuclear cells)
propagate leukemias in NOD/SCID mice [106]. In contrast,
Kong et al. found that CD38 is not useful for the identi-
fication of a c-ALL stem cell [107]. They observed that
CD34+/CD38−/CD19+ as well as CD34+/CD38+/CD19+
cells are able to establish leukemias after 4–15 weeks
when at least 5 × 103 cells were intravenously injected into
NOD/SCID/IL2r null mice [107]. Recently, Cox et al. found
evidence that the pediatric B-ALL stem cell resides within
a CD133+/CD19− subpopulation [108]. After injection of
743–50,000 CD133+/CD19− cells into NOD/SCID mice,
engraftment was observed after 8–10 weeks [108]. Another
potential B-ALL stem cell marker may be CD9. Nishida et
al. injected at least 1 × 104 CD9+ (cultured) ALL cells intra-
venously into a total of 20 NOG mice, and all mice died
within 45 days due to leukemia [109]. Despite the com-
mon notion that the ALL stem cell should be CD34+ cells,
this may not hold true for all B-lineage ALL variants and
also not for T ALL [110,111]. Recent data suggest that the
Notch pathway may be involved in CSC function in patients
with T-ALL [112]. Notch is already known to play a role in
normal T-cell development and T-ALL [113–116]. Whereas
in ALL, at least first attempts have been made to identify
a leukemic stem cells, there is very limited if any infor-
mation about NOD/SCID mouse-repopulating cells derived
from other lymphoproliferative neoplasms, such as chronic
lymphocytic leukemia (CLL) or other Non-Hodgkin’s Lym-
phomas (NHL) [117]. In one study, Nowakowski et al. found a
small CD5+/CD19+/ABCG+ population of CLL cells [111].
From gene expression profiling data and an increase of this
subpopulation of CLL cells after therapy, the authors con-
cluded that these cells may have stem cell like properties
[118]. More recent data suggest that the CLL cell with stem
cell-like properties may also reside within a small CD34+
subset of leukemic cells that co-express CD19 and CD5
(unpublished observation).
8.2. Multiple myeloma
Matsui et al. were the first to report on the existence of
myeloma-initiating stem cell fractions in multiple myeloma
patients [119]. They showed that that CD138+ fraction of
myeloma cells cannot undergo long-term proliferation but
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
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9
arise from clonogenic CD138-negative B cells [112]. In par-
ticular, CD138− cells were found to act clonogenic in vitro
and to produce myelomas in NOD/SCID mice [119]. In
2008 it was found that a combination of dexamethasone,
lenalidomide, bortezomib, and 4-hydroxycyclophosphamide
inhibits the growth of CD138+ multiple myeloma cells but
not the growth of CD138-negative myeloma precursor cells
in vitro. Several lines of evidence suggest that the phenotype
of myeloma stem cells is similar to that of normal memory
B cells (CD138−/CD20+/CD27+) [119–121].
9. Solid tumors
9.1. Head and neck squamous cell cancer
Despite combination therapy head and neck squamous cell
cancer (HNSCC) remains one of the most difficult challenges
in oncology. HNSCC resistance to various drugs has limited
the usefulness of chemotherapy in this disease. Recently,
CD44 has been identified as a potential marker of CSC in
HNSCC [122]. When 5 × 103 CD44-positive poorly to well
differentiated primary HNSCC cells were injected subcuta-
neously into NOD/SCID mice or Rag2␥DKO mice, these
cells gave rise to tumors within 10–16 weeks [122]. The
stem cell-related gene BMI-1 was demonstrated to be overex-
pressed in the CD44+ subpopulation of HNSCC tumor cells
compared to CD44− cells [122]. In vitro experiments per-
formed with various types of cancer cells suggest that CD133
may also be a marker for long-term proliferating cells, but
it remains unknown whether CD133 is indeed a stem cell
marker for HNSCC [123].
