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Embryonic and Cancer Stem Cells - two viewsof the same landscape
Unai Silván, Alejandro Díez-Torre, Lucía Jiménez-Rojo and Juan Aréchaga
Laboratory of Stem Cells, Development and Cancer. Department of Cell Biology &Histology and Analytical & High Resolution Biomedical Microscopy Core Facility. Facultyof Medicine & Dentistry. University of the Basque Country, E-48940 Leioa, Vizcaya
Spain
1. Introduction
According to the Cancer Stem Cell (CSC) theory, there is a small subset of neoplastic cells in
the tumors, which retain unlimited proliferative capacity. These cells give also rise to adifferentiated cancer progeny that does not have spreading potential but makes up the bulkof the tumor. Thus, CSCs would be the ultimate cause of tumor growth, maintenance andrecurrence (Figure 1).
CSCs are experimentally defined by the ability to recapitulate the heterogeneity of theoriginal tumor when transplanted into immunocompatible or nude mice. In 1867 Julius
Cohnheim proposed that tumors are derived from embryonal cells that rest in the adulttissues. Later on, in the middle of the following century, Furth and Kanh (1937) and Pierce
and Dixon (1959) proved the stem cell properties of a subset of cells in leukemia andtesticular germ cell tumors (TGCTs) and Till and McCulloch (1961) transplanted colony
forming units (CFU) from the bone marrow into lethally irradiated mice. Additionally, thegroup of Barry Pierce showed the in vitro modulation of cancer cell differentiation (Pierce &Verney, 1961) and the in vivo cloning of single embryonal carcinoma (EC) cells, proving theirpluripotency (Kleinsmith & Pierce, 1964). The differentiation of cloned leukemic cells(Pluznik & Sachs, 1965) and the reprogramming of embryonal carcinoma (EC) cells when
injected into early embryos (Brinster, 1974) were also outstanding results. All these
discoveries paved the way for the isolation of embryonic stem (ES) cells (Evans andKaufman, 1981; Martin, 1981) and one of the above groups also studied the differentiation of
CSCs from distinct tumors, like squamous cell carcinoma, chondrosarcoma andadenocarcinoma (Pierce & Wallace, 1971; Pierce, 1974; Pierce et al., 1977) seeding the conceptof cancer differentiation therapy (Pierce & Speers, 1988). A new progress in this matter wasdone with the isolation of CSCs in human acute myeloid leukemia (Lapidot et al., 1994) and,particularly, with the discovery of new markers for progenitor cells in several solid tumors,such as breast (Al-Hajj et al., 2003), brain (Singh et al., 2003) and colon (O’Brien et al., 2007)
cancer.
During the evolution of CSC research, there have been several technical improvements thathave undoubtedly contributed to the success in the engraftment of the tumor cells in thehost mice and the subsequent tumor formation. Firstly, it has been proved that the immune
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mutant mice with less effective immune systems increases the calculated CSC number
within the studied tumor. Similarly, CSCs have been shown to be more prone tosuccessfully form tumors when transplanted accompanied by either carcinoma-associatedfibroblasts or Matrigel® (Hwang et al., 2008, Quintana et al., 2008).

Fig. 1. Hierarchical and stochastic models of CSC in solid tumor growth. (A) According to
the stochastic model of cancer progression, every cell present in the neoplasia is able togenerate all the cell lineages of the tumor. Nonetheless, the malignant potential of the cells
may depend on external factors. (B) The CSC hypothesis states that only a small populationof cells, is responsible for tumor growth, due to their self-renewal capacity and unlimited
proliferative potential. As tumor progresses, distinct CSCs (CSC*) may originate due toadditional mutations or epigenetic modifications. Some of these new CSCs may undergo theepithelial-mesenchymal transition, retaining stem cell characteristics, giving rise tomigrating CSCs (mCSCs), the ultimate cause of metastasis
It is important to consider that the CSC model has not yet been proven true for all
the existing tumor types. Thus, it is also thinkable that some cancers follow the random orstochastic model that states that every cell within the tumor may have the ability to seedtumors under the required conditions. The importance of getting to a better distinction
between tumors that follows one model or the other lies on the increase of the effectivenessof cancer treatments, which will be condemned to failure if right cells are not targeted.

2. Cancer stem cell origin
Most cancers are believed to arise from a single cell that undergoes malignant
transformation triggered by genetic mutations or epigenetic modifications, followed byclonal selection of cells that gain the ability to adapt to the microenvironment. Although
CSCs have been described and even isolated in several tumor types, the identity of the precancerous cell that first acquires the tumorigenic potential is still a matter of debate. On one

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Embryonic and Cancer Stem Cells - two views of the same landscape373

hand, tissue-specific stem cells residing in normal tissues or organs, may acquire malignant
characteristics and turn into CSCs. This hypothesis is supported by the fact that tumorigenic
modifications require years to accumulate. Given that stem cells present the longest life spanin the organism, they are the ideal targets of the neoplastic process. Furthermore, adult stem
cells share many functional characteristics with CSCs, e.g. self-renewal and the potential todifferentiate into several cell types.
It is likewise possible that the first mutagenic "hit" affects somatic cells responsible for the
support of the specialized stem cell niche. This specific microenvironment is necessary forthe maintenance of the stem cell identity. Hence, alterations in one or more of the
supporting cells may cause that non-tumorigenic stem cells receive aberrant signals which
trigger their transformation into malignant cells.
Nevertheless, adult stem cells may not be the source of every type of cancer. CSCs could arise
from normal somatic cells, that as a consequence of genetic mutations are re-programmed
into “defective” stem cells. This is likely the case for T-lymphoid leukemia that develops fromT-cells in which the T-Cell Receptor (TCR) gene rearrangement has already occurred. This
genomic modification happens during T-Cell maturation in the thymus and is unique andirreversible. Interestingly, T-Cell leukemias present clonal TCR rearrangements and thus, it
seems clear that in this case tumors arise from somatic cells that undergo de-differentiation
and recover stem cell properties (Schmidt & Przybylski, 2001).
Experiments that comprise the analysis of cell lines cultured in 3D systems, have shown theability of certain cells to adopt a stem cell-like phenotype under particular culture
conditions. Liu and coworkers reported that embryonic fibroblasts from retinoblastoma (Rb)knockout mice, are able to form spherical structures that express specific stem cell markers
when cultured in suspension, and some cells in the spheres adopted CSCs characteristics(Liu et al., 2009). This process has also been reported in the 293T cell line, derived fromhuman embryonic kidney (Debeb et al., 2010). Furthermore, Meyer and colleagues havesuggested the possibility of reprogramming differentiated cancer cells into CSCs (Meyer etal., 2009). They have reported that non-invasive CD44+/CD24+ breast cancer cells are able
to give rise to malignant CD44+/CD24- progeny in vivo and in vitro.
In 2006, Takahashi and Yamanaka reported that the overexpression of a cocktail of 4transcription factors (Oct4, Sox2, c-Myc and Klf4) in adult fibroblasts turned them into
pluripotent stem cells (Takahashi & Yamanaka, 2006). Recent studies have demonstrated
that these so-called induced pluripotent stem (iPS) cells and ES cells exhibit similar gene
expression signatures and potentiality. Because they are genetically modified cells, the ideaof using these first generation iPS cells in therapeutic treatments has been seriously
questioned. Furthermore, two of the four genes used in the cocktail have been shown tobehave as oncogenes (Geoghegan & Byrnes, 2008). In order to solve these issues, newmethods for iPS generation have been established, which instead of transfecting cells, arebased on the use of recombinant proteins (Zhou et al., 2009).
Taken together, these experiments reveal the capacity of somatic cells to acquirepluripotency. Thus, it could also be possible that alterations in the expression of one or moreof those genes triggers the transformation of a given normal somatic cell into a malignant
cell capable of forming a tumor. Indeed, the overexpression of Oct4 can lead to epithelial
dysplasias by blocking the differentiation of progenitor cells (Hochedlinger et al., 2005).
Moreover, several tumor types, for example bladder carcinoma, lung adenocarcinoma,
ovarian carcinoma and testis tumors present abnormally higher levels of Oct4 whencompared to their normal counterpart tissues. Similarly, Klf4, Sox2 and c-myc appear

