title: Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes.
abstract: A normal fertilized human zygote contains two pronuclei, but zygotes may also display one, three, or even more pronuclei resulting from irregular insemination or meiotic division. Today diploid and triploid human embryonic stem cell (hESC) lines have been derived from tripronuclear (3PN) triploid zygotes, and an in-vitro fertilization (IVF) baby was born from a rescued diploid zygote by removing the extra male pronucleus of the 3PN zygote. However, whether hESCs can be derived from a rescued 3PN zygote is still unknown. Here, by microsurgical pronuclear removal, we restored 61 diploid zygotes from 3PN zygotes donated by 35 couples, and 11 blastocysts developed with a blastocyst rate of 18.0%, which seems higher than that of nonrescued 3PN zygotes according to previous reports. After the whole zona pellucida free embryos were plated onto feeder cells to grow and passage, 2 hESC lines (CCRM-hESC-22 and CCRM-hESC-23) were generated and both carried normal karyotype (46, XY). The hESC lines were then characterized by morphology, expansion in vitro, and expression of specific markers of alkaline phosphatase, OCT4, SSEA4, TRA-1-60 and TRA-1-81. Furthermore, the pluripotency of these 2 hESC lines was confirmed by in vitro embryoid body formation and in vivo teratoma production. Our study indicates that depronucleared 3PN zygotes can improve the blastocysts formation rate, and normal hESC lines can be derived from those corrected 2PN embryos. Based on their multi-directional differentiation potential in vitro, the established hESC lines could be applied to the developmental risk assessment for IVF babies born from restored zygotes.
authors.affiliation: State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Jiang
authors.forename: Lingbo
authors.initials: L
authors.lastname: Cai
authors.forename: Boxian
authors.initials: B
authors.lastname: Huang
authors.forename: Juan
authors.initials: J
authors.lastname: Dong
authors.forename: Aiqin
authors.initials: A
authors.lastname: Chen
authors.forename: Song
authors.initials: S
authors.lastname: Ning
authors.forename: Yugui
authors.initials: Y
authors.lastname: Cui
authors.forename: Lianju
authors.initials: L
authors.lastname: Qin
authors.forename: Jiayin
authors.initials: J
authors.lastname: Liu
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D059785:Karyotype; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015053:Zygote / Q000378:metabolism*
keywords: HUMAN EMBRYONIC STEM CELLS; MICROSURGICAL PRONUCLEAR REMOVAL; TRIPRONUCLEAR ZYGOTES