Glioblastoma Stem-Like Cells Approaches For Isolation and Characterization

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Journal of Cancer Stem Cell Research (2014), 2:e1007
2014 Creative Commons. All rights reserved ISSN 2329-5872

DOI: 10.14343/JCSCR.2014.2e1007
http://cancerstemcellsresearch.com
Review Article
Open Access
Glioblastoma stem-like cells: approaches for isolation and characterization

Erika S. Molina1, Micheli M. Pillat1, Vivaldo Moura-Neto2, Tamara T. Lah3, and Henning Ulrich1,
Departmento de Bioqumica, Instituto de Qumica, Universidade de S~

ao Paulo, S~
ao Paulo, Brazil, 2Instituto
3
Estadual do C
erebro Paulo Niemeyer, Rio de Janeiro, Brazi, Department of Genetic Toxicology and Cancer
Biology, National Institute of Biology, Ljubljana, Slovenia and Faculty of Chemistry and Chemical
Engineering, University of Ljubljana, Slovenia
CONTENTS
1. Introduction
1.1. Brain tumor glioblastoma multiforme (GBM)
1.2. Cancer stem cell (CSC) paradigm
1.3. GBM stem-like cells (GSCs)
2. Isolation methodologies of GSCs
2.1. SP and ABC transporters
2.2. Isolation based on biomarkers
2.2.1. CD133
2.2.2. ALDH
2.2.3. Aptamers
3. Enrichment of GSC cultures
3.1. Effects of medium and substrates
3.2. Effects of hypoxia
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7
3.3. Effects of endothelial cells

3.4. Effects of pericyte cells
3.5. Effects of immunosuppressed animals
4. Validation of the GSC phenotype
4.1. Sphere-formation assay
4.2. Tumor formation in vivo
4.3. Expression of biomarkers
4.4. Chemoresistance assay
4.5. Differentiation assay
5. Conclusions
Acknowledgments
References
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9
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Abstract: Glioblastoma (GBM) stem-like cells (GSCs) represent the most undifferentiated state of malignant cells with distinct
biological characteristics. The fraction of these cells within a glial brain tumor, ranging from 230%, is correlating with the
increasing WHO stage and poor prognosis of patients' survival. GSCs represent the least vulnerable, thus most preferential target
cell population to be exposed to various therapeutic modalities, although the underlying mechanisms of this resistance are not yet
fully understood. For the development of GSC-targeting therapies, further in depth studies are needed using enriched and stable
GSC cell populations. Here, we discuss the current approaches of GSC isolation and validation based on expression of stemness
and oncogenic markers as well as on functional assays. The enrichment of GSC phenotypes in established cell lines and/or
primary tumor cultures, achieved by different strategies, is reviewed, providing a comprehensive comparison of selected studies
and contemplating the characterization of the plethora of variants of reported GBM population exhibiting the GSC phenotype.
Keywords: Brain tumors, Glioblastoma multiforme, Glioblastoma stem-like cells, Cancer biomarkers, Tumor microenvironment.
ABBREVIATIONS:
Aldehyde dehydrogenase (ALDH)
Brain tumor initiating cells (BTIC)
Cancer stem cell (CSC)
Epidermal growth factor (EGF)
Epithelial to mesenchymal transition (EMT)
Fibroblast growth factor (FGF)
Glioblastoma multiforme (GBM)

Glioblastoma stem-like cell (GSC)
Iso-dehydrogenase 1 (IDH1)
Neural progenitor cell (NPC)
Neural stem cell (NSC)
O-6-methylguanine-DNA-methyltransferase (MGMT)
Temozolomide (TMZ)

Corresponding author: Tel.: C55 11 30918512 E-mail address:
henning@iq.usp.br Departamento de Bioqumica, Instituto de
Qumica, Universidade de S~ao Paulo Av. Prof. Lineu Prestes 748,
S~ao Paulo, 05508-000 SP, Brazil Tel.: C55 11 30919181
Received: September 8, 2014; Revised: October 21, 2014;
Accepted: October 22, 2014
1. INTRODUCTION
1.1 Brain tumor glioblastoma multiforme (GBM)
The most common primary brain tumors are derived from
genetic anomalies in glial development in the brain, comprising of astrocytoma and oligodendroglioma [1, 2].
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These are graded on a scale with increasing malignancy as

lower grade astrocytoma (WHO I and II), anaplastic astrocytoma (WHO III) and glioblastoma multiforme (GBM;
WHO IV), also termed glioblastoma (GBM). GBM is the
most aggressive of these tumors and ranks among the most
desperate of all human cancers. Survival of GBM patients,
although individually variable, has improved from an
average of 10 to only 14 months after diagnosis in the last
5 years, in spite of significant improvements in the standard
care with more targeted therapy (reviewed in [35]). In
addition to several clinical and histopathological parameters, as well as specific therapeutic approaches, patients
responses to therapy are heterogeneous. This is related to
insufficient data for prognostic and survival stratification.
More information about molecular fingerprints of tumors
as well as to better understanding of cellular origin of the
tumor-initiating cells will help to develop individualized
therapies for patients.
The classification of malignant gliomas is changing
rapidly, and novel schemes are being developed based on
their genetic landscapes [6] from next generation sequencing [7, 8], followed by epigenetics, proteomics and oncogenic miRNA data [9]. These are superimposed on the
histological origin of brain tumor-initiating cells (BTIC)
and their precursors evolving into glioma stem-like cells,
also termed glioma stem cells (GSC). Thus, in about 90%
of cases, GBMs arise de novo (primary GBM) without any
evidence of less malignant precursor lesions. Secondary
GBMs progress from low-grade diffuse astrocytoma or
anaplastic astrocytoma [2], occurring in younger patients
and having a lesser degree of necrosis and significantly
better prognosis. Although primary and secondary GBMs
are nearly indistinguishable in their histology, they differ
in their genetic and epigenetic profiles providing the stateof-the-art GMB classification. Decisive genetic markers
for secondary GBMs are iso-dehydrogenase 1 (IDH1)
mutations, which are absent in primary GBMs and associated with a hypermethylation (particularly of histones)
phenotype. IDH1 mutations are the earliest detectable
genetic alterations in precursors of low-grade diffuse
astrocytomas and in oligodendrogliomas. The former
acquire further TP53 and ATRX mutations, whereas the
latter lose the 1p/19q arm and are mutated in CIC and
FUBP1 genes. In contrast, primary GBM glial progenitor
cells overexpress epidermal growth factor (EGF) receptors and exhibit TP53 and PTEN mutations as wells as
LOH 10p and LOH 10q deviations. Therefore, recent
classification of glioma is based on the tumor metabolome
and epigenetic changes. As an example, methylation of the
O-6-methylguanine-DNA-methyltransferase (MGMT)
promoter silences expression of this gene by 40% in the
primary tumor, and more than 70% in the secondary GBM
[7]. As this enzyme acts by de-methylating alkyl groups,
epigenetic silencing of its expression increases GBM
sensitivity to treatment by DNA-alkylating agents, such
as commonly used temozolomide (TMZ) [10].
J Cancer Stem Cell Res http://cancerstemcellsresearch.com
E. Molina et al.
GBMs are histologically similar and featured by atypical mitotic nuclei often associated with necrosis and/or
angiogenesis. The standard treatment against GBM
includes surgery, radiotherapy and, in the last 5 years,
chemotherapy with TMZ [11]. However, since a population of the invasive cells is dispersed through surrounding
normal tissue, and GBMs reveal resistance against radioand chemotherapy, the effectiveness of the multimodal
treatment is limited, resulting in GBM reoccurrence.
1.2 Cancer stem cell (CSC) paradigm
The observed diversity of molecular fingerprinting might
originate from the genetically different glioma initiating
cells [3] also called cancer stem-like cells, here termed
glioblastoma stem cells (GSCs). Similar cells have been
found in many, although not in all human tumors. These
"roots of cancer" operate in a hierarchical fashion such as
conceptualized for normal stem cells [12]. The authors of
this work reviewed the evolution of the cancer stem cell
(CSC) model, which is going back to the 19th century to
Virchows embryonic-rest hypothesis, where cancer is a
consequence of the activation of dormant stem-like cells
in adult tissues. The basic understanding of the current
cancer stem cell theory is that heterogeneity within the
tumors is not a mere consequence of random mutations and
selection, leading to well recognized clonal evolution of
cancer [13], but results from an intrinsic hierarchy of tumor
cells [1416]. CSCs have unlimited capability to divide
symmetrically and asymmetrically. Asymmetric cell division occurs for originating progenitor cells together with a
reduction in the differentiation potential. The theory of a
hierarchical model of tumorigenesis has been widely
accepted, thus stating that only a small fraction of tumor
cells - the CSCs, are capable of initiating tumor growth or
renewing the tumor in the same or other organ after
incomplete surgical removal [17, 18]. When injected
orthotopically, CSCs are capable of forming tumors in
animal. Likewise, CSCs can directly or indirectly contribute to generation of metastasis [19]. However, the existence
and identification of these cells remains an open question,
as all the existing in vitro and in vivo using animal models
in fact represent artificial environments. Consequently it
cannot be ruled that the so named CSCs are just cells which
have adapted to altered growth conditions.
The above-described CSC model has a "static" hierarchical structure, largely based on the notion that normal
stem cells undergo oncogenic transformation. However, in
the past few years a number of publications have challenged this concept by demonstrating that in the tumor
environment progenitors, differentiated cells and even
transformed tumor cells may acquire the ability of selfrenewal through their de-differentiation, e.g. a reversal of
differentiation [20, 21], reviewed in [22], or by other
mechanisms, discussed in the following [23]. Moreover,
the finding that epithelial to mesenchymal transition
(EMT), which is a reversible process and can transiently
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Glioblastoma stem-like cells: Approaches for isolation and characterization
induce stem-like cells phenotypes, was crucial to expand

