Presently, the method of establishing human embryonic stem cells mainly contains blastula method, nuclear transplantation method and embryo blastomere method. Embryo destruction is involved in many ethical problems.OBJECTIVE:
To explore the feasibility of establishing a new human embryonic stem cell lines using frozen cleavage human embryos.DESIGN, TIME AND SETTING:
The cytological in vivo or vitro study was performed at the Reproduction Center, Shenyang Gynecology and Obstetrics Hospital from January 2005 to August 2006.MATERIALS:
A total of 20 ICR mice at pregnancy of 13.5 days were used to prepare mouse embryo fibroblast feeder layer. Two SICD mice aged 10 weeks were used for human embryonic stem cell in vivo differentiation study. Frozen-thawed cleavage stage human embryos donated by couples undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) were supplied by Reproduction Center, Shenyang Gynecology and Obstetrics Hospital.METHODS:
Frozen-thawed cleavage stage human embryos were cultured to the blastocyst stage by sequential culture method. Trophocytes were removed by immunosurgical method. Obtained inner cell mass was plated on mouse embryonic fibroblast feeder layer following mitomycin-C inactivation.MAIN OUTCOME MEASURES:
Cultured cells at various passages were determined in following parameters: growth characteristics, alkaline phosphatase staining, transcription factor OCT-4, phase specific embryonal antigen SSEA-4, SSEA-1, tumor rejection antigen TRA-1-60, TRA-1-81, differentiation potentials and karyotypes.RESULTS:
Nine blastocysts were obtained from 20 frozen thawed human cleavage stage embryos, and 7 inner cell masses were isolated by immunosurgery, and established two human embryonic stem cell lines. One line was cultured for 76 passages (20 months). Cultured human embryonic stem cell colonies were compact, flat and round, with 1-3 nuclei, and positive for alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, OCT-4, but negative for SSEA-1. Karyotype was normal, 46, XX type. These cells were subcutaneously infused into SCID mice to form tumors. Pathological sections showed mature teratoma. Short tandem repeat typing results demonstrated that formed teratoma had similar genetic background as human embryonic stem cells.CONCLUSION:
Established human embryonic stem cells from frozen-thawed blastocysts have the characteristics of human embryonic stem cells, and may be a good source of human embryonic stem cell establishment.