We confirmed the stemness of CCA-derived spheres by examining the expression of stemness genes essential for the proliferation, self-renewal and differentiation of stem cells, including CD13, CD24, CD44, CD90, CD133, ALDH1A1, epithelial cell adhesion molecule (EpCAM), Krüppel-like factor 4 (Klf4) and octamer-binding transcription factor 4 (OCT4). The gene discussed is KLF4; the disease is cholangiocarcinoma.