static the CSC model to the fluid CSC model [12], also
termed the CSC plasticity model. This model proposes
CSC plasticity, meaning that by dedifferentiation of a
variety of cell types within growing tumor mass even
various populations of CSCs may evolve by acquiring
additional mutations or epigenetic modifications. Several
authors propose that various CSC subclones can exist as
the result of evolutionary pressure of therapies, giving rise
to more aggressive secondary CSCs [24]. Taken together,
the clonal evolution and CSC hierarchical theory merge
into a so called fluid plasticity or convergence CSC model.
This results in several subclones of CSC within the same
tumors, depending on epigenetic and mutational events in
specific cells with self-renewal capabilities. These secondary aggressive CSCs may become dominant and drive
tumor formation or simply co-exists with other phenotypes
of CSCs. Such concepts represent challenges in order to
develop novel therapeutic approaches that would more
efficiently target the cancerous clonal population with
stem cell-like characteristics.
1.3 GBM stem-like cells (GSCs)
Recent research has revealed that GBM stem-like cells
play important roles in GBM pathogenesis. The first
evidence that GBM display a cellular hierarchy with
self-renewing and tumor-initiating cell types located at
the apex was provided by Singh et al [25, 26] and Galli et al
[27] followed by further relevant studies on this topic
(reviewed in [2830]. The origin of GBM stem cell has
been widely discussed and is now based on the fact that
genome analyses revealed the transcriptional fingerprints
of neural stem cells (NSC), their progenitors, as well as of
mesenchymal cells [31, 32]. Tumor initiating cell based
classification comprises four subtypes, such as classical,
mesenchymal, neural and proneural transcription profiles,
as summarized by Van et al. [3]. These GBM stem-like
cells (GSCs) self-renew, their hijacking molecular
mechanisms and expressing markers of NSCs [7, 33].
Over the past decade, a wide range of studies have shown
that several signaling proteins involved in neural development also play important roles in GBM pathogenesis, as
highlighted and discussed in the context of developing
treatments for GBM [34]. GSCs express the intermediate
filament protein nestin, the transcription factors Sox2 and
Oct 3/4, the RNA-binding protein musahi, transcriptional
repressor Bmi1 of the polycomb of proteins, proteins of the
Notch signaling pathway receptor, aldehyde dehydrogenase 1 (ALDH1) and among a few others also the extensively studied transmembrane protein, prominin-1
(CD133) (reviewed in [35, 36]). It has been suggested
that the resistance of GBMs towards radiation and chemotherapy is attributed to the GSC-like population
[28, 37, 38]. Hence, GSCs are proposed to persist as a
distinct population involved in tumor relapse and propagation and significantly impact the poor prognostics of the
GBM patients. Accordingly, GSCs have been pursued as a

promising target for the development of novel specific
diagnosis/prognosis therapies against GBM [39].
Although GSCs are increasingly studied for molecular
mechanisms, immunophenotyping or targeting purposes,
the GSC markers, functional assays and the culture conditions remain controversial challenging further investigations [40]. Different methods of GSC enrichment have
been proposed, either from GBM cell lines and/or primary
cultures [41], resulting in GSCs with rather different
phenotypes. Thus, the enrichment of GSCs by itself may
represent an artifact and contribute to misidentification of
these cells and to the discrepancies in their identification
and characterization.
2. ISOLATION METHODOLOGIES OF GSCs
Flow cytometry and fluorescence-activated cell sorting
(FACS) provide the opportunity to analyze various parameters in a single live cell as well as separating heterogeneous cell populations based on these characteristics.
Besides differential epitope expression, morphological
properties, such as cell size and granularity measured as
forward and side scatter parameters are assessed. Similar
to the identification of embryonic and adult stem cells,
measurements of specific clusters of cell surface markers
and enzymes responsible for metabolic activities are the
methods of choice in flow cytometric identification of
tumor stem cells, as illustrated in Figure 1 and detailed
below. However, the reliability of these tumor cell markers remains questionable. For instance, tumor cells in
general are known for genetic instability, and consequently proteome profiles of GSC biomarkers may be difficult to
analyze. Furthermore, epigenetic changes, induced by
tumor microenvironment, in particular when created in
the so called stem cell niches, induce expression of multipotency genes in GSCs [20, 23, 42]. It is worthwhile
mentioning that phenotypes may change under in vitro
culture conditions (reviewed in [43]).
As discussed above, GSCs are highly plastic. Consequently similar clones with different biomarker fingerprints without similar characteristics may exist even in the
same tumor tissue. Nevertheless, the strategies for CSC
identification, isolation and targeting discussed below,
have led to our current knowledge and novel strategies
of glioblastoma. To date, multi-parameter flow cytometry
has been developed for detection of a combination of
characteristics, which define a rare population, such as
cancer stem cells or circulating tumor cells in the blood
[44, 45]. Such rare populations can be identified in a single
experiment, following staining for side populations
(Hoechst dye exclusion by ABC transporters), ALDH1
in the presence of a substrate, which becomes fluorescent
following enzymatic cleavage, and CD markers, i.e.
CD133, and exclusion of dead cells (i.e. stained by 7aminoactinomycin-D, 7-AAD), such as proposed by
Greve and co-workers [43]. Optimization of fluorescence
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E. Molina et al.
Figure 1. GSC represent a promising target for treating GBM. Classical GSC enrichment consists of serial transplantation in vivo and/or
culture with defined medium supplemented with EGF and FGF-2, which might be improved by mimicking the micro-environment niche of GSC
through hypoxia and/or co-cultivation with endothelial cells. GSC isolation by FACS has been based on differential expression of CD133
followed by ALDH (Aldefluor) as more contemplating functional assessment of GSC phenotype. Additionally, aptamers have turned into
promising tools for targeting biomarkers allowing identification of specific binders against GSC populations.
probes as well as more knowledge on selection of molecular targets by screening for expression profiles will
improve the identification and separation of live cells.
Some of the pluripotency-coding genes, supposedly also
expressed by tumor stem cells, are intracellular antigens,
which cannot be targeted in live cells by flow cytometry.
Therefore, molecular beacons were developed for flow
cytometry detection of gene expression of these genes on
the mRNA level [46]. Applications of in vivo flow cytoJ Cancer Stem Cell Res http://cancerstemcellsresearch.com
metry and advanced imaging together with an automated

method for identifying and tracking rare cell events are
available for detection of circulating tumor cells in the
blood [47]. Galanzha and Zharov developed a highly sensitive method, by which the entire blood volume (5 L) of a
patient can be analyzed in vivo by photoacoustic flow
cytometry [48]. Further perspectives here are switching
from the identification of entire cells to the detection of
circulating cancer stem cells and associated microvesicles.
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2.1 SP and ABC transporters