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upregulated, either alone or together, in a variety of hematological malignancies and solid
tumors, including brain, breast, bladder, lung, pancreas, colon and kidneycancer (Schoenhals et al., 2009).
Interestingly, non-CSCs present in the tumor may as well gain stem cell properties due tothe acquisition of further genetic and epigenetic modifications and in response to changes inthe tumor microenvironment. Therefore, the cell population responsible for tumor growth ata given tumor stage, may not be the same during tumor evolution or metastasis (Roesch etal., 2010). In this regard, the previously mentioned CSC model proposes that CSCs are theonly cells within the tumor that can acquire the ability to spread and grow in distant sites.

3. Cancer types in which CSCs have been identified
Although the existence of CSCs has been reported in several tumor types, so far it has notbeen described any marker that exclusively labels CSCs. Normally, these cells are isolatedusing antibodies specific for normal stem cells of the same tissue from which the tumor is
originated. Flow-cytometry-based cell-sorting, enables the isolation of a side
population which is transplanted into host mice to test their tumorigenic potential andconclude whether indeed, it is enriched in CSCs. CD24, CD44, CD133, epithelial specificantigen (ESA) and ATP-binding cassette B5 (ABCB5) are some of the cell surface markers
that have been proved to be differentially expressed by those CSC enriched populations(Table 1).
Tumor typeSurface markers used to purify the CSCs

References

Accute myeloid leukemia

CD34+/CD38-

Lapidot et al., 1994

Breast cancer

CD44+/CD24-/CD24low

Al-Hajj et al., 2003

Brain cancer

CD133+

Singh et al., 2003

Head and neck squamouscell carcinoma

CD44+

Prince et al., 2007

Colon cancer

CD133+

O’Brien et al., 2007

Pancreatic adenocarcinoma

CD44+/CD117+

Li et al. 2007

Melanoma

ABCB5+

Schatton et al., 2008

Ovarian cancer

CD44+/CD24+/ESA+

Zhang et al., 2008

Prostate cancer

CD133+/ 2 1integrin/ CD44+

Maitland & Collins,
2008

Table 1. Distinct surface markers have been extensively used to isolate CSC-enrichedsubpopulations from different cancer types
Leukemia and TGCTs were the first tumor types in which CSCs were experimentallydescribed (Furth & Kanh, 1937; Pierce & Dixon, 1959). Furth and Kahn proved that a single
cell from a murine cancer cell line was able to transmit leukemia. Regarding TGCTs,
Kleinsmith and Pierce (1964) dissociated teratocarcinoma-derived embryoid bodies andtransplanted single cells into host mice. Around 2.5% of the grafted cells had the ability toform new tumors, albeit their differentiation potential was highly variable. Later on, Stevens

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Embryonic and Cancer Stem Cells - two views of the same landscape375(1968) managed to generate testicular teratocarcinomas by grafting 3 and 6-day embryos
into testicles of adult mice and to serially transplant the formed tumors. It was not until 1994
when surface markers were used to isolate an enriched fraction of tumor-initiating cells. In
that year, Lapidot and coworkers (1994) separated a subpopulation of CD34+/CD38- cells
from human accute myeloid leukaemia (AML) patients and verified their CSC properties
after transplanting them into severe combined immune-deficient (SCID) mice observing thatthey were able to recapitulate the disease.
Regarding solid tumors, it is likely that additional technical issues, such as the difficulty inobtaining homogeneous single cell suspensions for their further separation, delayed theachievement of a well-characterized CSCs population from a human tumor. The first solid
malignancy from which CSCs were isolated was breast cancer. Al-Hajj and colleagues
described a CD44+/CD24-/low cell population that was significantly enriched in tumorinitiating cells (Al-Hajj et al., 2003). Shortly after these findings, CSCs of brain tumors werealso isolated using CD133 as a marker and characterized by the expression of other markers
for non-pathological neural stem cells (Singh et al., 2003) . Interestingly, different pathologic
subtypes of brain tumors, like medulloblastomas, astrocytomas and gangliogliomas, sharecommon CSCs. However, the self-renewal capacity of CSCs varies depending on the tumor
subtype and its aggressiveness.
Prince and colleagues (2007) developed an immunodeficient mouse model to test thetumorigenic potential of different populations of cancer cells derived from primary humanhead and neck squamous cell carcinoma (HNSCC). They reported that a population of CD44+
cancer cells was capable of giving rise to new tumors that reproduced the original tumor
heterogeneity and could be serially passaged. Moreover, these CSCs had a primitive cellularmorphology and did express the basal cell markers Cytokeratin 5 and 14. On the contrary,CD44- cells did not show tumorigenic potential and were similar to differentiated squamous
epithelium as assessed by the expression of differentiation markers such as Involucrin.
Later on, it was established that in colon cancer, CSCs are characterized by the expression of
the CD133 marker (O'Brien et al., 2007). In humans, colon cancer-initiating cells represent
only around 0.06% of all cells within the tumor, but 0.4% of them are CD133+. Thus, it is stillnecessary to find additional cell surface markers in combination with CD133 in order tofurther purify the CSC fraction from this type of cancer.
In 2007, Li and colleagues analyzed the tumor initiating ability of different subpopulations
from primary human pancreatic adenocarcinoma. They reported that those pancreaticcancer cells with a CD44+/CD24+/ESA+ phenotype, that represent around 0.2 to 0.8% of
pancreatic cancer cells, had a 100-fold increased tumorigenic potential compared to nontumorigenic cancer cells. Furthermore, the genetic analysis of pancreatic CSCs revealed anincreased expression of the signaling molecule Sonic Hedgehog (Li et al., 2007).
Schatton and colleagues (2008) identified an ACBB5+ subpopulation of melanoma cells that
represents 0.0001% of the total tumor cells and shows high capacity to re-establish themalignancy after xenotransplantation into mice. In addition, they proved that the specifictargeting of these CSCs using monoclonal antibodies against ABCB5, resulted in inhibitionof tumor growth. However, Quintana and collaborators (2008) have recently shown that theamount of CSCs in human melanoma may change dramatically depending on theconditions of the xenotransplantation assay. This finding raises the question whether tumors
in which CSCs are rarely detected, may in fact have a higher number of these cells. For
example, tumor microenvironment as well as site of inoculation may have an influenceon the tumorigenic potential of presumptive CSCs. The frequency of CSCs in humanwww.intechopen.com

