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surface levels of CD271 as a stem cell marker (reviewed in [137,138] ). However, CD271 expression has also been
reported to inversely correlate with hypoxia (HIF-1α expression) in skin samples from human melanomas
analysed histologically at different stages of progression [139] and likely reflects a delay in its expression after
hypoxic induction. CD271 was noted to be highly expressed on the cells in the dermal nests of the biopsied
melanomas from human skin samples [139,140] .

In this regard, hypoxia and HIF activation induce Oct4 gene expression, which will in turn activate
[141]
CD271 . Thus, transfected gene expression of OCT4 caused melanoma cell dedifferentiation, acquiring
features of CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers,
ABCB5 and CD271 [141] . Mechanistically, Oct4-induced dedifferentiation was associated with increased
expression of endogenous stemness factors, OCT4, NANOG and KLF4, and changes in global gene
expression enriched for general transcription factors. The reverse study, applying RNAi-mediated
knockdown of Oct4 in the dedifferentiated melanoma cells, led to diminished CSC phenotypes [141] .
Moreover, OCT4 expression in melanoma cells was induced by hypoxia and its expression was detected in
a sub-population of melanoma cells from clinical samples [141] . Overexpressing CD271 on human melanoma
-
cell lines was shown to reduce their invasive and metastatic traits, whereas CD271 melanoma cells showed
less adhesive, more highly proliferative and invasive traits in vitro and in vivo [142] . Mechanistically, similar
to EPCAM, it was found that the CD271 ICD regulated proliferation [143] . CD271, originally expressed in the
epidermis of skin reconstructs, also disappeared when melanoma started to invade the dermis [142] . Thus,
CD271 has been identified as a suitable CSC marker, at least in the case of melanoma.

CD35 (Cripto-1)
Mouse and human Cripto-1 proteins range in sizes from 24 to 36 kDa and are found in complexes associated
with other proteins of between 14 to 60 kDa. Cripto-1 is glycosylated at N- and O-linked asparagine and
serine residues respectively [144] . It is a member of the epidermal growth factor (EGF)-related family of
growth factors, also known as the EGF-Cripto-1-FRL-1-cryptic (CFC) family. The EGF-CFC proteins are all
associated with the cell surface and their structures including an amino terminal region, a modified EGF-
like domain, a preserved CFC cysteine rich domain and a short hydrophobic COOH-terminus for attaching
to the glycosylphosphatidylinositol (GPI) membrane anchor [144] . The soluble, biologically active form of
Cripto-1 generates from the detachment of the GPI by GPI-phospholipase D such that Cripto-1 can act either
as a soluble ligand or a cell-membrane anchored protein [144] .

Cripto-1 was found highly expressed in a subpopulation (up to 60%) of human embryonal carcinoma
cells which showed CSC-like properties, expressing OCT4, SOX2, and NANOG. Cripto-1 has a role in
the maintenance of stem cells and their malignant progression [145] as a multifunctional modulator during
embryogenesis, as well as being important for oncogenesis [144,146] . In the process of embryogenesis, Cripto-1
plays an important role in association with the TGFb ligand, Nodal [147] . Cripto-1 is highly expressed in many
different human tumors, findings consistent with its role as a marker of the undifferentiated CSCs [148-150] .

Cripto-1 is one of the master regulators of embryonic development along with the Notch/CSL, and WNT/
b-catenin systems [151] . Hence, Cripto-1 is a surface marker for the identification of stem and quiescent
cancer cells including the CSC phenotype in colorectal cancers and similar to the other CSC markers LGR5,
EPCAM and CD271 described above, Cripto-1 expression is also up-regulated by hypoxia when HIF-1α
binds to the hypoxia response elements [145,152] within the promoter of human or mouse Cripto-1 genes [153] .
Hypoxia can enhance production of beating cardiomyocytes and modulates the differentiation of mouse
ESCs and Cripto-1 is required for the hypoxia-induced differentiation of mouse ESCs into cardiomyocytes,
helping to regulate stem cell self-renewal and proliferation [153] . It follows that Cripto-1 could be a potential
marker for sub-populations of tumor cells with stem-like properties.