9.2. Colon CSC
Since the gastrointestinal tract is an organ-system with
a high turnover of cells where proliferation and cell self-
renewal take place, it was of great interest to learn about
the location, biology, and phenotype of colon CSC. Sev-
eral lines of evidence suggest that immature colon cells (and
presumably also CSC) are located in colon crypt bottoms.
With regard to the phenotype, first reports pointed to CD133
as a potential CSC marker antigen colon cancer [26,44]
(Table 2). When CD133-positive cells were injected into
the kidney capsule or subcutaneously into NOD/SCID mice,
colon tumors developed after several weeks [26,44]. Later,
Dalerba et al. found that CD44 is another potential marker for
CSC in colon cancer patients [45]. When CD44+/EpCAM+
cells were injected subcutaneously into NOD/SCID mice
tumors developed after 20 weeks in these mice [45]. Fur-
thermore, they found that the CSC population is further
characterized by coexpression of CD166 [45]. Recently, it
was reported that CD133+ cells derived from colon spheres
are clonogenic cells and can form adenocarcinomas in a
mouse xenotransplant model [28]. These CD133+ sphere-
derived cells in part co-expressed CD24, CD29, CD44, and
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CD166. Interestingly, of all markers tested, only CD24 was
found to enrich for cells with even higher clonogenic activ-
ity. All in all, several surface markers, including CD44 and
CD133 have been discussed as potential markers of colon
CSC (Table 2). With regard to CD44, this may also hold true
for colon adenomas. In fact, it has been shown that the colon
adenoma cell strain LT97 consists of a CD44+ and a CD44−
portion, and that both subfractions differ in their growth
kinetics and expression of stem cell markers [52]. In particu-
lar, CD44-positive LT97 cells attach and grow for unlimited
time periods, whereas CD44− cells are slowly growing cells
(Fig. 2) [52]. Moreover, in contrast to CD44− LT97 cells,
CD44+ LT97 cells display nuclear beta-catenin and express
beta-catenin target genes, such as ephrin B receptor (ephB2)
and the musashi1 antigen (msi1) [52]. These stem cell anti-
gens are also detectable in colon CSC [124]. All in all, CD44
may not only be a CSC marker for established colon ade-
nocarcinomas, but also for CSC in preneoplastic lesions, i.e.
colon adenomas which may have implications for the biol-
ogy of tumor development. In fact, colon carcinoma CSC
may develop from adenoma CSC by subclone selection after
accumulation of further hits [125,126].
9.3. Liver CSC
A number of previous studies suggest that hepatic can-
cer cells derive from immature progenitor cells in the
liver [127]. There are four candidates for liver CSC: bone
marrow derived cells, oval cells, hepatocytes, and hep-
atopancreatic stem cells [128]. Recent data suggest that
the so-called small oval cells in hepatocellular carcinomas
(HCC) are involved in the carcinogenic process and dis-
play stem cell markers [129,130]. Since these small oval
cells are also found in the normal liver, the hypothesis
was raised that HCC arise from normal liver progenitor
Fig. 2. Correlation between expression of CD44 and proliferative poten-
tial of LT97 cells. The adenoma cell strain LT97 was cultured in complete
medium and passaged once every 10–14 days. Expression of CD44 on LT97
cells was determined by flow cytometry in early (passage up to 20), inter-
mediate (passage 20–30), and late (passage above 30) cultures. The figure
shows the percentage of CD44+ cells (black bars) and the plating efficiency
(hatched bars) over time (early, intermediate, late passage).
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
cells by maturation arrest. Functional analysis confirmed
that distinct cell subsets present in HCC exhibit stem
cell-like properties including long-term survival (immor-
tality), transplantability, and resistance to (chemo)therapy.
More recently, several markers that may indicate stem cell
function in HCC cells have been identified. These mark-
ers include CD133 and CD90 [131,132]. When 5 × 103
CD45− /CD90+ cells, isolated from tumor specimens or blood
samples of HCC patients, were injected into the liver of
SCID/Beige mice, tumors developed within 12–16 weeks in
these mice.