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melanoma reported by this group is indeed much higher than reported for any other cancer
type that follows the CSC model. Although efforts were made to characterize thosemelanoma-seeding cells, they could not find phenotypic differences between tumorigenic
and non-tumorigenic cell populations.
Zhang and colleagues (2008) have also identified the surface phenotype of CSCs fromovarian adenocarcinoma. These cells are characterized by a higher expression of CD44 andCD117 (c-kit), Bmi-1, stem cell factor, Notch-1, Nanog, nestin, ABCG2 and Oct4 whencompared to non-malignant ovarian tumor cells (Zhang et al., 2008). Finally, Maitland andCollins (2008) have reported thattumor-initiating cells are enriched in a
CD133+/ 2 1integrin+/CD44+ subpopulation from human prostate cancer. Moreover,prostate CSCs have a unique genetic fingerprint that makes them useful to predict the tumor
staging and clinical outcome. Remarkably, these putative CSCs do not express androgen
receptor, which makes them refractory to the widely used androgen-based therapies(Maitland & Collins, 2008).

4. CSC and ES cell similarities
Three different types of non-malignant stem cells have so far been described in vivo: ES cells,
chord blood/placental stem cells and adult stem cells. Among them, ES cells are the only
ones that show pluripotency, being capable of giving rise to cell derivatives of the threegerm layers: ecto-, endo- and mesoderm. ES cells are obtained from the inner cell mass(ICM) of the blastocyst and can be cultured in vitro using specific conditions to prevent theirdifferentiation. In turn, adult and chord blood/placental stem cells are multipotent and candifferentiate into a limited number of cell lineages (Rogers & Casper, 2003).
The ability of cancer cells to grow indefinitely led to the belief that CSCs were similar to
adult stem cells. However, detailed gene expression analysis of both cell types reveals that
actually, CSCs share more characteristics with ES cells. In fact, long before the CSC
hypothesis was enunciated, it was observed that tumor and embryo development sharemultiple common features. For instance, many tumors have been histologically classified
due to their differentiation state, being this characteristic also relevant to prognosis. This is
the case of TGCTs that are characterized by the presence of embryonic and extra-embryonictissues, together with embryonal carcinoma (EC) cells, a population of pluripotent stem
cells. EC cells, the CSCs of TGCTs, are considered the pathological counterpart of ES cells,
due to their ability to lose their malignant phenotype and participate in normal embryo
development when transplanted into blastocysts. In the resulting chimeric mice EC cells
appear in tissues derived from the three germ layers, including germ cells (Brinster, 1974;
Mintz & Illmensee, 1975). Based on these similarities, EC-derived post-meiotic neurons havebeen implanted in damaged regions of the brain in a clinical trial (Hara et al., 2008).
Nonetheless, due to the karyotypic instability of EC cells, these regeneration therapies havesome safety concerns.
4.1 Gene signature in CSC and ES cells
A key goal in cancer research is to identify the molecular mechanisms by which CSCs arise
and acquire their stem-like characteristics. Nowadays, extensive databases, mainly obtainedby microarray analysis, have been generated, offering the possibility of comparing theexpression profile of a huge number of both tumor and normal cells or tissues. Based on thefact that ES and cancer cells share properties such as self-renewal and differentiationwww.intechopen.com

Embryonic and Cancer Stem Cells - two views of the same landscape377

capacity, there has been a recent increased interest in finding out whether CSCs and ES cells
present a similar gene signature. As previously mentioned, Oct4, Sox2, c-Myc and Klf4
comprise the gene cocktail that induces pluripotency in somatic cells by a process known as"somatic cell reprogramming" (Takahashi & Yamanaka, 2006). Thus, it is believable thatalterations in the expression of one or more of these ES-defining genes may trigger thetransformation of a normal somatic cell into a malignant CSC.
Oct4 is the commonly used synonym for POU5F1 (POU class 5 homeobox 1). Thistranscription factor is active from the fertilized oocyte throughout the whole preimplantation
period of embryo development. Oct4-deficient mouse embryos fail to form the ICM, lose
pluripotency and differentiate into trophectoderm. Therefore, the level of Oct4 expression inmice is crucial for regulating pluripotency and early cell differentiation since one of its mainfunctions is to maintain the undifferentiated state of the embryo (Zaehres et al., 2005).
Moreover, an Oct4 overexpression leads to epithelial dysplasias by blocking the differentiationof progenitor cells (Hochedlinger et al., 2005). It is likewise known that several tumor types, as
for example bladder carcinoma, lung adenocarcinoma and testis tumors among others, present
increased levels of Oct4 expression compared to their normal counterpart tissues (Schoenhals
et al., 2009). It has been recently reported the existence of a subpopulation of cells from ovariancancer that express Lin 28 and Oct4 genes, both highly expressed in human ES cells. In fact, theup-regulation of these genes in tumor samples is correlated with advanced tumor grade (Penget al. 2010). Interestingly, the CSC population isolated from ovarian cancer has an up-regulated
expression of Oct4 (Zhang et al., 2008). The expression of these two factors seems to be
essential for cell growth, since their inhibition using iRNA results in a significant reduction incell growth and survival (Peng et al., 2010).
Sox2, also known as Sry (sex determining region Y), is a key transcription activator during
early embryonic development and its activity is also important in adult stem cells, since it
has been reported to maintain the proliferative potential of neural stem cells (Episkopou,
2005). Oct4 forms an heterodimer with Sox2 that drives the expression ofseveral pluripotent-specific genes, including Nanog, FGF-4, UTF1, Fbx15 and Lefty1,
together with Oct4 and Sox2 themselves. The expression of at least three of these genes(Nanog, Sox2 and Oct4) is essential to maintain the pluripotent ES cell phenotype. With thepossible exception of Lefty1, the expression level of each of the genes regulated by the
Oct4/Sox2 complex is substantially reduced upon differentiation of both ES and EC cells,
due to the down-regulation of Sox2 and Oct4 (Boer et al., 2007). It has also been reported
that the CSCs of multiple myeloma express high levels of Sox2, together with Oct4 and cMyc, being the latter a well-known proto-oncogene. c-Myc was first described in Burkitt's
lymphoma patients, and its function has been proved to be crucial for early embryo
development and adult stem cell maintenance. For example, antisense DNA inhibition of cMyc expression in preimplantation mammalian embryos results in developmental arrest at theeight-cell morula stage (Paria et al., 1992). Over-expression of c-Myc has been described inseveral hematological malignancies, such as leukemia, lymphoma, smoldering myeloma
and multiple myeloma. Wang and colleagues (2008) studied the importance of thistranscription factor in CSCs using glioma CD133+ cells as a model. Inhibition of c-myc
using lentivirally transduced short hairpin RNA (shRNA) resulted in cell cycle arrest in theG(0)/G(1) phase, reduced proliferation and increased apoptosis. Furthermore, glioma CSCs
with decreased c-Myc expression levels failed to form neurospheres in vitro or tumors whenxenotransplanted into brains of immunocompromised mice. Overall, c-Myc seems to play aessential role in the regulation of the stem cell characteristics of CSCs. Finally, the transcription

