Almost all CD133-related experiments carried out to date have made use of two mAbs AC133 and AC141 which were originally generated to aid in the purification and characterization of hematopoietic stem and progenitor cells and which subsequently led to the identification of the CD133 surface protein antigen [1
]. Despite increasing reports of the successful use of the AC133 and AC141 mAbs to identify and purify CSC populations, there are several factors that need to be considered when interpreting these results (). First, there is very little information available about the molecular nature of the epitopes bound by the two antibodies. The AC133 and AC141 mAbs are widely reported to bind glycosylated epitopes on CD133, but the evidence supporting these claims is not well documented in the literature, consisting of unshown data [2
] in the case of AC133 mAb and a brief reference to unpublished data [5
] in the case of AC141 mAb. The exact location of the modified amino acid residues on CD133 targeted by AC133 and AC141 mAbs has not been described. Despite the fact that the AC133 and AC141 mAbs are commonly used interchangeably for analysis and cell purification, discordant expression of the AC133 and AC141 epitopes was observed in marrow precursor and peripheral blood stem cells from patients with myelodys-plastic syndrome and acute myelogenous leukemia [42
]. Additionally, several studies have demonstrated that the AC133 and AC141 epitopes can be downregulated independently from the CD133 protein or mRNA [5
], and the tissue distribution of CD133 mRNA was found to be much more widespread than expression of the AC133 epitope [2
]. An additional complicating factor is the presence of alternatively spliced CD133 isoforms. The human CD133 gene contains at least 37 exons spanning 152 kb on chromosome 4, and several alternatively spliced forms of CD133 have been described in both human [44
] and mouse [46
]. For example, a CD133 isoform (termed AC133-2) lacking exon 4 results in a deletion of nine amino acids in the N-terminal extracellular domain [45
]. Interestingly, AC133-2 was shown to be the predominant CD133 isoform expressed on hematopoietic stem cells and presumably recognized by the AC133 and AC141 mAbs [45
]. Although currently unknown, alternatively spliced CD133 isoforms lacking the AC133 or AC141 epitopes could exist. Thus, given these complexities, it may be incorrect to call AC133 or AC141 epitope-negative cells “CD133-negative” without proper verification of CD133 protein or mRNA levels. Indeed, it is possible that the glycosylation status of CD133 may be a more specific marker of the CSC phenotype than CD133 protein levels. Another issue is that the AC141 mAb displays cross-reactivity with cytokeratin 18 [47
], which could be a complicating factor with fixed cells or cells with damaged membranes such as those in immunohistochemistry studies. In addition, because the nature of the glycosylated epitopes have not been defined (i.e., sugar structure and peptide context), it is formally possible that the AC133 and/or AC141 mAbs recognize glycosylated epitopes on molecules other than CD133. Despite the uncertainty surrounding the target epitopes and specificity of the AC133 and AC141 mAbs, they have nonetheless been extremely useful reagents for CSC research. However, these factors should be kept in mind, particularly when analyzing the results of experiments carried out with “CD133-negative” cells purified with the AC133 and AC141 mAbs.
Anti-CD133 antibodies other than AC133 and AC141 that target unmodified extracellular CD133 epitopes are commercially available. For example, the rabbit polyclonal Abs H-284, mouse mAb 32AT1672, and rabbit mAb C24B9 were produced by immunizing with peptides corresponding to predicted extracellular regions of CD133 (). Although the use of these antibodies for isolation of CD133-positive cells has not been reported in the literature, they could be suitable for this application. By comparing the results of CSC isolation experiments done with anti-CD133 antibodies that target unmodified extra-cellular CD133 epitopes to the results obtained with the glycosylation-specific AC133 and AC141 mAbs, it should be possible to determine if there is a correlation between the glycosylation status of CD133 and the CSC phenotype.
Commercially available anti-CD133 antibodies recognizing nonglycosylated extracellular epitopes that could be suitable for CSC isolation