9.4. Pancreas CSC
In pancreatic cancer, CSC have also been described.
These pancreatic cancer stem cells express the cell sur-
face markers CD44, CD24, and EpCAM, and represent
0.5–1.0% of all pancreatic cancer cells [16]. When ≥100
CD44+/CD24+/EpCAM+ cells were injected into the pan-
creas of NOD/SCID mice, tumors developed within 4 weeks
in these mice [16]. Another report suggested that CD133
is expressed on pancreatic CSC [12]. When 500 CD133-
positive pancreas tumor cells were injected into the pancreas
of NMRI-nu/nu mice, tumors developed within 3 weeks [12].
Pancreatic CSC display high levels of genes involved in self-
renewal, such as the Sonic Hedgehog (SHH) antigen and
BMI-1 [133,134]. In addition, CD133+/CXCR4+ cells have
been described to be responsible for metastasis formation in
pancreatic cancer in a model employing the L3.6pl cell line
[12].
9.5. Breast CSC
In 2003, Al-Hajj et al. identified a NOD/SCID mouse-
repopulating breast cancer cell subpopulation. These cells
express CD44, the breast/ovarian cancer-specific antigen
B38.1, and epithelial-specific antigen ESA (= EpCAM) [13].
In contrast to non-repopulating breast cancer cells, breast
CSC coexpress CD44 but display only low amounts of or
lack CD24. When ≥100 CD44+/B38.1+/CD24− cells were
injected into the mammary fat pad of NOD/SCID mice, pal-
pable tumors developed within 12 weeks, whereas none of the
CD44−/B38.1− cells developed tumors in these mice. Other
studies have suggested that high aldehyde dehydrogenase lev-
els may be indicative for enhanced malignant and metastatic
potential and thus may help in the identification of breast CSC
[60]. Most consecutive studies were performed in cell line
models, whereas only very little is known about expression of
surface antigens or targets in primary breast CSC. One inter-
esting question will be to learn whether breast CSC express
members of the ERBB family of oncogenic receptors. In fact,
ERBB receptors are major targets of therapy in this type of
cancer. Data published so far suggest that mammary CSC
express EGFR and ERBB2/Her2 but lack estrogen receptor
alpha [135,136]. This may be due to several different mecha-
nisms such as gene silencing by epigenetic events [137,138].
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Whether this holds true for all breast cancer variants remains
at present unknown.
9.6. Brain tumor stem cells
Hemmati et al. characterized and isolated stem cells from
human paediatric brain tumors of different pathologic sub-
types, including glioblastoma and medulloblastoma [27]. The
authors reported that a minority of the brain tumor cells
were able to form neurospheres and to grow to tumors when
xenotransplanted into rat brain. These neurosphere-derived
cells were found to be Lin-negative and expressed CD133
as well as nestin [27]. More recently, Singh et al. confirmed
that brain CSC reside within the CD133+/nestin+ fraction
of brain tumor cells in a NOD/SCID mouse model [15]. In
fact, when as few as 100 CD133+ glioblastoma cells were
injected into the brain of NOD/SCID mice, tumors devel-
oped after 12 to 14 weeks in most animals. Bao et al. found
that L1 CAM may also serve as a marker for brain CSC
since L1 CAM is overexpressed on CD133+ cells, and tar-
geting of L1 CAM reduced the tumorigenicity of CD133+
cells [43]. Ehtesham et al. showed that glioblastoma pro-
genitors in glioma-derived spheres may express CXCR4
and that binding of its ligand CXCL12 results in increased
growth of neurosphere cells [139]. Recently, Read et al.
found that CD15+ cells are enriched for CSC in medulloblas-
tomas [140]. After intracranial implantation of 3 × 105 cells,
Ptc+/− mice develop tumors within 50 days.