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factor Klf-4 (Krüppel-like factor 4), necessary for somatic cell reprogramming, is also requiredfor ES cell self-renewal and maintenance of pluripotency (Zhang et al., 2010). Klf4 appears upregulated in most hematological malignancies, such as acute lymphoblastic leukemia, hairycell leukemia and multiple myeloma, being yolk sac tumors, a germ cell tumor subtype, one of
the few solid tumors that present elevated levels (Schoenhals et al., 2009).
A better understanding of the acquisition and regulation of the self-renewal andproliferation potential by CSCs will improve the design and generation of anti-neoplastic
drugs and accelerate the discovery of novel molecular targets for clinical application. Thisknowledge may as well help to use the existing treatments in a more CSC-specific andeffective manner.
4.2 Signaling pathways shared between CSCs and ES cells
Besides the gene signature that defines the ES cells, there are some signals that can
extrinsically regulate several stem cell properties such as self-renewal. Many studies showthat embryonic development and tumorigenesis share common regulatory mechanisms.
Thus, it would not be surprising to find out that the cellular process, which leads to thegeneration of both, ES and CSCs, is regulated by the same factors. Hereby, we list the mostimportant pathways that modulate ES function, mentioning their relationship with CSCs.
The Notch signaling pathway is highly conserved in mammals, playing an important role inembryonic development and adult tissue repair. It is known that Notch signaling is downregulated during the cellular differentiation, being a promoter of stem cell survival,
proliferation and undifferentiation of some adult stem cells, like for example hematopoietic
and brain stem cells. It has been reported that the inhibition of this signaling pathway in somecancer types, such as medulloblastoma, reduces their proliferation in vitro and their capacity offorming tumors when transplanted into mice (Fan et al., 2006). These authors showed theblockage of Notch signaling by gamma-secretase inhibitors (GSI) reduced neurosphere growthand clonogenicity in vitro, whereas the activation of this pathway increased tumor growth.
Furthermore, the expression of glioblastoma CSC markers such as CD133, Nestin, Bmi1, andOlig2 was reduced when Notch signaling was blocked. Xenograft transplantation experiments
revealed that GSI-pretreated cells did not form tumors and even the implantation of drugimpregnated polymer beads in the tumor beds also effectively reduced tumor growth andsignificantly prolonged survival (Fan et al., 2010). Thus, it is likely that Notch pathway
inhibition depletes CSCs through reducing proliferation and increasing apoptosis associatedwith decreased AKT and STAT3 phosphorylation.
Regarding the Wnt pathway, it is known to play an essential role in cell proliferation andstem cell maintenance. Sato and coworkers (2004) demonstrated that the overactivation of
the Wnt/b-catenin pathway was responsible of the maintenance of pluripotency in ES andadult stem cells, as assessed by the expression of markers such as Oct4, Nanog and Rex1.
Mutations within this signaling pathway occur frequently in human cancers. In fact,
experiments in mice have shown that the overactivation of the Wnt/b-catenin pathway
promotes oncogenic transformation of different cell types. For instance, mice thatoverexpress Wnt1 under de MMTV (mammary tumor virus) promoter develop salivary andmammary gland tumors and the accumulation of b-catenin due to mutations in APC gene
may be linked to the appearance of colorectal tumors in humans (Markowitz et al., 2009).
Fibroblast growth factors (FGFs) are intercellular signaling molecules that, among otherfunctions, participate in embryonic development, affecting several cell functions such as
proliferation, differentiation, survival, adhesion and migration (Szebenyi & Fallon, 1999).

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The FGF signaling pathway regulates the specification events that occur in the early embryo,when the cells composing the ICM of the blastocyst give rise to the three germ layers. Since
ES cells are derived from the ICM it is believable that these cells conserve the same
pathways. One of the most interesting members of the FGF pathway is FGF-2, also known asbasic-FGF or bFGF. It has been reported that elevated expression levels of FGF-2 in the
microenvironment of metastatic prostate tumors induce chemotherapy-resistance in themalignant cells. It has been speculated that the dysregulation of this factor in tumor cells is
acquired as an adaptation to gain self-renewal and unlimited proliferative ability, two of themain features that define ES cells (Villegas et al., 2010).
During normal embryo development, the Hedgehog (Hh) signaling pathway is related tothe epithelial-mesenchymal transition (EMT) by means of the up-regulation of the Ecadherin repressor SNAIL1. EMT is related to tumor progression in correlation with the lossof the epithelial characteristics and the acquisition of metastatic phenotype (Hay, 1995).
According to the CSC theory, metastatic cells retain stem cell properties, thus cells that
undergo EMT have to conserve stem cell properties. To describe these cells, Brabletz andcolleagues (2005) proposed the term migrating CSCs (mCSCs).
A better knowledge of the pathways that regulate ES cell functions is crucial to understandhow CSCs self-renewal, survival, proliferation and metastasis are regulated. This will help
to target specifically those stem cells that may be the source of recurrent tumors and are able
to escape from the majority of the currently used cancer therapies (Takebe & Ivy, 2010).