GSCs can be also identified within side population (SP)
assays using flow cytometry. This SP cell population is
obtained by the efflux of Hoechst fluorescent dyes revealing stem cell-like characteristics. This dye-efflux, presumably due to the exclusion of the dye triggered by an ATP
binding cassette (ABC) transporter, was also identified in
cells from several tumors exhibiting stem-like genes,
tumorigenicity and multi-drug resistance [49, 50]. In the
C6 glioma cell line, SP cells are described as enriched in
stem-like cells [51]. To date, the SP is employed as gating
criteria for GSC enrichment by flow cytometry with
following demonstration of self-renewal, multi-lineage
differentiation and tumorigenicity [52] or decreased
migration [53]. However, as a drawback to GSC enrichment and purification based on Hoechst exclusion, recent
evidences indicate that, in fact, the GBM SP cells do not
contribute to self-renewal or tumor initiation and thus is
not enriched in GSC and not tumorigenic [54] but consists
of brain endothelial cells [55]. In fact, recent evidence
shows that the three present ABC transporters ABCA1,
MRP4 and MRP5 are differentiation instead of stemness
markers [56]. Taken together, SP flow cytometry is not
recommended as GSC enrichment methodology.
2.2 Isolation based on biomarkers
2.2.1 CD133
The surface marker prominin-1 CD133 is widely used as
phenotypic markers for enrichment of CSCs, including
GSCs, although it is also expressed by hematopoietic,
endothelial and neural progenitors cells [5758] showing
that in the GBM SC population, membrane bouding
expression of CD133 was restricted to tumor cells bearing
the extracellular CD133 epitope AC133 [59]. Furthermore, CD133 may also be expressed by differentiated
tumor cells with changed conformation as result of differential glycosylation, thus masking the epitope recognized by the anti-AC1333 antibody [59, 60]. CD133
expression is discussed controversially, regarding its use
as criterion for GSC enrichment, since GBM tumorigenesis is not only driven by CD133-positive cells. Indeed,
some CD133-negative cells comprise the most undifferentiated and aggressive cells placed in the apex of the
hierarchy [61]. In view of that, the polycomb protein
EZH2 has been indicated to be a more specific marker
for CSCs than CD133 [62]. The enrichment of CSCs or
GSCs based on EZH2 expression remains to be proven.
2.2.2 ALDH
Aldehyde dehydrogenases (ALDH) are a family of cytosolic NAD(P)C-dependent enzymes that catalyze the oxidation of a wide spectrum of endogenous and exogenous
aliphatic and aromatic aldehyde substrates to their corresponding carboxylic acids [63]. High expression of isoenzyme ALDH-1 was for long correlated with resistance to
highly reactive alkylating agent based therapies, such as
derivatives of cyclophosphamide. The enzyme detoxifies

alkylating agents by converting them into less reactive
molecules and favoring survival of leukemia tumor [64]
and GBM cell populations [65, 66]. ALDH-1 has been
proposed to also mediate the resistance of GBM against the
current standard chemotherapy compound TMZ [67]. Consequently, ALDH inhibition enhances cytotoxicity of alkylating agents for GSCs [68]. ALDH-2 and ALDH-3 prevent
cytotoxicity such as ALDH-1 [65]. However, only ALDH1A1 is involved in the metabolic oxidation of retinal, also
known as retinaldehyde or vitamin A aldehyde, to the active
metabolite retinoic acid (RA) [69]. Retinal promotes differentiation of GSCs, although it is not sufficient to antagonize differentiation inhibition by growth factors such as
EGF and fibroblast growth factor (FGF)-2 [70]. ALDH-1
has thereby been proposed as a CSC and GSC marker [71
73]. Interesting, hypoxia induces or up regulates ALDH-1
expression in established and primary GBM cell lines [74].
Controversially, ALDH-1A1expression was also considered a marker of astrocytic differentiation in brain tumors
correlating with longer survival of GBM patients [75].
Membrane-permeable fluorescent substrates for ALDH,
such as i.e. dansyl aminoacetaldehyde (DAAA) allow
identification and isolation of ALDH positive cells by flow
cytometry [76]. However, the DAAA-based method does
not only require further fractionation steps to yield highly
purified stem cells and dye excitation with UV light could
be mutagenic to the purified cells. A better alternative is the
stable fluorochrome Bopidy aminoacetaldehyde diethylacetal (BAAA-DA), which when converted into the fluorescent ALDH substrate (BAAA) is excited by visible
light, yielding brighter emission than other fluorochromes,
which could be detected using green fluorescence (FL-1)
channel of a standard flow cytometer [77]. Hence, Aldefluor assays can be used in combination with other fluorescent labels such as R-phycoerythrin (PE), PE-tandem
conjugates, PerCP, Cyanine-5 (Cy-5) and allophycocyanin
(APC), which are detected in the FL-2, FL-3 or FL-4
channels. Aldefluor assays employ the ALDH inhibitor
diethylamino-benzaldehyde (DEAB) to set up the fluorescent background of negative control populations [78]. In
addition, due to the highly expression of the multiple drug
resistance (MDR) pump by stem-like cells [79]. Aldefluor
assays also contain a MDR inhibitor to avoid the efflux of
the fluorescent product better resolving ALDH positive
from negative discrete cell populations [78, 80]. Remarkably, all refinement provided by Aldefluor assays are
improving ALDH-activity studies by fluorescence-activated cell sorting (FACS), supporting them as a tool marker
for isolation of NSCs [81] and GSCs [82, 83] as well as for
other CSCs [84, 85]. ALDH-1A2 and ALDH-2 activities
are also measured by the Aldefluor assay [86].
2.2.3 Aptamers
Despite the ongoing controversial discussion on the diagnostic value of tumor stem cell markers and whether stem
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cells are a stable component of the tumor cell population