9.7. Prostate CSC
The isolation of CSC in prostate cancer is difficult due
to the heterogeneity of prostate tumors and the small sam-
ple size. It is unclear whether prostate CSC derive from
the basal or luminal layer [141]. However, selection of
cells with a CD133+/alpha 2 beta 1 integrin+/CD44+ phe-
notype resulted in enrichment for prostate cancer-initiating
cells [142–145]. Moreover, CD44+ prostate cancer cells
have recently been described as more invasive and shown
to have increased Hedgehog signalling and activation of the
PI3K/AKT signalling pathway compared to CD44− cells
[146–148]. In 2008, Hurt et al. observed stem cell-like prop-
erties of CD44+/CD24− cells derived from prostate cell lines
(LNCaP and DU145) [51]. The expression of androgen recep-
tors on prostate CSC is controversial [142,147,149]. The gene
expression profile of prostate cancer stem cells can also be
related to Gleason grade and patient outcome [150].
9.8. Sarcoma
Recently, Suva et al. identified for the first time a Ewing
sarcoma cancer stem cell by sorting with magnetic beads for
CD133+ cells [151]. After cell isolation from primary Ewing
sarcoma tissue, these CD133+ cells were found to establish
Ewing sarcomas with parental tumor phenotype and hierar-
chical organization in NOD/SCID mice [151]. The authors
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
11
were also able to show that these tumors again contained
Ewing sarcoma-initiating stem cells when re-transplanted
into secondary recipient mice [151].
9.9. Other solid tumors
In gastric cancer up to now only CD44 is described as
a potential CSC marker [152]. Another group postulated
that some gastric tumors may even arise from bone mar-
row cells [153]. Lung CSC are not well characterized so
far. In one paper, Kim et al. describe bronchioalveolar stem
cells (BASCs) as potential lung CSC, whereas in another
paper, lung CSC are identified as CD133+ cells [154,155].
When 1 × 104 EpCAM+/CD133+ cells from small and non-
small-cell lung cancers were injected subcutaneously, tumors
developed within 3 weeks in SCID mice [155]. This obser-
vation has recently been confirmed for non-small-cell lung
cancer cells by Tirino et al. [156]. As in many other organs,
there is also evidence for the expression of CD44 as well as
CD133 on the surface of ovarian CSC [157–159]. Baba et
al. injected 4–5-week-old BALB/cAnNCr-nu/nu mice with
sorted CD133− (left flank) and CD133+ (right flank) A2780
cells. They observed that CD133+ cells formed tumors that
grew to a larger size and appeared within shorter time than
tumors from CD133− cells isolated from the same parental
cell line [158]. On the other hand, Kusumbe et al. showed
that CD133+ cells in ovarian cancer are only responsible for
the development of the tumor vasculature by giving rise to
endothelial cells. By contrast, CD133+ ovarian cells were not
tumorigenic in this study [160]. In one report CD133+ cells
from anaplastic thyroid cancer showed reconstitution of the
tumor in NOD/SCID mice after injection of at least 10,000
CD133+ cells [161].
10. Melanoma stem cells (MSC)
So far, little is known about tumor-initiating cells in skin
cancer patients. In fact, CSC have only been investigated
and partly characterized in malignant melanomas. Fang et al.
described that melanoma spheres can be grown from pri-
mary melanoma cells, and that melanoma sphere-derived
cells exhibit long-term growth, multilineage potential, and
tumor-initiating potential in SCID mice [24]. They found that
a subpopulation of cells in these spheres is CD20+/CD45−
cells that co-express melanoma antigens. This CD20+ frac-
tion of melanoma cells, when cultured separately, was found
to form larger spheres than CD20− cells, and showed
long-term proliferation in vitro. Based on these data, it
is tempting to speculate that melanoma stem cells reside
within the CD20+ fraction of cells, although this has not
formally been proven by mouse experiments using primary
melanoma cells sorted for CD20+ and CD20− cells. In addi-
tion, the expression of CD20 on melanoma cells has not
been confirmed in other studies. Another marker that has
been discussed as a potential stem cell marker for human
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melanoma is CD133 [162]. It has been described that only
0.2–0.8% of primary metastatic melanoma cells express
CD133 [162]. Monzani et al. observed that 40–50 days
after subcutaneous injection of 3.5 × 105 CD133-positive
cells into NOD/SCID mice, these mice develop detectable
melanoma lesions, whereas the CD133-negative fraction did
not form tumors in NOD/SCID mice [162]. These data sug-
gest that melanoma stem cells may reside within a CD133+
fraction of the clone, at least in metastatic melanomas [163].