5. Testis germ cell tumors as a CSC model
Early pathologists noted that certain germinal tumors contained a mixed population of
adult tissues, but looked like a “caricature” of them (Aréchaga, 1993). Therefore, thesemalignancies were named teratocarcinomas, being “terato” a prefix meaning “monster”.
Testis germinal cell tumors (TGCTs) form a heterogeneous group of neoplasias that areusually classified according to their histological aspect. Teratocarcinomas are characterized
by the presence of embryonic and extra-embryonic tissues together with a population ofpluripotent stem cells, named EC (Kleinsmith & Pierce, 1964). EC cells represent the mostaggressive cell population in germ line tumors and are as well responsible for thetransplantability of the tumors between immunocompatible animals. Interestingly thesecells are frequently referred as the “malignant counterpart” of ES cells, due to the similarfeatures that both cell types share (Aréchaga, 1993, Andrews et al., 2005). For example, aspreviously noted, when injected into a blastocyst, EC cells lose their malignancy andparticipate in the normal development of the embryo, just like ES cells (Brinster, 1974).
Furthermore, it has been described that when cultured, ES cells acquire karyotypic changes
that resemble those of EC cells, being their general in vitro behavior also similar.
TGCTs derive from a precursor lesion called carcinoma in situ (CIS) of the testis, which isfound forming a single row at the basement membrane of seminiferous tubules. It was
described for the first time when “atypical spermatogonia” were found in testis samplesof patients that later developed TGCTs (Skakkebaek, 1972). It is known that CIS cells are
already present in a latent state at the moment of birth, but it is not until puberty, when
triggered by hormonal changes, the cells start to proliferate and to invade the testis stroma(Díez-Torre et al., 2004).
Two possible origins for the CIS of the testis have been proposed. Taking in considerationthat germ cell tumors appear mostly in the gonads and that the transplantation of mouseembryo genital ridges into testis originates tumors (Stevens, 1967), it seems that CIS cells

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result from germ cells that are unable to differentiate correctly. However, ES cell origin is
supported by the fact that these tumors can be also generated by the transplantation of early
embryos (Stevens, 1968) and that the gene expression pattern and the differentiation
potentiality of the tumor cells is similar to that of the ES cells. Furthermore, the existence of,in terms of histology, identical tumors that appear in extra-gonadal locations, suggests its
non-germinal origin. However, it is possible that these rare neoplasias develop fromprimordial germ cells (PGCs) that are retained during their migration towards the gonads.
In fact, extra-gonadal germ cell tumors mostly arise in the body mid-line, which is the paththat PGCs follow in their migration.

Fig. 2. Technique of cell transplantation into the seminiferous tubules. (A) Scheme showing
the three different approaches for cell transplantation into the seminiferous tubules. The
sharpened micropipette can be inserted directly in the seminiferous tubules (I), through theefferent ducts (II) or into the rete testis.(B) Time lapse photographic series of the
microinjection of a blue-colored cell suspension. The microinjection pipette is inserted
through the efferent ducts in this case. The process finishes when almost all tubules arefilled. Scale bar represents 1mm

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Embryonic and Cancer Stem Cells - two views of the same landscape381

Since non-human mammals rarely develop TGCTs until now no suitable animal models areavailable to study this malignancy. Leroy Stevens (1973) made one of the first attempts toestablish an animal model of TGCTs describing an inbred subline of mice termed 129-terSv
which shows a high incidence of those tumors. However, most of the spontaneous tumors
were teratomas, the differentiated and benign variant of teratocarcinoma, and thus notadequate for the study of human TGCTs, which generally are malignant (Stevens, 1973).
In the middle 90s of the last century Brinster and Zimmerman (1994) developed the techniqueof cell transplantation into the seminiferous tubules of azoospermic animals. Their initialapproach consisted of microinjecting a spermatogonia-enriched cell suspension into single
seminiferous tubules. Later on, two more effective approaches were described, namely the
microinjection into the rete testis and through the efferent ducts (Figure 2 A and B; Ogawa etal., 1997). Using this technique, it was possible to determine that germinal cells transplanted
from one animal into another are capable of nesting in the seminiferous epithelium anddifferentiate into fertilization-competent spermatozoa. At the same time, Brinster andAvarbock (1994) tested the ability of the seminiferous compartment of reprogramming ES cells
and driving their differentiation towards the germ cell lineage. However, ES transplantation
resulted in tumors. Later on, a TGCT model using this technique was developed. It consists ofthe transplantation of EC cell lines into the seminiferous tubules of germ-depleted mice (Li etal., 2008a). The experiments involved the transplantation of two human lines derived from
TGCTs, the JKT-1 seminoma cell line and the 833K EC cell line. Transplantation of bothresulted in tumors that expressed TGCT markers.

Fig. 3. Histology of an experimental testis teratocarcinoma. (A) Normal testicular tissue iscomposed of dense packed seminiferous tubules. (B) Cryostat section of a mouse testis few
minutes after transplantation of autofluorecent ES cells (arrow) into the seminiferous
tubules. (C) 5 weeks after transplantation teratocarcinoma tumors are formed in thetransplanted testis, (D) Most of the neoplastic tissue fluorescent. Scale bars represent 50µm

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Besides the similarities between EC and ES cells (Andrews et al., 2005), several studies havealso shown that ES cells share many phenotypic and genetic similarities with the CIS of thetestis (Almstrup et al., 2006; Rajpert-De Meyts, 2006; Looijenga, 2009). For example, it has
been estimated that nearly 50% of the genes up-regulated in human ES cells are also
expressed in CIS cells. Among these genes pluripotentiality-related and undifferentiationrelated genes can be found. These observations led us to establish a TGCT model based in
the transplantation of ES cells into the seminiferous tubules, with the belief that this model
mimics more accurately than others the early stages of TGCT development (Silván et al.,2009a; Silván et al., 2010a). To follow the fate of the transplanted cells, we generated a stable
GFP-transfected ES cell line, named AB1GFP. Short after the transplantation ES cells can beseen in the lumen of the seminiferous tubules (Figure 3 A and B). Around 24 to 36 hours
later, ES cells are found integrated in the seminiferous epithelium, close to the basal
membrane. Interestingly, this localization is similar to that of the spontaneous CIS of thetestis. Five weeks after the ES cell transplantation, mature teratocarcinoma are completely
formed (Figure 3 C). Most of the structures of the formed tumors were found to be derived
from the transplanted cells, as can be determined by their fluorescence (Figure 3 D).