and not merely a transient phenotype within a certain tissue
niche, attempts have been made to diagnostically and
therapeutically target the above-discussed marker epitopes
by high-affinity and specificity ligands. In general, cell
surface and transmembrane proteins expressed by various
types of tumor stem cells, including CD44, CD47, CD123,
EpCAM (CD326), CD133 and IGF receptor I, as well as
antibodies interfering with notch-receptor signaling have
been used for identification of monoclonal antibodies of
possible therapeutic value (reviewed by Naujokat, 2014).
Aptamers, evolved from combinatorial DNA or RNA
oligonucleotide libraries by an in vitro selection process
called SELEX, systematic evolution of ligands by exponential enrichment) against a target epitope compete with
antibodies for many applications. Their synthetic nature,
not involving animals in any process, as well as the
easiness of chemical modifications for increasing stability
in biological solutions including plasma have turned them
into promising agents for diagnosis and therapy [88,
89, 90]. There are two alternatives in using aptamers for
identification and isolation of GSCs: (1) Selection of
aptamers against their putative biomarkers using purified
or recombinant expressed proteins. (2) Developing aptamers against GSC-like cells by Cell SELEX. This recently
developed technique uses intact live cells expressing the
target epitope(s) and exposes them to the combinatorial
library for aptamer selection (reviewed in [91]). The
differences between the epitopes expression on the cell
surface of a tumor-stem like cells, vs. an untransformed or
non-stem cell of the same histologic origin can be
exploited for obtaining tumor stem cell-specific aptamers.
Aptamers were created with success as specific binders
of GBM populations. First, aptamers following reiterative
cycles of Cell-SELEX were identified binding to human
GBM cell lines. Kang and co-workers [92] first used the
procedure to isolate GBM cells, obtaining a set of aptamers not interacting with non-neoplastic astrocytes and
other tumor types. Another approach to identify GBM
cells was to raise aptamers against the EGF variant III
(EGFRvIII), one of the most common mutants in GBM
[93]. These aptamers were used as diagnostic tools, along
with highly specific radionuclide molecular imaging of
GBM in vivo [94]. However, they were developed for
targeting a heterogeneous tumor population and not stem
cells within this cell mixture.
The development of nuclease-resistant 20 -fluoropyrimidine-modified RNA aptamers, which recognize CD133 and
AC133 epitopes with high specificity [95] is a significant
progress towards targeting CSCs. DY647-fluorescence
labelled anti-CD133 aptamers were verified regarding their
diagnostic potential in imaging and flow cytometry. Furthermore, these aptamers were effective in penetrating
tumorspheres and internalized by tumor cells, opening a
possibility for loading of the aptamer with a cytotoxic
compound or si-RNA for down-regulation of oncogenes
E. Molina et al.
relevant for cell survival. Along the same line, Kim et al.
[96] enriched CD133-positive cells by immunobead separation from mice GSC xenografts, and validated these for
their tumor-initiating capacity in another animal model.
Then, the authors used CD133-negative cells as well as
non-neoplastic neural progenitor cells as negative control
for a subtraction process for removal of all common
aptamer binders to both cell types. As already shown for
the aptamers selected by Shigdar et al. [95], the aptamers
bound to proliferating and tumor-initiating cells with dissociation constants in the subnanomolar range. The authors
concluded that these aptamers may gain therapeutic importance, if conjugated to tacytotoxin. Indeed, paclitaxelloaded nanoparticles, reducing GBM proliferation [97],
have been shown to cross the blood brain barrier [98]. In
addition, aptamer penetration of brain tumors can be
facilitated by liposomes, such as those used as vehicles
for doxorubicin-loaded aptamers into brain tumors in
animal models [99, 100].
3. ENRICHMENT OF GSC CULTURES
3.1 Effects of medium and substrates
Since the first experimental studies supporting in vitro selfrenewal and capacity of GSCs to differentiate into neuronal
and glial cells, GSC enrichment has been essentially
performed in vitro using defined medium. Originally,
GSCs were identified by their capacity to form three
dimension cellular clusters, termed tumorspheres, similar
to neurosphere cultures of NSCs and NPCs [101, 102].
Spheres culture is considered to keep the undifferentiated
state of the cells by minimizing stimulation from the
environment, such as adhesion to substrates, but also
avoiding the access of the cells in the core of the sphere
to differentiation factors [103]. Classically, GSCs arive in
adherent cultures with defined medium following 20 to 40
days after plating [25, 27, 104, 105]. Based on this, tumorspheres formation has been obtained faster through anchorage-independent culture, such as in dishes previously
treated with poly-Hema (poly2-hydroxyethyl methacrylate) [105, 106], gelatin [83] and agar [107]. In fact,
tumorigenicity correlates with suspension growth [108].
In addition, three-dimensional culturing models are also
available, including gelatin foam cultures, for the study of
GSCs [109]. However, special care should be taken regarding suspension cultures [110], since poly-Hema derived
spheres might not be truly three dimensional clone derived,
but rather reflect cell aggregates. In fact, the formation of
spheres as predicative property of CSC enrichment has
been questioned, as no correlation was found with the
expression of GSC markers [111]. Indeed, the technique
of tumorsphere cultivation presents some limitations, such
as differentiation and cell death, occurring in the sphere
environment [112]. Alternatively, GSC enrichment has
also been performed using adhesive cultures, which would
provide uniform access to growth factors, thus avoiding
differentiation [113, 114]. Phenotypic differences are
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15:21:44
observed within adhesive surfaces [115], such as in dishes

previously treated with matrigel [116], laminin [113], collagen [117, 118] or extracellular matrix [82]. On the other
hand, organotypic cultures in the presence of ECM and
vascular elements are also available, especially for migratory assays, as this model represents a more accurate brain
matrix micro-environment, in which cells migrate [119].
GSC enrichment performed in adhesive as well as nonadhesive cultures supports a major role for medium composition in this process [113, 118]. GSC culture medium,
similar to that for NSCs, is supplemented with 20 ng/ml of
EGF, 20 ng/ml of FGF-2 and sometimes with the 5 ng/ml
of leukemia inhibitory factor (LIF). The mitogenes EGF
and FGF are for long known for inducing renewal and
expansion and notably increasing the frequency of spheres
formed by undifferentiated cells [120, 121]. On the other
hand, LIF has also been shown to enrich GSCs based on the
transforming growth factor-beta (TGF-b)-mediated promotion of symmetric expansions inducing self-renewal
and preventing differentiation [122], also increasing the
expression of core stemness transcription factors and thus
the number of NPCs [123]. Furthermore, medium supplemented with B27 and/or N2 enhances proliferation of
multi-potent NPCs [124].
Interestingly, the basis behind the use of the defined
medium is that self-renewal and multipotency of stem-like
cells would be induced and sustained with a supplement
medium switch from serum to EGF and FGF [125, 126].
EGF induces proliferation of adult mouse brain NSCs,
generating spheres responsive to FGF-2. Then, upon stimulation by FGF-2, undifferentiated cells proliferate undergoing symmetric or asymmetric divisions and give rise to
either unipotent (neuronal) or bipotent (neuronal/astroglial)
progenitors [127]. The EGF-responsive mammalian
embryonic neural precursor is a stem cell [121]. Later,
other evidences supported that FGF-2 responsiveness was
sufficient to isolate progenitors found in the adult mammalian spinal cord [128] and that NSCs responsive to FGF-2
arise earliest in embryonic development [129]. GSC marker
expression was increased upon cultivation stimulation with
FGF-2 compared to EGF [130]. In fact, blockade of EGF
receptors reduced sphere growth, even in the absence of
EGF in the culture medium. Both EGF and FGF enhanced
GSC proliferation and sphere size. However, the absence of
both mitogens in GBM culture was already demonstrated to
originate multipotent spheres that were capable of selfrenewal and also forming highly invasive tumors [131].
3.2 Effects of hypoxia
Lowering oxygen concentrations promotes GSC enrichment. Although the standard normoxic condition uses 20%
of oxygen, this oxygen level is considered as hyperoxic
compared to physiological brain oxygenation. In fact,
hypoxia is the physiological environment of adult brain
tissue, ranging from 1% to 5% of oxygen [132, 133]. Solid
tumors including GBM are less oxygenated, ranging from
physiological levels to below 0.1% in necrotic areas [134,