In line with this hypothesis, several melanoma cells lines
display CD133 [164,165]. Other studies have shown that cer-
tain drug-transporters that (when expressed) are indicative
of chemoresistance, may be expressed on melanoma stem
cells (and stem cells in other tumors). Among these trans-
porters are MDR1, ABCG2 and ABCB5 [166]. Schatton et al.
described that the ABCB5+ fraction of primary (freshly iso-
lated) melanoma cells is enriched for melanoma-repopulating
stem cells, whereas ABCB5-negative cells are less capable
of initiating melanomas in NOD/SCID mice [54]. ABCB5+
melanoma cells were found to co-express other potential
stem cell markers including ABCB1, TIE1, nestin, and CD44
[54,167].
However, the melanoma-initiating potential of single
(stem) cells may greatly depend on the microenvironment
and the mouse strain employed to demonstrate repopulation.
Quintana et al. have recently shown that up to 30% of all
melanoma cells are melanoma-repopulating cells (indepen-
dent of their phenotype) when interleukin-2 receptor gamma
null NOD/SCID mice are used, whereas tumorigenicity of
the same cells is much lower when NOD/SCID mice with
an intact interleukin-2 receptor are employed [23]. These
data suggest that the frequency of melanoma-initiating cells
may be relatively high, at least in a severely immunocom-
promised host. Whether the same cells (all these cells) are
also able to repopulate melanomas in an immunocompetent
host or in patients remains unclear. In fact, the stem cell
function of a given tumor/melanoma cell may not only be
predetermined by intrinsic factors (genetic and epigenetic
stem cell programs) but also be microenvironmental factors
(factors defining the stem cell niche) and also the immune
system (immunosurveillance, apoptosis-induction by killer
cells, tumor cell phagocytosis).
The observation that up to 30% of melanoma cells may
have the potential to repopulate melanomas in NOG mice
and thus have stem cell function, has changed our views on
what types and subpopulations of melanoma cells indeed are
repopulating and non-repopulating cells. Unfortunately, only
a few markers can clearly discriminate larger subpopulations
of melanoma cells with different potential to metastasize and
to form new tumor lesions in patients. One such marker is
the erythropoietin (EPO) receptor. In fact, the EPO receptor
is only expressed in trace amounts in normal melanocytes,
but is expressed in melanoma cells [168–171]. Whereas in
primary melanomas, only a small subpopulation of cells dis-
play the EPO receptor, in metastatic melanomas up to 30% of
all cells express this antigen [168]. Moreover, it has recently
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
been described that EPO receptor expression is associated
with disease progression and the metastasizing potential of
melanomas [169,172]. Finally, EPO has been described to
initiate signalling and promote survival in human melanomas
[171]. Whether indeed the EPO receptor is a functionally rev-
elant antigen expressed specifically on melanoma initiating
cells is currently under investigation.
