Zito et al describe a high percentage of CD133+ cells in ARO and KAT4 cell lines and conclude that these cells have stem-like properties. As they discuss, these cell lines have been recently questioned for their origin. Particularly, it has been recently reported that these and other thyroid cancer cell lines have short tandem repeat (STR) profiles identical to that of the HT-29 colon cancer cell line (Schweppe et al., 2008). Thus, the finding that CD133 is present in ARO and KAT4 could fit with reports describing this glycoprotein as a marker for colon cancer (O'Brien C et al., 2006; Ricci-Vitiani et al., 2007; Todaro et al., 2007). In contrast, Zito et al show that the cell lines they use express thyroid specific factors such as thyroid transcription factor-1 (TTF1) and oncofetal fibronectin (onfFN) (Figures 2 and 7). Actually, these factors could be not specific for the thyroid gland. TTF-1 positivity, which is a typical feature of epithelial cells of the thyroid gland and the lung, has also been reported in primary and metastatic colonic adenocarcinoma using the SPT24 Novocastra antibody clone (Penman et al., 2006) while onfFN has been identified in association with several malignancies including, apart from thyroid tumors, advanced epithelial ovarian cancer (Menzin et al., 2000), human mammary tumors (Loridon-Rosa et al., 1990), hepatomas and sarcomas (Matsuura and Hakomori, 1985). Similarly, paired box gene 8 (PAX8) that Zito et al describe positive in ARO and KAT-4 without providing any picture, was shown to be expressed in ovarian and breast cancers. Thus, the presence of such markers in ARO and KAT-4 could not confirm that these cells are of thyroid origin.
Moreover, the medium used for propagation of CD133 positive cells (RPMI plus growth factors) should be not adequate for the expansion of cells with stem-like features.
In addition, immunocytochemistry of CD133 in ARO cell line shows a polarized localization of the antigen on the apical surface of the cells. As indicated by Corbeil et al. polarization might be related to proliferation. CD133 is concentrated in surface domains corresponding to the spindle pole region during metaphase. In telophase and cytokinesis, it is either equally or unequally distributed between the two nascent daughter cells (Bauer et al., 2008) . The concentration of CD133 in cleavage furrow during cytokinesis is consistent with the enrichment of microvilli like structures in neuroepithelial cells (Dubreuil et al., 2007). Since the CD133 positive cells shown by Zito el al are dividing and proliferating better than CD133 negative cells (Figures 1C, 4A and 4D), they cannot be considered stem cells which are believed to be quiescent. Thus, the cells shown in the picture could not be considered stem cells but dividing cells. We have provided evidence that ARO CD133+ cells are dividing cells by the co-expression with and the most of the cells that are positive are in cytokinesis. Zito et al showed that CD133 subpopulation accounted for almost 60% within two different anaplastic cell lines, this high amount of stem cell-like compartment could be attributed to the proliferation rate of the cells.
Besides, not all the CD133 positive cells could have stem-cell like properties. In order to verify this, a clonogenic assay based on the ability of a single cell to grow into a colony would had carried out. Finally, the study presented by Zito el al should be validated in in vivo animal models to ascertain that CD133 confers tumorigenicity based on the use of cells derived from human thyroid biopsy specimens and not questionable cell lines.


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