Fig. 4. Experimental teratocarcinomas showed different histological patterns ofdifferentiation, such as cartilage (A), neural (B), epidermoid (C) and adenomatous areas (D).
Scale bars represent 50µm
This transplantation procedure mimics better than others the early stages of TGCT
development. At the histological level, experimental teratocarcinomas highly resembled thespontaneous TGCTs, showing structures derived from the three germ layers (Figure 4 A-D).
In addition, regions with undifferentiated appearance (Figure 3C), which would be similar

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Embryonic and Cancer Stem Cells - two views of the same landscape383

to the EC component of spontaneous non-seminoma tumors, could as well be found (Silván
et al., 2010a).
Although TGCT treatment has a high success rate (Gerl et al., 1996; Bosl, 1999), early
diagnosis and identification of the causes of the high incidence among young men are still
unknown. The experimental model that we have developed allows the study of the preinvasive state of testicular teratocarcinomas and is potentially useful for the screening ofnovel therapeutic drugs, such as inhibitors of angiogenesis. Furthermore, prior to thetransplantation procedure, donor cells can be modified, up- or down-regulating one or moregenes, thereby providing a functional assay to evaluate the effect of these genes in EC
transformation. Hence, we believe this model can help in the study of the cellular andmolecular mechanisms involved in CSC establishment.

6. CSCs, ES cells and hypoxia
It is well known that adult tissues contain stem cell populations for cell renewal and anincreasing number of evidences support that these cells are localized in specific niches
characterized by low oxygen levels (Mazumdar et al., 2009). Since stem cells are the only celllineage that is not replaced during the whole animal's life span, it is likely that the hypoxic
microenvironment, where these cells are found, protects them from potential damages
caused by oxygen (Diabira & Morandi, 2008). In vitro studies that tested the effect of oxygen
showed a direct relationship between oxygen levels and the regulation of stem cellproliferation, differentiation and survival. More precisely, hypoxia has been reported toincrease the proliferation of neural crest stem cells (Morrison et al., 2000), stimulate thesurvival of chondrocytes (Schipani et al., 2001) and disrupt adipocyte differentiation (Yun etal., 2002). Applying observations to the cancerous tissue, it is likely that CSCs are as welllocated in low oxygenated regions. In fact, due to their rapid growth and defective
vascularization, solid tumors frequently present regions with reduced oxygen supply andnecrosis. Consequently, tumor hypoxia has been correlated with bad prognosis factors, suchas malignancy stage and resistance to radio- and chemotherapy (Jubb et al., 2010; Bertout etal., 2008).
Cellular response to hypoxia leads to several changes in gene expression triggered by agroup of transcription factors known as Hypoxia Inducible Factors (HIFs). These factors,
which belong to the basic helix-loop-helix (bHLH) and PAS (PER-ARNT-SIM) families, arecomposed by two subunits: one of them is variable (HIF-1α, -2α or -3α) and oxygen sensitive,
but rapidly stabilized in response to hypoxic conditions. The other subunit, HIF-1 , also
known as ARNT (aryl hydrocarbon receptor nuclear translocator), is constant andconstitutively expressed (Wang & Semenza, 1995). When the cell is exposed to low
oxygenation conditions, degradation of the alpha subunit is inhibited, binds the subunit
and the resulting complex translocates into the nucleus. There, it activates specific genes
through the recognition of promoter regions known as hypoxia response elements (HREs).
The up-regulation of these hypoxia-related genes mediates a number of changes at both
cellular and systemic level.
Oxygen availability regulates several physiological processes, such as cell proliferation,differentiation and migration, being low oxygen levels, one of the main causes of cellularstress. The relationship between hypoxia and tumor growth was first reported as theradioprotective effect of anoxia in normal tissues was demonstrated using whole-bodyanoxia in newborn rodents (reviewed by Gray et al., 1953). Later on, the study of ex-utero

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mouse embryo growth demonstrated that hypoxia is needed for a proper embryonicdevelopment (Morriss & New, 1979). In fact, during early development ES-precursor cells
are exposed to a hypoxic microenvironment due to the absence of vasculature in early
embryos (Mitchell & Yochim, 1968). Because of this common hypoxic environment, HIF
factors play a critical role in normal development and tumor growth, invasion andmetastasis (Harris, 2002).
The study of HIF knockout mice has demonstrated the key role of this transcription factors
during the embryonic and fetal development. HIF-1α and HIF-1 null mice embryos die as
the result of defects in vascular development (Ryan et al., 1998, Maltepe et al., 1997). The
effects of HIF-2α disruption range from embryonic lethality due to aberrant vasculature(Peng et al., 2000) to postnatal lethality because of multiorgan failure and alteredmitochondrial metabolism (Scortegagna et al., 2003). The relationship of hypoxia with solid
tumor progression has been repeatedly reported in clinical studies that prove that thosepatients with hypoxic tumors have a significantly poorer clinical outcome (Vaupel, 2008; Liuet al., 2010; Jubb et al., 2010). The causes of this worst prognosis include an increased ability
for tumor invasion and metastasis and a higher resistance to radio- and chemotherapy(Brizel et al., 1999; DeClerck & Elble, 2010) as the result of a hypoxia-dependent expressionof drug-resistance genes (Wartenberg et al., 2003), the selection of apoptosis-resistant clones(Graeber et al., 1996) and the disruption of DNA repairing mechanisms (Chan et al., 2009).
The link between low oxygen levels and tumor progression, together with the identificationof CSCs and the known role of hypoxia as a key component of the stem cell
microenvironment has lead to the hypothesis that hypoxia maintains the undifferentiatedstate of CSCs and thus contributes to cancer growth, invasion and metastasis. Stem cellniches constitute the adequate environment for the maintenance of the undifferentiated stateand for regulating stem cell differentiation into specific cell lineages (Moore & Lemischka,2006). It has been recently speculated that hypoxia may regulate stem cell localization andmaintenance inducing the expression of paracrine factors in a HIF-dependent manner. Thatis the case of endothelial progenitor cells. Hematopoietic stromal cells expressing the SDF-1
chemokine attract the stem cells expressing CXCR4 at low oxygenation conditions (Ceradini
et al., 2004). This example illustrates the importance of stroma cells in the regulation of thestem cell microenvironment. The differentiation status of the stroma cells is crucial for the
maintenance of stem cells. In bone marrow, for instance, when osteoblasts are removed
during early differentiation, there is a critical reduction in bone marrow-derived
hematopoietic stem cells (Visnjic et al., 2004). Nevertheless, this effect on hematopoiesis issignificantly reduced when osteoblasts are removed at later stages of differentiation (Corral
et al., 1998). Further evidences, such as the expression of hematopoietic stem cellmaintaining factor angiopoietin-1 by undifferentiated CD146+ osteoprogenitor cells but notin differentiated cells, point to immature stroma cells as key supporters of the stem cellniche (Sacchetti et al., 2007). The crosstalk between tumor cells and the surrounding stroma
cells has a crucial effect on tumor growth, invasion and metastasis (Diez-Torre et al., 2010).
The importance of this interaction suggests that stroma cells may provide the adequate
microenvironment to maintain CSCs. In fact, hypoxia may regulate CSC maintenance anddifferentiation through three different mechanisms and can act directly on CSCs inhibiting
them to differentiate. Low oxygen levels can also induce or maintain an immature state intumor stroma cells and, finally, it can up-regulate the expression of paracrine factors thatmediate the interaction between tumor and stroma cells in a way that stimulates CSC
homing, proliferation and invasion.