11. CSC plasticity—the Hydra Model of CSC
development
A number of previous and more recent data suggest that
neoplastic stem cell clones display substantial plasticity and
are often composed of several different subclones. In many
cases, subclone formation may precede the development of
a frank neoplasm, and only a few (or even only one) of
these subclones may progress to an overt malignancy. This
assumption is consistent with the multi-hit theory of cancer
development [173–176] and would predict that neoplastic
stem cells detectable in the NOD/SCID or NOG (NSG)
mouse assay would not all be able to repopulate all com-
ponents (cell subclones) in a given neoplasm. Rather, this
model predicts early formation of subclones with different
transformation potential, and each subclone must be expected
to contain subclone-specific stem cells. A good example
for the presence of multiple subclones in one neoplasm is
the coexistence of two histologically different hematopoi-
etic disorders in one patient. Another example is CML,
where during treatment with imatinib, one or more different
subclones exhibiting imatinib-resistant BCR/ABL mutants
may be selected (by treatment), and each of these subclones
may progress into a clinically relevant leukemia (subclone-
specific progress) [91,177,178]. Whereas many of these
subclones bearing (drug-resistant) BCR/ABL mutations may
be present (as small clones) before imatinib is started, some
of these subclones may progress to leukemia during imatinib
therapy which may point to clonal instability and a potential
effect of the drug on subclone formation. Similar observations
of stem cell plasticity have been made in other hematopoi-
etic malignancies and may also apply to non-hematopoietic
neoplasms [179].
Recent data suggest the stem cell plasticity may even
involve the supportive stroma or tumor/leukemia-associated
angiogenesis. In particular, it has been described that
endothelial cells in lymphomas and other neoplasms are of
monoclonal origin [180]. Other studies suggest that cultured
bone marrow stroma cells and mesenchymal progenitors in
patients with Ph+ CML are of clonal origin [181]. However,
other studies suggest that stromal cells and mesenchymal pro-
genitor cells in patients with CML are non-clonal cells and
not derived from the Ph+ clone [182,183].
The above described heterogeneity and plasticity of CSC
populations in various neoplasms may be one reason for
the complexity of CSC evolution and function, and may
also explain why it is difficult to design effective treatment
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approaches sufficient to eliminate all these cells and sub-
clones in cancer/leukemia patients [184–186].
12. Strategies for the successful elimination of CSC
Several different strategies have been considered to inhibit
growth and/or survival of CSC, with the ultimate goal to elim-
inate all CSC in these malignancies. These concepts include
relevant surface targets, signal transduction molecules, and
certain survival molecules expressed in CSC (Fig. 3). Many
concepts still relate to myeloid or lymphoid neoplasms,
whereas so far, only a few treatment strategies have been pre-
sented for solid tumors and melanomas. Moreover, whereas
much is known about the expression of various surface and
cytoplasmic drug targets in solid tumors and leukemias, only
very little is known about expression of the same targets in
neoplastic stem cells (Fig. 3).
In myeloid neoplasms, potential surface targets include
the IL-3R alpha chain CD123, the Mylotarg-receptor Siglec-
3 (CD33), CD44, and the tyrosine kinase receptor KIT
(CD117). Target expression can be exploited by selecting
blocking antibodies or by constructing antibody–toxin con-
jugates, cytokine–ligand–toxin conjugates, or antibody–drug
conjugates. A good example for a ligand–toxin con-
jugate is the IL-3-diphteria toxin fusion protein [187].
