Abstract
Human embryonic stem cells (hESCs) during long‐term culture acquire chromosomal changes similar to those occurring in tumorigenesis. This was raised concerns about the progression from hESCs to malignant cells. This study aimed to investigate the changes in chromosomes, cell phenotype, and genes in culture‐adapted hESCs to ascertain whether tumorigenic transformation occurred. By cytogenetic analysis we found progressive karyotypic changes from simple to complex in __ch__HES‐3, one of the hESC lines established in our laboratory, during a long‐term suboptimal culture. We further compared __ch__HES‐3 cells at different karyotypic stages in cell surface markers, in vivo differentiation, cell cycle, apoptosis, and gene expression profiles. We found that the karyotypically aberrant __ch__HES‐3 had higher S‐phase fraction in cell cycle distributions and antiapoptosis ability. In vivo differentiation of karyotypically normal __ch__HES‐3 resulted in relatively mature teratoma, whereas karyotypically aberrant __ch__HES‐3 formed immature teratoma (grade III), in which more primary neural epithelium was revealed by pathological analysis. The microarray analysis and real‐time PCR results showed that some oncogenes were upregulated in karyotypically aberrant __ch__HES‐3 cells, whereas the genes related to differentiation were downregulated, and that Wnt signal pathway was activated. In conclusion, __ch__HES‐3 cells underwent deregulation of self‐renewal and dysfunction of related genes in long‐term culture adaptation, leading to malignant transformation. © 2008 Wiley‐Liss, Inc.