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Embryonic and Cancer Stem Cells - two views of the same landscape385

Karnoub and colleagues (2007) reported that mouse stroma cells surrounding human breast
cancer xenografts contain mesenchymal stem cells able to form fibroblastoid colonies in vitro
whereas this ability is absent in those cells obtained from adjacent normal stroma. The same
study demonstrated that the presence of bone marrow-derived mesenchymal stem cells is
sufficient to induce the acquisition of a highly aggressive phenotype by poorly metastatic
human breast carcinoma cells when injected in host mice. Thus, immature stroma cells may
be recruited by the tumor microenvironment, being these undifferentiated cells involved in
the acquisition of an aggressive phenotype by breast carcinoma cells.
Surprisingly, it has been reported that brain CSCs are preferentially located in the proximityof tumor associated endothelial cells. Although this result may partially contradict the
hypoxic niche-theory, in vitro co-culture experiments have shown that endothelial cells
secrete paracrine factors that promote CSC growth and stemness. Thus, the role ofendothelial cells within the tumor stem cell niche could be independent of their vascularfunction (Calabrese et al., 2007). This idea is in concordance with the well known
abnormality of tumor-associated blood vessels that lead to the formation of hypoxic oranoxic regions into the solid tumors and the presence of endothelial precursors without
vascular functionality near the tumor cells (Silván et al., 2010b). Further research on theinteractions between CSCs and endothelial cells in tumor hypoxic regions could provide
valuable information for a better understanding of the tumor stem cell niche.
The role of hypoxia in the induction and maintenance of CSC undifferentiation has been
recently studied in two different tumor types, neuroblastoma and breast carcinoma(Axelson et al., 2005). In both tumors, a correlation between poorly differentiated regions
and tumor aggressiveness has been observed. In the case of neuroblastoma, the analysis ofgene expression profiles of cultured tumor cells and neuroblastoma xenografts has
demonstrated that hypoxia down-regulates the expression of mature neuron marker genes
whereas induces the expression of c-Kit and Notch, genes associated with a neural crest-likephenotype (Jögi et al., 2002). Similar effects have been reported in ductal breast carcinoma(Helczynska et al., 2003) and cultures of EC cell lines (Silván et al., 2009b). However, the roleof hypoxia in tumor progression is not limited to the maintenance of the undifferentiatedstate of CSCs, it has also been shown that low oxygen availability increases tumor cellproliferation and apoptosis resistance in lung adenocarcinoma (Chen et al., 2007a),
melanoma (Bedogni et al., 2008) and thymus lymphomas (Bertout et al., 2009) through thealteration of Notch1 signaling. The relationship between hypoxia and tumor progression is
also mediated through the overlapping of HIFs and some oncogene signaling pathways,
such as cMyc and p53 (Mazumdar et al., 2009).
All these observations point out that oxygenation level plays a critical role in the stem cell
microenvironment, and that, together with other niche components such as stromal cell
contacts, extracellular matrix proteins, growth factors and temperature, is directly involvedin CSC behavior, and thus, cancer progression and metastasis.

7. Importance of CSCs for cancer therapy
Many of the currently used cancer therapies are directed against rapidly dividing cells,
which represent most of the tumor cell population. However, in a significant number ofcases these therapies fail to eliminate the stem cell fraction of the tumor, what leads to tumor
relapse and the selection of a more aggressive and therapy-resistant cancer cells and thus
implies a worst clinical outcome. The development of therapies that specifically target CSCs

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seems to be needed in order to achieve the complete tumor remission and prevent
metastasis. The identification of the different cancer cell populations through DNA andtissue microarray analyses, the study of in vitro obtained cancer spheroids or the selection of
more aggressive and therapy-resistant tumor cell lines by successive xenografttransplantation, are some of the strategies that are being followed with the aim ofidentifying those agents that selectively eliminate CSCs (Ischenko et al., 2008). It has to beconsidered that CSCs share many features with normal adult stem cells, and therefore, it has
to be checked that the new therapies are effective against CSCs without being harmful for
healthy adult progenitor cells. The results obtained by recent research works support thefeasibility of this objective.
As we have previously mentioned, recent evidence demonstrates that the stem cell fractionof several tumor types show a higher resistance to radio and chemotherapy (DeClerck &Elble, 2010; Elliot et al., 2010). Different mechanisms have been suggested to explain theacquisition of this resistance. One of these explanations is related to the slow proliferation
rate shown by stem cells, which stay in the G0 phase of the cell cycle during long periods oftime. This feature protects them from drugs that target actively dividing cells, the so-called
cell-cycle specific agents. Moreover, CSCs show an increased expression of adenosine
triphosphate-binding cassette proteins, which are known to outflow chemotherapeutic
drugs. In fact, one of these proteins, the breast cancer-resistance protein (ABCG2), has beenused to identify and isolate the side population of breast and other cancer types by flow
cytometry due to its ability to extract Hoechst dye from the cell (Kim et al., 2002).
Additional mechanisms used by CSCs to escape chemotherapy are the up-regulation of
drug metabolizing enzymes, for example the ALDH1 enzyme that metabolizes
cyclophosphamide (Smalley et al., 2005), and the over-expression of survival promoting
factors, such as the apoptosis inhibitors Survivin and Bcl-2 family proteins (Litingtung et al.,1999). Obviously, some of these stem cell markers have a prognostic significance. The
expression of ALDH1 in breast carcinoma, for example, has been associated with poor
clinical outcome and an increased risk of recurrence (Ginestier et al., 2007)
The results of several studies on the resistance of CSCs to radio- and chemotherapy suggestthat the limited success of the current therapies in some tumor types could be related totheir inability to target the CSC population. Indeed, it has been recently reported thatchemotherapy leads to an increase of the breast CSC population, characterized by aCD44+/CD24- phenotype (Yu et al., 2007; Li et al., 2008b). Interestingly, in one of theseneoadjuvant therapeutic trials, it was shown that targeting Her-2 with Lapatinib® produces areduction of the CSC population and that this effect leads to a significantly increased
pathologic complete response rate (Li et al., 2008). Since Her-2 is a known stem cell
regulator, this observation constitutes important evidence in favor of the CSC hypothesis
and suggests that the effectiveness of Her-2 inhibitors, like Trastuzumab® and Lapatinib®,
could be connected with its ability to target the CSC fraction.
The encouraging results obtained with Her-2 inhibitors point to other proteins and signaling
pathways that regulate self-renewal of stem cells, such as Wnt, Notch, Bmp4 and Hh, as
potential targets for future therapeutic compounds. In this regard, targeting of Hh signaling
by Cyclopamine®, a steroid-like compound, has been shown to inhibit the growth of ovariancarcinoma (Chen et al., 2007b) and the EMT in pancreatic cancer cell lines, leading to asignificant reduction of in vitro invasiveness and metastatic potential in vivo (Feldmann etal., 2007). Interestingly, a combined treatment with Cyclopamine® and EGFR inhibitor not
only resulted in a reduction of tumor cell invasion in vitro but it also increased the rate of

