[Establishment of two human embryonic stem cell lines from cleavage arrested embryos]

To determine whether cleavage developmentally retarded embryos have not cleaved during a 24 hour period could develop into blastocysts and produce hESC cell lines.

A total of 120 such embryos were cultured to blastocyst stage by sequential culture. Blastocysts formation rate and quality of blastocyst were detected under microscope. The relation between blastocyst formation rate and blastomere number, the fragment of blastomere and blastomere symmetry were analyzed by stepwise Logistical regression analysis. Inner cell masses (ICMs) were isolated by immunosurgery. Colonies derived from the ICMs were passed every 4 - 7 days and the derivatives were passaged and identified.

A total of 22 blastocysts were obtained from 120 embryos. The blastulation rate was 18.7%. Early blatocyst, blastocyst, full blastocyst, expanded blastocyst, hatching blastocyst and hatched blastocyst accounted for 5.9%, 23.5%, 35.3%, 23.5%, 5.9%, and 5.9% respectively. The grade of ICM and trophoblast was mostly scored C or B. Blastocyst formation rate was related to cell number and blastomere symmetry but not fragment. Immunosurgery resulted in the formation of 7 ICMs and 3 primary colonies, which produced 2 cell lines. The cell lines satisfied the criteria that characterize pluripotent hESC cells. Undifferentiated cells were positive for AKP, SSEA-4, TRA-1-60, and TRA-1-81. It could continue to proliferate in vitro and form embryoid bodies when cultured in suspension. It had capability to form teratoma in SCID mice. Both cell lines had normal karyotypes after 45 and 34 passages respectively.

Our results suggest that a subset of developmentally retarded embryos can form blastocysts and give rise to hESC cell lines.