Most current research on cancer stem cells (CSCs) associated with human tumorshas focused on the molecular and cellular analysis of hematopoietic lineagemarkers (e.g., CD44, CD138, and CD 133), which can also serve as important CSC markers in a variety of cancers. However, these markers are generallyexpressed at late stages in embryonic development. Oct-3/4, a member of thefamily of POU-domain transcriptional factors, and alkaline phosphatase (ALP) areknown to be expressed in the inner cell mass of blastocysts, germ cells, andpluripotent embryonic stem cells. We thus consider Oct-3/4 and ALP to bepromising markers for CSC. Herein, we examined expression of Oct-3/4 and ALPusing 6 established human pancreatic carcinoma cell lines. RT-PCR analysisrevealed the presence of Oct-3/4 and ALP mRNA in those cells.Immunocytochemical and cytochemical staining revealed that both Oct-3/4 andALP proteins are present as mosaics in PANC-1 cell line, one of those 6 cell lines(23% and 19%, respectively). However, Oct-3/4-positive PANC-1 cells did notexhibit overt ATP-binding cassette transporter G2 (ABCG2) activity, as revealedby Hoechst 33342 dye exclusion assay. Transfection of PANC-1 cells with anOct-3/4 promoter-directed, enhanced green fluorescent protein (EGFP) constructconfirmed the presence of Oct-3/4-positive cells. These findings indicate that inPANC-1 cells there are at least 2 subset populations, namely Oct-3/4-positive andALP-positive cells. However, it remains unknown whether expression of these 2markers overlaps. Enrichment of Oct-3/4- or ALP-positive cells by gene transferand subsequent drug selection will be helpful for further characterization of thesecells as possible CSCs.