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Embryonic and Cancer Stem Cells - two views of the same landscape387

apoptotic death in prostate carcinoma cells (Mimeault et al., 2006) and several humanpancreatic cancer cell lines (Hu et al., 2007). Moreover, it has been reported that
Cyclopamine® increases the cytotoxic effect of both radio- and chemotherapy in different
pancreatic cancer cell lines (Shafaee et al., 2006). The inhibition of Hh signaling may haveinteresting applications in the treatment of glioma. This idea is supported by the fact that thetreatment with this drug targets CSCs in vitro and leads to a reduction of glioma tumor
formation in vivo (Clement et al., 2007). Another interesting effect caused by the blocking of
the Hh pathway has been observed in multiple myeloma. In this poor prognosis tumor, theinhibition of Hh significantly reduced the myeloma stem cell clonal expansion and,additionally, stimulated the complete differentiation of these stem cells without affecting the
growth of other plasma cells (Peacock et al., 2007).
Some promising results related to blocking of the Notch signaling pathway have been
already obtained. The treatment of medulloblastoma cells with GSI-18, a inhibitor of Notch
signaling, produces a remarkable reduction of the CD133+ stem cell population and thecomplete eradication of the side population detected by flow cytometry in medulloblastoma
cell mass (Fan et al., 2006). Interestingly enough, the suppression of the stem cell fraction
correlates with the loss of tumorigenic potential of the cell mass when transplanted into
nude mice. Given that Notch signaling has been reported to participate in pancreaticdifferentiation, the targeting of this pathway might also be effective in the treatment ofpancreatic cancer (Murtaugh et al., 2003).
There are several studies that emphasize the importance of Bmp4 has a potential target for
anti-CSC therapies, this possibility is linked to the role of Bmp4 in the regulation of adultstem cell expansion (Hua et al., 2006). It has been reported, for example, that Bmp4 induces
a decrease in CD133+ glioblastoma CSC population in vitro and in vivo (Piccirillo et al.,2006).
PTEN is another factor known to participate in the maintenance of stem cell populations
and thus a potential target for cancer therapies. It is a tumor suppressor gene whose
expression is usually down-regulated in many tumor types (Di Cristofano & Pandolfi, 2000).
PTEN activity is mediated through the inhibition of PI3K/AKT signaling pathways, which
include the downstream target mTOR, and in this way modulates several processes related
to cell proliferation, growth and survival (Seeliger et al., 2007). A study performed withadult hematopoietic cells demonstrated that the conditional deletion of PTEN in these cells
induces an expansion of leukemic cancer cells whereas significantly reduces the proportionof normal hematopoietic stem cells. Additionally, the treatment with Rapamycin®, an mTOR
inhibitor, in order to overtake the PTEN deletion, leaded to the recovery of normal stem cell
population and a reduction in CSCs (Yilmaz et al., 2006).
A different strategy to target CSCs based on the cytotoxic effect of IFN-a has been recently
reported (Moserle et al., 2008). More precisely, it has been demonstrated that IFN-a presents
a remarkable antiproliferative and pro-apoptotic activity on ovarian cancer cells containinga high proportion of side population cells. Similarly, in vivo gene therapy with human IFN-α
leaded to a significant regression of large side population containing tumors, whereas notevident effects were observed in those tumors with a poor side population fraction. This
result may have an important therapeutic significance since the IFN-a activity seems
specifically target tumors that usually present a higher resistance to conventional treatments
and, thus, with high number of CSCs.
It has also been reported that the resistance to radiation of glioblastoma CSCs correlateswith an over-expression of DNA damage response genes (Bao et al., 2006). In fact, treatment

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Embryonic Stem Cells - Recent Advances in Pluripotent Stem Cell-Based Regenerative Medicineof this malignancy with radiotherapy leads to an increment of CD133+ glioblastoma cells. A
similar observation has been reported for colorectal cancer, in which the CSC population isenriched after chemotherapy. Moreover, the CSCs that remain are responsible for tumor
relapse and the acquisition of a more aggressive phenotype (Dylla et al., 2008). Colorectal
CSC resistance to chemotherapy agents like 5-fluorouracil and Oxaliplain® seems to be
mediated by CSC-derived IL-4, that acts as an autocrine apoptosis inhibitor (Todaro et al.,2007). Indeed, the pre-treatment of the colorectal tumor with IL-4 blocking antibodies results
in a significant increase in the treatments effectiveness.
Finally, it is important to note that those CSCs present in the tumor at one particular time
point, may change during the progression of the malignancy, resulting in a “moving
target” (Clarke et al., 2006; Roesch et al., 2010). Thus, the adequate therapy of a neoplasia
may depend of a balanced situation of CSCs rate of proliferation, stroma reaction anddegree of differentiation of the tumor parenchyma. In conclusion, the development of novel
therapies that target specifically the CSC population, may be effective for the treatment of
those cancer types that can be interpreted according to the CSC theory.

8. Acknowledgements
This work was supported by a research project from the Spanish Ministry of Science andInnovation (BFU 2007-66610) and a Research Group Grant (IT560-10) from the regional
Basque Government to J A. U S and L J-R had pre-doctoral fellowships from the Universityof the Basque Country and A D-T a post-doctoral fellowship from the Juan Gangoiti Barrera
Foundation of Bilbao, Spain.

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Embryonic Stem Cells - Recent Advances in Pluripotent Stem CellBased Regenerative Medicine
Edited by Prof. Craig Atwood

ISBN 978-953-307-198-5
Hard cover, 410 pages
Publisher InTech

Published online 26, April, 2011

Published in print edition April, 2011
Pluripotent stem cells have the potential to revolutionise medicine, providing treatment options for a wide
range of diseases and conditions that currently lack therapies or cures. This book describes recent advancesin the generation of tissue specific cell types for regenerative applications, as well as the obstacles that need to
be overcome in order to recognize the potential of these cells.

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