[Human embryonic stem cell lines preliminarily established in China]

Abstract

Objective: To establish embryonic stem (ES) cell lines from human fertilized ovum in China.

Methods: Fourteen human oocytes obtained from a volunteer were cultured in Earlg's medium and fertilized in vitro. Four of the morulae formed from the fertilized ovi were frozen and five continued to develop into blastocysts. The pellucid zones of the 5 blastocysts were removed and their inner cell masses (ICM) were taken out and inoculated onto the feeder layer of mouse embryonic fibroblasts inactivated with mitomycin C. The cell clones formed from the three surviving ICM were selected and dispersed mechanically and the cells were inoculated onto new feeder layers to produce more cell clones. When multiple clones were produced, they were dissociated with dispase into smaller cell masses which were inoculated onto new feeder layer again for subculturing. During different periods of cell culture, alkaline phosphatase activity (AKP) staining, stem cell surface marker test, and karyotyping were conducted. The suspension of stem cells subcultured for 5 months were inoculated to the legs of severe combined immunodeficiency (SCID) mice subcutaneously to observe the teratoma formation.

Results: Out of the 9 fertilized oocytes, 5 developed into blastocysts. The ICM of three from the five blastocysts survived and developed into embronic stem cell lines, named CHE1, CHE2, and CHE3 respectively. These stem cells remained undifferentiated after subculturing for 7 months. The stem cell line CHE3 had been grown for 40 passages in vitro, CHE1 for 36 passages, and CHE2 for 32 passages. AKP staining was positive at the passages 5, 15, and 30 for the 3 cell lines. The surface markers of human stem cell, SSEA-4, SSEA-3, TRA-1-60, and GCTM-2 were positive at passage 25, 30, 40 for CHE3, and at the passages 21, 22 and 30 for CHE1 and CHE2. But all cell lines did not express SSEA-1, a marker for mouse ES cells. All cell lines stably retained a normal karyotype. After routine passage by 5 months, human ES cells from 3 cell lines were injected subcutaneously into SCID mice. Six weeks after inoculation of the 3 cell lines teratomas were formed in all mice. Histological examination revealed that they contained various tissues derived from all three embryonic germ layers.

Conclusion: Human embryonic stem cell lines were successfully established in China.