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.

Improved Efficiency of Microsurgical Enucleated Tripronuclear Zygotes Development and Embryonic Stem Cell Derivation by Supplementing Epidermal Growth Factor, Brain-Derived Neurotrophic Factor, and Insulin-Like Growth Factor-1

“…However, since all hESC lines have chromosomal abnormalities, the risks involved in this method need to be evaluated [33]. In our previous study, we also indicated that the diploid hESC lines can be derived from 3PN zygotes when one pronuclear is removed [8], and Jiang et al subsequently proved our results [9]; however, the lower efficiency limited the development of this method. Here, we have developed a new culture system to promote the application of 3PN zygotes.…”

“…However, the lower developmental efficiency of this process limited its application in the clinical setting, and only about 10% of the repaired 3PN zygotes could develop into the blastocyst stage [8,34]. Although Jiang et al showed that 25 r2PN embryos could develop into blastocysts using this method, the fact that only 2 hESC lines were derived from 11 blastocysts indicated the lower quality of the blastocysts obtained [9].…”

“…Jiang et al also indicated the lower efficiency of hESC lines derivation using similar r2PN embryos (2 cell lines from 11 blastocysts) [9]. In another study, Lerou et al derived hESC lines using poor-quality embryos such as early arrested or highly fragmented embryos and suggested a statistical correlation between the developmental stage of such poor-quality embryos and the yield of hESC lines [55].…”

“…Based on these findings, Fan et al derived the first hESC line using five blastocysts developed from 3PN zygotes repaired by microsurgery [8]. Subsequently Jiang et al obtained 2 hESC lines using 11 blastocysts generated by the microsurgical method [9]. Since the efficiency (*20%) was significantly lower than when normal fertilized blastocysts were used (*40%-50%), the use of polyspermic zygotes in the field of hESCs has been limited.…”

Derivation of normal diploid human embryonic stem cells from tripronuclear zygotes with analysis of their copy number variation and loss of heterozygosity

“…The 1PN embryos had approximately 48.7% normal diploid chromosomes (Yan et al, 2010), whereas the aneuploidy incidence in 3PN zygotes was approximately 70À80% (Staessen and Van Steirteghem, 1997). Despite these aneuploidy frequencies, hESCs derived from 0-, 1-, and 3PN zygotes can possess a normal karyotype and express typical stem-cell surface markers (Heins et al, 2004;Suss-Toby et al, 2004;Lavon et al, 2008;Chen et al, 2012a;Jiang et al, 2013). Indeed, our hESCs Zh19 and Zh20 carried normal karyotypes even though they were derived from 3PN zygotes, which are not usually regarded as suitable for continued culture in assisted-reproduction systems; how these 3PN zygotes generated hESC lines with normal karyotypes remains unclear.…”