Objective: To establish a protocol for culturing of human embryonic stem cells (HUES4) without any animal-derived feeder cells and to investigate the karyotype stabilities of HUES4 cells after long-term cultivation.
Methods: HUES4 cells were cultured on mitomycin C treated MEFs or human foreskin fibroblast feeder cells. The pluripotency of the ES cells was analyzed by immunocytochemistry staining to detect the expression of pluripotent marker, karyotype of the ES cells at passage 27, 34, 41, 44 and short tandem repeat (STR) at passage 27 were analyzed.
Results: The HUES4 cells cultured on human feeder cells were positive for alkaline phosphatase activity, SSEA-4, TRA-1-60 and TRA-1-81 staining, but negative for SSEA-1. Analysis of karyotype at different passages suggested an abnormal karyotype 46, XY, t(9;15)(q22;q26) mosaicism occurred in HUES4, and the ratios of abnormal increased with passage.
Conclusion: HUES4 could be cultured without animal-derived feeder cells and the incidence of abnormal karyotype might be increased with long-term culture.