135]. Hypoxia regulates gene expression of hypoxiainducible factor 1 (HIF-1), a transcription factor composed
by an oxygen-regulated HIF-1a and a constitutive HIF-1b
subunit. HIF-1a has a half-life of few minutes and destabilizes in increasing oxygen levels by the action of proline
hydroxylase which utilize O2 in the range of 0.1 to 21% as
a cofactor [136]. HIF post-translational modified within
several domains interacts with the von Hippel-Lindau
(pVHL) followed cytoplasmic translocation and ubiquinin-mediated for proteasome degradation [137, 138]. On
the other hand, HIF-1a stabilized in hypoxic conditions
translocates to the nucleus interacting with co-activators,
such as cAMP, and binding to the hypoxia response
element located (HRE) in the promoter or the enhancer
regions of target genes [139]. To date, three HIF isoforms
have been described, including HIF-2a and HIF-3a.
HIF-1a and HIF-2a expression was increased in GSCs
exposed to hypoxic conditions together with other stem cell
markers such as CD133, Bmi-1 and nestin [140]. Whereas
HIF-1a is ubiquitously expressed, including by all hypoxic
tumor cells, HIF-2a participates in cell reprogramming
towards a cancer stem cell phenotype maintaining the GSC.
HIF-1a target genes are regulated in a tissue-specific
fashion. More than 100 downstream genes were identified
as regulators of multiple physiological responses to oxygen
deprivation, in general shifting the metabolic balance from
oxidative phosphorylation toward glycolysis. HIF-1a is
required for the proliferation, survival and angiogenesis
[126]. Overstabilization of HIF-1a supports GBM resistance to TMZ [141]. Moreover, HIF-1a interaction with
the activated Notch intracellular domain attenuates differentiation-promoting effects of bone morphogenetic proteins (BMPs), in particular of BMP-2. In NSCs and NPCs,
increasing O2 levels degrade HIF-1a, thus promoting
differentiation or apoptosis [142]. In GBM, knock down
of HIF-1a at the RNA expression level reduced migration/
invasion phenotypes beyond the ability to form tumorspheres [143]. A proposed mechanism is that HIF-1a
drives the initial response to hypoxia whereas HIF-2 a
drives the chronic response. The switch from HIF-1a- to
HIF-2a-dependent signaling pathways plays divergent,
however complementary roles during the hypoxic response
reprograming GBM cells towards stemness, increasing
aggressive tumor growth and invasiveness [144, 145].
However, HIF-2a stability is unaffected in physiological
oxygen levels. Moreover, evidences support that HIF-2a is
essential only by GSCs expressed and not by NPCs [146].
HIF-2a is overexpressed by GSCs acting as a mediator of
tumor plasticity and tumorigenesis [126, 146]. A underlying mechanism could be that HIF-2a upregulates expression of stem-related genes such as Oct4 [147].
Remarkably, hypoxic microenvironment contributes to
the GSC phenotype and increases the number of stem-like
cells in tumor populations. Low 1% oxygen level enriches
the percentage of CD133-expressing cells [148] without
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affecting N-glycosylation of this epitope [149]. A culture in

hypoxia for 48 hours resulted in a four-times increase of the
number of CD133C cells [150]. Hypoxia-mediated HIF-1a
activation results in a time-dependent increase in CD133
mRNA and protein levels with the peak of protein expression after 48 hours [149, 150]. The enrichment of GSCs
from GBM primary cultures or the cell line U-87 under 1%
oxygen condition results in tumorspheres smaller than the
ones obtained in conditions of 20% oxygen. In agreement,
expression of Ki-67, a marker of proliferation, is reduced in
conditions of 1% oxygen, whereas expression of stemness
markers, such as CD133, podoplanin, Bmi-1 and nestin, is
increased. Sox-2 expression, in particular, is increased only
in tumorspheres from primary cultures [140]. In terms of
enrichment, 1% of oxygen increases the stem-like cells
over fivefold with the percentage of CD133-positive cells
being threefold or more enriched. In the same line, oxygen
levels reduced to 7% enhance the stem-like phenotype of
CD133-positive cells [151]. In addition to promoting
proliferation, hypoxic conditions induced angiogenesis
known to be a prerequisite of tumor expansion. Angiogenesis is favored by the association of GSCs and endothelial
cells in perivascular niches [152]. Regulatory functions of
such niche on GSCs is proposed in analogy to the ones in
the control of NSC proliferation [153].
The effects of hypoxia on in vitro cell culture have
gained scientific interest. Precise control of oxygen levels
is essential for accurately interpreting obtained results. To
date, hypoxic cell culture might be performed by different
strategies. The most widely used incubator is based on a
chamber flushed with 1% O2/5% CO2/94% N2 [154].
However, this chamber suffers frequently from the leakage
of hypoxic conditions. As a main drawback of this
approach, every time the incubator door is opened, a certain
amount of oxygen needs to be consumed for re-establishing
initial conditions. Alternatively, a hypoxic environment
might be created by putting the cell dishes inside a subchamber with oxygen controller, which allows studies
exploring multiple oxygen levels for each chamber to be
placed inside a regular cell culture incubator. However, this
alternative also implies in discontinuous oxygen levels and
difficulties of reproducing exact hypoxic conditions.
For improving experimental conditions, hypoxic glove
boxes emerged allowing cell manipulation without
affecting oxygen levels. However, as the incubation and
manipulation cells in the same humid station implies
risk of contamination of cultures, the glove box station
allowing hypoxic manipulation might be enclosed in a
hypoxic incubator. Since exposure to room air also
occurs, disrupting hypoxic condition during regular
microscopy, a hypoxia microscope chamber should be
added the workstation. In this sense, appropriate modular interconnected closed-hoods accommodating
microscopes, centrifuges, cell sorters, bioreactors are
receiving more attention as these ensure unprecedented
stable and continuous hypoxia, completely isolated from
E. Molina et al.
the outside environment. In parallel, easy to use and

low-cost alternatives have been developed, as, for
instance, inflatable chambers made of transparent plastic
materials allowing real-time studies of hypoxia under
microscopy [155]. Finally, as the oxygen level is typically controlled in the gas phase differs from that
supplied to the cells submerged in the growth medium;
pericellular oxygen level monitoring might be used for
better accuracy. This can be achieved with sensor chips
embedded in a conventional tissue culture flask [156].
3.3 Effects of endothelial cells
Vascularization is a diagnostic hallmark of GBM. At least
five interlinked pathways by which GBM achieves neovascularization, have been described. A step of vascular
co-option between brain and tumor vasculature, is followed by angiogenesis as second step. Vasculogenesis is
the third mechanism sustained by the differentiation of
circulating bone marrow-derived cells (BMDCs), known
as endothelial progenitor cells (EPCs). These cells are
suggested to promote a supportive role and might be
incorporated into tumor vasculature. The fourth mechanism of neovascularization, termed vascular mimicry, is
defined as the ability of tumor cells to form functional
vessel-like networks. The fifth mechanism is based on the
trans differentiation of GBM cells into an endothelial
phenotype [157]. However, tumor cells involved in vascular mimicry are endowed with stemness plasticity,
providing a source for transdifferentiation of GSCs
towards the endothelial phenotype. The presence of
GBM-derived vasculatures highlights the plasticity of
GSCs, although the underlying mechanisms remain to be
elucidated [158, 159].
Hypoxia is involved in all steps of the neovascularization
process. HIF activation has been described to up-regulate
VEGF expression and thus to promote angiogenesis [160],
but also to increase levels of SDF-1, which in turn promote
vasculogenesis recruiting CXCR4 positive BMDCs to the
tumor site. However, unlike in normal vascularization, the
emerging vasculature is often abnormal. In these pathological angiogenesis, the excess of vascular proliferation
together with the lack of structure-giving pericytes contributes to the formation of tortuous and dilated blood
vessels that are poorly organized and hyperpermeable
(reviewed in [161]). Hence, vaso-occlusive and plasma
coagulation impairs the blood supply perpetuating the
hypoxic microenvironment, which in turn regulates HIF
expression and activity thus leading to a vicious circle of
induction of abnormal vasculature growth in GBM [162].
At the same time, hypoxic exosomes secreted by GBM cells
induce endothelial cells to liberate growth factors and
cytokines accelerating tumor growth [163].
In this context, GSCs are maintained within the vascular niche, in which the endothelial cells play an important role to sustain the GSC-like phenotype and GBM
propagation. The endothelial secreted factors, which
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activate self-renewal pathways, including Hedgehog

