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Stem/progenitor cells are involved in the regeneration of the renal tubules after damage due to a toxic insult. However, the mechanism involved in the regeneration of the tubules by the stem cells is not well understood due to the lack of immortal cell lines that represent the stem/progenitor cells of the kidney. A previous study from our laboratory has shown that the immortalized cell line RPTEC/TERT1 contains two populations of cells, one co-expressing CD24 and CD133, the other expressing CD24 only. The goal of the present study was to determine if both these populations could be sorted into separate independent cultures and if so, determine their characteristic features and response to the nephrotoxicant cadmium. The results of our study show that both the populations of cells could grow as independent cultures and maintain their phenotype after extended sub-culture. The CD133+/CD24+ co-expressing cells formed multicellular spheroids (nephrospheres), a characteristic feature of stem/progenitor cells, and formed branched tubule-like structures when grown on the surface of matrigel, whereas the CD133−/CD24+ cells were unable to form these structures. The CD133+/CD24+ cells were able to grow and undergo neurogenic, adipogenic, osteogenic, and tubulogenic differentiation, whereas the CD133−/CD24+ cells expressed some of the differentiation markers but were unable to grow in some of the specialized growth media. The CD133+/ CD24+ co-expressing cells had a shorter doubling time compared to the cells that expressed only CD24, and were more resistant to the toxic effects of the heavy metal, cadmium. In conclusion, the isolation and characterization of these two cell populations form the RPTEC/TERT1 cell line will facilitate the development of studies that determine the mechanisms involved in tubular damage and regeneration particularly after a toxic insult.CD24 and CD133 proximal tubule progenitors repopulate the nephron after cell death.The RPTEC/TERT1 cell line was cell sorted for these two markers.CD133+/CD24+ cells showed tubulogenic, adipogenic and osteogenic differentiation.CD133−/Cd24+ where not capable of differentiation.CD133+/CD24+ cells were more resistant to cadmium compared to CD133−/Cd24+.