An example for an antibody–drug conjugate is the
CD33-targeting fusion molecule gemtuzumab–ozogamcin
(Mylotarg) [81–83] (Fig. 1). It has also been reported that
an unconjugated blocking CD123 antibody can effectivity
reduce leukemia cell growth in mice [188] and even in a few
patients with AML [189]. So far, only a few studies have
attempted to demonstrate functional significance of target
expression in leukemic stem cells. In one study, Mylotarg was
found to induce apoptosis in CD34+/CD38−/CD33+ stem
cells in AML [65] (Fig. 1). A novel and promising marker
for stem cell targeting in AML may be CLL-1 that is able
to discriminate between normal and leukemic stem cells in
the bone marrow [86]. Another promising marker is CD96
since Hosen et al. showed that CD96 is specific for AML
stem cells. He showed that CD96+ AML cells can engraft
irradiated Rag2(−/−) gamma(c)(−/−) mice [85]. However,
so far no targeted drugs specific for CD96 or CLL-1 have
been developed. Several different CD antigens are employed
as targets of therapy in B-cell Non-Hodgkin’s lymphomas,
including CD20, CD22, CD23, CD25, or CD52. Clinical tri-
als have shown that antibodies directed against some of these
antigens (especially CD20 and CD52) can improve treat-
ment and prognosis in these patients. It is therefore tempting
to speculate that some of these antigens are also expressed
on immature lymphoma-initiating tumor cells. Studies are
ongoing to define the CD antigen profile in lymphoid stem
cells in various NHLs. An interesting aspect is that some
of the lymphoid markers, like CD20, are also expressed on
non-hematopoietic CSC. In solid tumors, cell surface targets
include members of the ErbB receptor family, IGF receptors,
TGF␤ receptors, and FGF receptors [190–193]. A general
problem with surface targets is that CSC subclones often
exhibit or develop resistance against antibody-based or other
drugs. Several different mechanisms of drug resistance have
been discussed, including expression of multi-drug resistance
gene products like MDR1 or reduced antibody-binding. In
addition, subclones that do not express the surface CD anti-
gen may be selected by antibody-based therapy. Therefore,
antibody-therapy is usually combined with conventional or
with other targeted drugs.
Apart from surface markers, also intracellular targets
have been identified in CSC. Among those, signal trans-

Fig. 3. Expression of molecular targets in and on neoplastic cells and their progenitors. Whereas much is known about target expression profiles in more mature
neoplastic cells (left part of panel), little is known about expression of molecular targets in leukemic (neoplastic) stem cells (right panel part “?”). ST, signal
transduction; TK, tyrosine kinase(s); TF, transcription factor(s); TSG, tumor suppressor gene(s); DR, death receptors.

Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
ONCH-1372; No. of Pages 20
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14 A. Schulenburg et al. / Critical Reviews in Oncology/Hematology xxx (2010) xxx–xxx
duction molecules and survival molecules may represent
most promising target antigens. These drugs include tyro-
sine kinase inhibitors, farnesyl transferase inhibitors, and
other kinase inhibitors including PI3 kinase and mTOR
blockers [194–196]. In some myeloid neoplasms like CML,
BCR/ABL tyrosine kinase inhibitors have been used with
considerable success [197]. More recently tyrosine kinase
inhibitors have also been employed in clinical trials in solid
tumors and melanomas [98,196,198–209]. Among survival-
related targets, most promising molecules may be members
of the BCL-2 family (BCL-2, MCL-1, BCL-xL, others) and
various Heat shock proteins (HSP32, HSP70, HSP90, others)
[210–212]. Studies are ongoing to define whether these tar-
gets are expressed in CSC in various hematopoietic or solid
tumors. Likewise, it has been described that the HSP32, also
known as heme oxygenase 1, is expressed in CSC in various
leukemias and solid tumor models [213,214].
Despite intensive research and some progress in targeted
therapy in solid tumors, only a few targeted drugs have pro-
duced convincing results in clinical trials, which may be due
to resistance and the complexity of the signalling cascades
in cancer cells and CSC. This is mainly due to (the various
forms of) resistance as well as CSC plasticity with target-
negative subclone formation. Therefore, it seems reasonable
to combine targeted drugs (antibodies, small molecules) with
each other or with conventional therapy (chemotherapy) in
order to overcome drug resistance in CSC.
Finally, provided that specific antigens of CSC are iden-
tified, an attractive approach would be to induce specific
immune responses and to establish vaccination therapy
approaches in these malignancies.
13. Concluding remarks and future directions
The emerging concept of neoplastic stem cells and CSC-
related targets may offer new insights into the biology and the
pathogenesis of various malignant disorders and new possi-
bilities for the design of targeted drug therapies. Since CSC
display considerable heterogeneity and plasticity, eradication
of these cells and thus cure may only be reached when combi-
nations of anticancer drugs (therapies) are applied. Therefore,
treatment designs aiming at cancer/leukemia stem cell elim-
ination need to take all potential targets and all relevant stem
cell subclones into account. There is hope for the future
that such novel treatment approaches will improve therapy
in cancer patients.