[164] and the Notch pathway [165, 166], at the same time
sustain mTOR activation, which in turn suppresses autophagy and apoptosis of GSCs [167]. Both, endothelial cells
and GSCs, express nestin and the cell surface antigen
CD133 [168]. Almost 75% of nestinC cells co-express
CD133 while less than 0.1% of nestinC cells coexpress the
endothelial marker CD34 [116]. In addition, GBM SP
cells, characterized by their efflux properties using flow
cytometry were identified as brain endothelial cells [55].
Co-culture of GBM and endothelial cells results in a
GSC population with higher proliferation rates and
subsequently formation of bigger spheres and more
aggressive tumors than observed with GSCs in monoculture [116]. On the other hand, co-culture of GSCs and
endothelial cells also augments number and proliferation of endothelial cells [126]. The majority of GSCenrichment studies employing co-culture conditions
were done with immortalized human brain endothelial
cells (hCMEC) [167, 169], umbilical vein endothelial
cells (HUVEC) [164], microvascular endothelial cells
(HMVEC) [126], primary endothelial cells [116] and
primary bovine endothelial cells [117]. In general, transwell inserts are used to co-culture endothelial and GSCs
ensuring free diffusion of signaling molecules without
direct cell contact [116, 117, 126, 164]. Alternatively,
conditioned media from 72 h endothelial cell culture in
serum-free medium might also be employed [167].
Endothelial cells cultivated in three-dimensional system
enhanced secretion of IL-8, upregulating IL-8 cognate
receptor CXCR1 and CXCR2 activities with the consequence of enhanced GSC migration, growth and stemness properties [169].
3.4 Effects of pericyte cells
Pericytes are perivascular contractile stromal cells that
surround the wall of endothelial cells regulating blood
flow [170]. In GBM, it was demonstrated that pericytes
mediate co-option of modified pre-existing blood vessels
supporting the expansion of the tumor margin [161].
Besides, pericytes present mesenchymal stem cell (MSC)
phenotypes, exhibiting neural multi potential activity
[171, 172], also mediating immunosuppression effects
on GBM. [173]. Interestingly, although GSCs have been
suggested to differentiate into endothelial cells, recent
evidence indicates that GSC preferentially differentiate
into pericytes, attempting to support vasculature function
and tumor growth [174, 175]. Besides, endothelial cells
have been proposed to promote paracrine stimulation of
pericytes, specifically in the context of hypoxia. In this
context, hypoxic exosomes secreted by GBM cells
enhance pericyte vessel coverage and induce endothelial
cells to secrete factors stimulating pericyte PI3K/Akt
pathway activation and migration [163]. Endosialin
(CD248), not expressed by endothelial cells, is a marker
of closely associated pericytes in GBM [176].
3.5 Effects of immunosuppressed animals

One of the initially described characteristics of GSCs is
tumorigenicity in vivo [26]. Considering that GSCs
present a distinct capacity for tumor growth and propagation, these cells have been enriched by serial
passages in immune-compromised animals. The enrichment of human GSCs in vivo is based on tumor xenoengrafting into immunosupressed animals (generally
mice and rats). The congenitally athymic and hairless
nude mouse immunodeficient in functional T cells has
been routinely used in tumorigenesis assays and shown
to be suitable for GSC transplantation due to its
impaired capability of rejecting human xenografts
tumors. On the other hand, the severe combined immunodeficient (SCID) mouse carries a genetic defect preventing functional development of T and B cells due to
dysfunction in the TCR and BCR recombination
machinery. However, the SCID mouse retains normal
numbers of natural killer (NK) cells [177], which might
interfere in the rejection of xenografts, as NK cells
promote selective lysis of particular tumor targets
[178]. An appropriate model for analysis of GSCs has
been created by the inclusion of the non-obese diabetic
background (NOD-SCID) with reduced NK cell function. Properly, injection of anti-NK cell antibodies
before transplantation enhances the engraftment of
human xenografts [179]. Followed by the NOD/
SCID/g cnull (NOG) strain, with the above-noted deficiencies including cytokine production incapability, the
NOG strain has been considered a more appropriate
animal recipients and especially suitable for GSC transplantation [180].
Intracranial GSC grafting has been widely performed
using stereotactic approach, whereas few studies have
explored subcutaneous or intraperitoneal engrafts.
Engrafting cells into a mouse brain is a critical step as
the site of injection determines the location of the tumor
bulk and requires special care for in fact reaching the
brain tissue [181]. Alternatively to stereotactic procedures, an implantable guide screw was developed to
establish intracranial xenografts in mice, allowing a large
number of animals to be engrafted [182]. Later, the guide
screw method was modified incorporating an infusion
pump that allows up to 10 animals to be simultaneously
intracranial injected with tumor cells [181]. In general
the number of intracranial injected cells was 105 cells per
animal, although tumor formation was also observed with
injection of 2 102 to 103 cells after 12 weeks [183].
Injection of 5 103 to 104 cells resulted in tumor
establishment after 8 weeks. Tumor formation in vivo,
a functional method for defining GSCs, is widely used
since for enrichment of these cells. Cells are replicated by
serial passages re-injecting them into secondary mice
[26, 159], which also serve as a validation assay. Xenograft tumor excision later allows further histological and
molecular characterization.
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10
4. VALIDATION OF THE GSC PHENOTYPE

4.1 Sphere-formation assay
Tumor cells have less requirements for proliferation, in
general being less independent from growth factor stimulation and anchorage when compared to normal cells.
However, not every cell within the tumor present the same
independence in order to survive and propagate. On the
other hand, GSCs are considered to be capable of clonogenic proliferation as anchorage independent spheres
under serum withdrawal, as similarly observed for NSCs.
Also similar to NSCs, the frequency of GSCs has been
predominantly assessed by primary spheres assays. The
studies on GSCs usually refer to GBM primary tumors
cultured with defined medium supplemented with EGF
and FGF. Ignatova et al. [106] cultured tumor cells using
non-adhesive dishes previously treated with poly-Hema
and detected after 18 days a yield of GSC frequency from
0.05% to 1.26% (n 10) in the GBM population. Moreover, the authors of this paper reported that the size of
formed tumorspheres is heterogeneous (<50 cells or 50 to
2000 cells), and some tumors do not present no clonogenic
cells at all. In another study published by Galli et al. [27],
tumor cells cultured on adhesive surfaces for 20 to 40 days
yield a GSC frequency of 0.5% to 31% (n 12).
Another work reports that GSCs could by isolated
following only 7 days of culture on adhesive surfaces
with a yield of nearly 20% based on FACS separation of
CD133-positive cells [184]. On the other hand, a subpopulation of 25% of CD133C GSCs isolated by marker
formed spheres after 42 days in culture whereas no sphere
formation was observed in CD133 cells by Beier et al
[104]. The work of Wang et al. (2010) agreed with the
observation that CD133 cells were not capable of forming tumorspheres, while one of CD133C cells actually
contributed to tumorsphere formation. Another study
resulted in the observation that small spheres (50 cells)
were frequent and that 5% of plated cells were able to form
secondary spheres, with around 70% of the sphere-forming cells being CD133C [104]. Qiang et al. [105] confirmed that CD133C cells presented higher colonyformation capacity including more G0/G1 phase cells.
GBM cell lines cultured using non-adhesive surfaces
and presenting no ALDH activity (ALDH) do not yield
tumorspheres whereas GBM cell lines presenting ALDH
activity (ALDHC) exhibit tumor sphere formation capacity [83]. A recent study supports this observation as
ALDHC cells isolated by FACS from primary GBM
cultures on adhesive surfaces, previously treated with
extracellular matrix, yield tumorspheres after only 1 day
whereas ALDH cells did not. Regarding hypoxic conditions, tumor cell culture in 1% of O2 for 5 days increased
almost by three-fold the capacity of the tumor cells to form
spheres [150]. Furthermore, hypoxia affects spheroid
diameters and numbers forming tumorspheres. Sphere
diameters were in average 50 mm in 1% O2 and 100 mm
E. Molina et al.
in 7%. However, hypoxia favored a higher proliferation

