[Establishment of two human embryonic stem cell lines from cleavage arrested embryos].
OBJECTIVE
To determine whether cleavage developmentally retarded embryos have not cleaved during a 24 hour period could develop into blastocysts and produce hESC cell lines.


METHODS
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.


RESULTS
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.


CONCLUSIONS
Our results suggest that a subset of developmentally retarded embryos can form blastocysts and give rise to hESC cell lines.
Qian
Kun
K
Reproductive Medicine Center, Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology, Wuhan 430030, China.
Chen
Hong
H
Zhang
Han-Wang
HW
Jin
Lei
L
Li
Yu-Feng
YF
Zhang
Su-Ming
SM
Zhu
Gui-Jin
GJ
D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002970:Cleavage Stage, Ovum; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D007150:Immunohistochemistry; D016015:Logistic Models; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism
D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D012333:RNA, Messenger; D055746:Stage-Specific Embryonic Antigens