Conflict of interest
All the authors declare that they have no proprietary, finan-
cial, professional or other personal interest of any nature or
kind in any product, service and/or company that could be
construed as influencing the position presented in the review
entitled “Neoplastic stem cells: Current concepts and clinical
perspectives”.
Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001
Reviewers
Dr. Dominique Bonnet, Cancer Research UK, London
Research Institute, Haematopoietic Stem Cell Laboratory, 44
Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom.
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19
Biographies
Axel Schulenburg graduated at and is now working at the
Medical University of Vienna, Bone Marrow Transplantation
Unit. His research focus is Cancer stem cells.
Kira Brämswig studied medicine in Würzburg and Vienna
and has M.D. and Ph.D. degrees. She is now working
at the Oncology Department of the Medical University
of Vienna. Scientifically she is focused on tumorangio-
genesis.
Harald Herrmann obtained his M.D. in 2008 and is now
a Ph.D. student at the Medical University of Vienna. He is
enrolled in the Ph.D.-program malignant diseases and focuses
his research on stem cells in myeloid leukemias.
Heidrun Karlic studied biology and biochemistry at
the University of Vienna with a Ph.D. graduation and
is since July 1987 senior scientist at the Ludwig Boltz-
mann Institute for Leukemia Research and Hematology,
Hanusch Hospital, Vienna Austria. Her research focus is
epigenetics.
Irina Mirkina studied molecular biology at the Russian
Academy of Sciences (RAS), Moscow, Russia from 1996
to 1999 with a Ph.D. She is now working on melanoma
stem cells as a postdoctoral scientist in the laboratory of the
Medical University of Vienna.
Rainer Hubmann studied molecular biology at the Uni-
versity of Vienna and holds a Ph.D. He is now a postdoc
at the Medical University of Vienna and is working on the
regulation and function of NOTCH2 in B-CLL.
Sylvia Laffer studied biology at the University of Vienna
from 1986 to 1992 and has a Ph.D. She is now working on
cancer stem cells for the Ludwig Boltzmann Cluster Oncol-
ogy.
Brigitte Marian studied pharmacy from 1972 to 1977 at
the University of Vienna and has a M.Sc. and a Ph.D. in
science. She was a research fellow at the Memorial Sloan
Kettering Cancer Center, New York, USA and is now an
Asc. Prof. at the Institute of Cancer Research at the Med-
ical University Vienna. Her research is focused on Colon
Cancer.
Medhat Shehata is a postdoctoral scientist at the Medical
University of Vienna and is involved in research of Chronic
lymphocytic leukemia.
Clemens Krepler obtained his M.D. in 2000 and is now
a specialist degree in dermatology and venereology at the
Medical University of Vienna. He is enrolled in the Ph.D.-
program malignant diseases and focuses his research on stem
cells in melanoma.
Hubert Pehamberger is full professor and head of the Der-
matology Department of the Medical University of Vienna.
His research focus is melanoma.
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20 A. Schulenburg et al. / Critical Reviews in Oncology/Hematology xxx (2010) xxx–xxx

Thomas Grunt studied biology from 1976 to 1982 at
the University of Salzburg. He has Ph.D. and master
degrees. He is now professor at the Oncology Laboratory in
Vienna.
Ulrich Jäger is full professor and head of the Hematology
Department of the Medical University of Vienna. His research
interests are lymphatic malignancies.
Christoph Zielinski is full professor and head of the Oncol-
ogy Department at the Medical University of Vienna.
Peter Valent is Asc. Professor for experimental hematol-
ogy at the Medical University of Vienna. He is working in
the research of stem cells in hematologic malignancies and
mast cells.

Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev
Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001