rate [140]. On the other hand, conditioned media from
immortalized human brain endothelial cells yield larger
tumorspheres size [169].
Neurosphere assays for quantification of NSCs in vivo
[185] remain an useful and low-cost tools for assessing
GSC populations. However, the capacity to form tumorspheres has been criticized as a prediction for CSC enrichment [111]. Tumorsphere formation is not predictive for
CSC expression markers, at least not for all types of cancer
cells. Pastrana and coworkers critically evaluated the
sphere-formation as an assay for stemness and pointed out
that this assay may not detect quiescent stem cells. In
contrast, the proposed assay detects cells with either
tumorigenic properties in vivo or already dividing ones
[186]. It is noteworthy that the source of multipotencial
NSCs relies on a relatively quiescent cell and not on the
proliferating population [187]. For the determination of
self-renewal capacity, cultures should be plated as single
cells to ensure that tumorsphere formation is only due to
truly clonogenic proliferation instead of cell fusion and/or
aggregation, which might occur even at very low culture
density. On the other hand, as cell survival and proliferation are directly affected by paracrine stimulation, very
low culture density conditions suffer from poor dispersion
of signaling molecules in the medium [186]. In this case, a
good option to improve the sphere-formation assay is to
plate cells with previously conditioned and filtered medium. Additionally, a software specially suitable for comparing populations depleted and enriched in stem cells is
available [188].
4.2 Tumor formation in vivo
GSCs are usually functionally defined by their tumorigenic
property in immunosupressed animals. As self-renewal is a
key feature of stemness, GSC identity relies on serial
transplantation capability. Singh and co-workers showed
that after 5 weeks of reinjection of 1,000 CD133C cells in
NOD-SCID mouse brains (n 3) the generated tumors
recapitulated the phenotype of the original patient primary
tumor whereas CD133 cells took 12 weeks after transplantation to exhibit tumor formation [26]. Of note, the first
sign of neurologic impairment in mice was observed 810
weeks after transplantation of GSCs. Galli and co-workers
observed that GSCs gave rise to tumors that mimic GBM,
which did not occur in the U-87 MG cell line. Moreover,
GSCs presented migratory capacity typical for GBM,
which was also not observed with U-87 MG cells [27]. In
contrast, Pollard et al. [113] demonstrated tumor engraftment using a minimum number of 100 cells, whose isolation was not based on CD133 expression, but on exploring
adhesive culturing. Indeed, Qiang et al. pointed out that
seven weeks of culture on non-adhesive surfaces generated
larger subcutaneous tumor xenografts than compared to
those obtained in cultures on adhesive surfaces [105].
Compared with CD133C cells enriched in the presence of
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primary endothelial cells, xenografts were generated within

7 days of injection of 1,000,000 cells [116]. Co-culture with
endothelial cells increases tumor formation by a factor of
six compared to the monoculture. After 11 weeks, animals
intracranially injected with 100,000 cells formed tumors
with a size of 110 mm3, whereas previous co-cultures with
endothelial cells resulted in 600 mm3 tumors [169]. In mice,
co-injection of GL261glioma and b.END3 endothelial cells
resulted in larger allografts when those observed following
transplantation of glioma cells alone [164].
Alternative methods for animal experimentation
include GBM growth in semi-solid medium employing
agar or agarose suspension [189, 190]. Agarose-suspension culture is for long known to be one of the best and low
cost in vitro assays correlating with tumorigenicity
[108, 189, 190], still used for GSC validation [191
193]. Alternatively, GSC enrichment has also been performed using agarose as non-adhesive surface [194].
4.3. Expression of biomarkers
GSCs can be validated based on expression patterns of
classical NSC markers, correlating with the progression of
WHO grades astrocytomas [195]. Among them, the transcription factor Octamer 4 (OCT-4), a marker of stem cell
pluripotency [196], has been employed. OCT-3/4 promotes migration and invasion of GBM [197], whose
knockdown was described to result in CSC apoptosis
[114]. In stem cells, OCT-4 expression was shown to be
upregulated by HIF-2a during hypoxia, increasing its
mRNA level four times [150]. However, in another studied
OCT-4 expression could not be detected in GSC, although
performed with GSC enriched in adhesive cultures previously treated with laminin [126]. On the other hand,
SRY (sex determining region Y)-box 2, also known as
SOX-2, which is a encoding group of transcription factors,
is also found in NPCs and are also used as GSC validation
marker [83]. Musashi encodes for various RNA-binding
proteins which contribute to the maintenance of stemness
during CNS development and are also applied in the
validation of the GSC phenotype [83, 150]. Nestin is an
intermediate filament protein specific for glia, usually
applied as a multipotent marker in NSC characterization
and also in the validation of the GSC phenotype [198].
Nestin-knockout embryos revealed reduced self-renewal
with no overt defects in cell proliferation or differentiation, surprisingly uncoupled from nestins structural
involvement in the cytoskeleton [199]. Nestin has been
found to be highly expressed in invasive GBM xenografts
delineating tumor infiltration [200]. However, as neurogenesis precedes gliogenesis during development, the
more undifferentiated stem cell-like phenotypes are thus
negative for nestin expression and found nestinC later
[112]. In line with this observation, nestin mouse NPCs
gave raise to neurons and glia [201]. In addition, 75% of
nestinC cells in GBM coexpressed CD133 [116]. However, in GSCs after 5 days of differentiation with serum,
11
nestin expression levels were still at a high level, whereas

CD133 was much lower expressed [105]. CD133 is the
most used marker for GSC phenotype validation. However, a controversial discussion remains about the suitability of CD133 expression for validating and targeting
GSCs. Beier and co-workers supported evidences that
CD133C GSCs maintain only a subset of primary GBMs
[104]. At the same time, CD133 derived GBM cells
formed tumors in nude rats beyond the efficiency of
CD133C cells [61]. In addition, CD133 has been presented
as marker of bioenergetic stress in GBM rather than for
being a marker protein of their stem-like properties [202].
However, considering that surface phenotype of stem cells
may remain intact despite decay in functional activity, a
more functional assay would be appropriate, as those
employing ALDH expression. According to Choi and
co-workers ALDHC cells range from 0.328.9% in GBM
primary cultures. Furthermore, near 40% of ALDHC cells
overlapped with CD133 expression whereas only near
0.3% of CD133C cells exhibit ALDH activity [82].
4.4 Chemoresistance assay
GSCs are proposed to be chemoresistant, a feature explicating GBM recurrence after conventional therapy [38].
Attempting to predict this chemoresistant phenotype, in
vitro chemotherapeutic drug assays assess the chemosensitivity of CSCs [194]. Temozolomide (TMZ) is a chemotherapeutic alkylating agent included in the standard
therapy against GBM. Although experimental data demonstrate that TMZ preferentially depletes GSCs [203], and
that primary GBM cell response to therapy occurs in
patient-specific fashion and independent of GSC phenotype [204], the majority of studies regarding the susceptibility of GSCs points at a chemoresistant phenotype of
GSCs towards alkylating agents [205]. TMZ induces
apoptosis and senescence in GBM cells cultured as tumorspheres, a phenotype also used for GSC validation [206].
In fact, conversion of differentiated GBM cells into GSCs
could take place upon treatment with TMZ [207].
Recently, TMZ was described to down-regulate P-glycoprotein expression, subsequently promoting survival of
GSCs by the Wnt3a/glycogen synthase-3 kinase/b-catenin
signaling pathway [208]. In line with this observation,
BMP2-induced differentiation sensitized GSC to TMZ
therapy [141]. However, GSCs are not uniformly resistant
to TMZ. In fact, CSCs are neither resistant nor susceptible
to chemotherapy per se. DNA repair mechanisms restore
the integrity of alkylated DNA bases thus contributing to
drug resistance and subsequent tumor reoccurrence.
O6-methylguanine-DNA-methyltransferase (MGMT),
a DNA repair enzyme, possesses detoxifying functions
during TMZ treatment, when the MGMT gene is nonmethylated, conferring a strong intrinsic resistance to GSC
[209]. However, MGMT methylatio stauts alone sill does
not predict the TMZ response with high precision as
atypical TMZ-resistant GSC presented both methylated
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12
and unmethylated forms [210]. TMZ resistance of GSCs

via regulation of MGMT expression is promoted by c-Jun
N-terminal kinase (JNK) and Ras-Raf-MEK-ERK signaling pathways, whose inhibition enhances cytotoxicity of
TMZ on GBM [211213]. In addition to MGMT, another
DNA repair protein, ALKBH2, was shown to mediate
TMZ resistance in human GBM [214], however its specific
effects on GSCs remain unknown. Recently, microRNA125b has been shown to play a role in the resistance of
GSCs against TMZ through down regulation of Bak1 and
PIAS3 (protein inhibitor of activated STAT3) expression
[215, 216]. In view of that, detection of microRNA-125b
expression levels may be useful for validation of the GSC
phenotype.
4.5. Differentiation assay
GSCs are postulated to recapitulate the heterogeneity of
the parental tumor in immunosupressed animals due to
their multipotency capacity. As the differentiation property is a key feature of stemness, differentiation assays
attempt to evaluate the functional GSC phenotype.
Similar to assessing stemness in NSCs, the assay is
done by cultivating GSC under pro-differentiation conditions. Thus, upon induction of differentiation, cells are
going to express specific genes and proteins of the three
cell types of neural lineage (neurons, oligodendrocytes
and astrocytes). These results suggest that initial cells
have stem cell-like characteristics in vitro. Differentiation progress is usually evaluated based on expression
determination of cytoskeleton-associated proteins such
as neuron-specific b3-tubulin (TuJ1) and MAP2, or glial
cell-specific GFAP [217]. Moreover, glycoproteins, postulated to determine stem cell fates, are differentially
expressed during differentiation, and used to determine
the differentiation status of GSCs [218]. The loss of the
CD133 epitope, ABC transporters, and ALDH-1 also are
evidence for CSC differentiation [56, 60, 73].
The glial differentiation of GSCs, evidenced by increased
expression of GFAP, can be induced by all-trans retinoic
acid (RA) GSC cultures were cultured in growth factor-free
medium [70]. Similarly, BMPs promote also astro-glial
differentiation of GSCs [219]. Interestingly, BMPs were
already demonstrated to sensitize GSC for TMZ by affecting HIF-1a stability and MGMT expression [141]. Lee et al.
[217] showed that GBM-derived GSCs cultured with N2
supplement, RA or 10% fetal bovine serum alone, within a
month progressively differentiate losing their NSC markers
nestin, SSEA-1 and Sox2 and developing morphologies and
immunohistochemical staining patterns (GFAP, TuJ1 and
MAP2), consistent with phenotypes of glial and neuronal
lineages. Finally, in vivo experiments with immunosupressed animals provide a proper environment for differentiation of GSCs, recapitulating GBM mass with a variety
of cell types [26]. Therefore, since differentiation property
is a key feature of stemness, this process might be applied in
the validation of GSC phenotypes.
E. Molina et al.
5. CONCLUSIONS
A number of methods have been used to identify GSCs.
Emerging evidence suggests that GSCs do not comprise
a fixed entity, but a state-dependent phenotype of the
micro-environmental niche. In this sense, diverging strategies to obtain GSC hinders inter-laboratory comparisons
when not giving rise to conflicting results. Enrichment and
isolation methods proposed to obtain GSC are evolving,
allowing mimicking in vivo niches to enrich GSC culturing in vitro and introducing novel markers for cytometry
isolation. Hypoxic and co-cultivation with endothelial
cells enriches the cell population with the expected GSC
phenotype. In line with this, the isolation of GSCs based on
expression of CD133 remains a valuable approach, while
ALDH expression has turned into a promising strategy for
GSC isolation, contemplating functional and viable
assessment of the GSC phenotype (see Figure 1 for a
comprehensive scheme). On the other hand, validation
assays are usually performed by measuring the capacity of
forming tumorspheres, stemness marker expression and
tumorigenesis in vivo. However, these strategies for validation might overlap with those used to enrich and/or
isolate GSCs, such as in the endless m
obius ribbon paradigm. Similar to this paradigm, the overlap of the methods of enrichment/isolation with the ones to validate the
obtained GSC is presented in an endless cycle potentially
introducing bias into the process to obtain bona fide GSC.
In this sense, differentiation assays, together with chemoresistance assays, assess more interesting predictive functional features of GSC, although precautions regarding the
expression of DNA repair genes should be taken in
account in order to interprete the latter test. Remarkably,
little knowledge exists on quiescent GSCs, the subset of
GBM cells responsible for the production of transient
populations of highly proliferative cells. Notably, the
hierarchy of markers during NSC versus NPC stage transition remains obscure, especially considering the high
plasticity of this process, with more committed NPCs
reverting back to a more primitive NSC state. Interestingly, ALDH expression has been suggested as marker for
distinguishing between NSCs and NPCs. On the other
hand, the sphere frequency assay is more suitable for
progenitor cell than for stem cell activity. In summary,
the scheme presented in Figure 2 puts together how
methods used for GSC enrichment and isolation might
interfere with the parameters used for GSC phenotype
validation. Investigation of GSC as a relatively recent field
relies on the establishment and optimization of universal
methods focusing at isolation of GSC and robust multiparameter characterization
ACKNOWLEDGMENTS
HU acknowledges grant support from FAPESP and CNPq,
Brazil. TLS acknowledges Slovenian Agency for
Research (ARRS) for supporting the work by granting
the Program P1-0105-0245 to TTL. TLS is grateful for a
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Figure 2. Multi-parameter panel for GSC enrichment, isolation

and validation. The GSC panel summarizes usually employed
methods for isolation and enrichment of GSCs compared to the
ones for GSC-phenotype validation. GSC assessment usually takes
into account differential biomarker expression, sphere formation
and tumorigenesis in vivo capacities, which might overlap with the
parameters of the chosen methods for GSC enrichment and/or
isolation methods. Robust GSC validation assays include other
functional aspects, such as chemoresistance and multipotent differentiation capacities.
visiting professor fellowship granted by the Sciences

without Frontiers Program of the CNPq. ESM and MPP
are grateful for fellowship support by the Brazilian funding agencies FAPESP and CNPq, respectively.
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Journal of Cancer Stem Cell Research (2014), 2:e1007

2014 Creative Commons. All rights reserved ISSN 2329-5872

Glioblastoma stem-like cells: approaches for isolation and characterization

Departmento de Bioqumica, Instituto de Qumica, Universidade de S~

3.3. Effects of endothelial cells

Glioblastoma multiforme (GBM)

These are graded on a scale with increasing malignancy as

Glioblastoma stem-like cells: Approaches for isolation and characterization

induce stem-like cells phenotypes, was crucial to expand

GBM patients. Accordingly, GSCs have been pursued as a

metry and advanced imaging together with an automated

Glioblastoma stem-like cells: Approaches for isolation and characterization

2.1 SP and ABC transporters

derivatives of cyclophosphamide. The enzyme detoxifies

cells are a stable component of the tumor cell population

Glioblastoma stem-like cells: Approaches for isolation and characterization

observed within adhesive surfaces [115], such as in dishes

physiological levels to below 0.1% in necrotic areas [134,

affecting N-glycosylation of this epitope [149]. A culture in

the outside environment. In parallel, easy to use and

Glioblastoma stem-like cells: Approaches for isolation and characterization

activate self-renewal pathways, including Hedgehog

3.5 Effects of immunosuppressed animals

4. VALIDATION OF THE GSC PHENOTYPE

in 7%. However, hypoxia favored a higher proliferation

Glioblastoma stem-like cells: Approaches for isolation and characterization

primary endothelial cells, xenografts were generated within

nestin expression levels were still at a high level, whereas

and unmethylated forms [210]. TMZ resistance of GSCs

Glioblastoma stem-like cells: Approaches for isolation and characterization

Figure 2. Multi-parameter panel for GSC enrichment, isolation

visiting professor fellowship granted by the Sciences

[9] Riddick G, Fine HA. Integration and analysis of genome-scale

Glioblastoma stem-like cells: Approaches for isolation and characterization

3A1 (ALDH3A1) for the enhancement of cyclophosphamide

J Cancer Stem Cell Res http://cancerstemcellsresearch.com

Glioblastoma stem-like cells: Approaches for isolation and characterization

J Cancer Stem Cell Res http://cancerstemcellsresearch.com

Glioblastoma stem-like cells: Approaches for isolation and characterization

cancer stem-like cells in a glioblastoma model after primary

[214] Johannessen TC, Prestegarden L, Grudic A, Hegi ME, Tysnes

J Cancer Stem Cell Res http://cancerstemcellsresearch.com

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