Triploid and diploid embryonic stem cell lines derived from tripronuclear human zygotes.
ABSTRACT Human embryonic stem cells (hESCs) are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. The need for new hESC lines motivated our attempts to find a new resource for the derivation of hESC lines. The aim of this work was to establish more hESC lines from abnormal fertilized zygotes and to meet the emerging requirements for their use in cell replacement therapies, disease modeling, and basic research.
A total of 130 tripronuclear human zygotes was collected 18-20 h post-insemination and cultured in a modified culture medium. The inner cell mass of 12 blastocysts were isolated by a mechanical method in order to establish embryonic stem cell lines.
We established four hESC lines derived from 130 trinuclear zygotes, one of which was triploid and the others were diploid. The efficiency of deriving hESC lines is 3.08 %. The ratio of deriving triploid and diploid hESC lines is 1:3. All of these hESC lines exhibited similar markers of undifferentiated hESCs and had the typical morphology of hESCs, a capacity for long-term proliferation, and pluripotent differentiation potential both in vivo and in vitro.
These abnormal zygotes, which otherwise would have been discarded, can serve as an alternative source for normal euploid hESC lines.
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ABSTRACT: Human embryonic stem cells (hESCs) hold great promise for future clinical cell therapies because of their unique potential to differentiate into all human cell types. However, the destruction of normal fertilized embryos and derivation of hESCs for research has resulted in polarized ethical debates, with most of the controversy centered on embryo destruction. Therefore, because of less ethical controversy surrounding them, abnormal fertilized zygotes that are usually discarded are a potential feasible resource for the derivation of hESCs Microsurgery on human polyspermic zygotes can contribute to the derivation of human ESCs, but the efficiency is much lower. Here we reported a culture system to enhance the development competence of such microsurgical human polyspermic zygotes by EGF-BDNF-IGF-1 combination, which eventually resulted in the increased derivation efficiency of human embryonic stem cells from them. We found that the developmental efficiency of microsurgical enucleated tripronuclear embryos cultured with the EGF-BDNF-IGF-1 combination was significantly increased compared with control group. More importantly, when the microsurgical enucleated tripronuclear embryos were cultured in medium supplemented with EGF-BDNF-IGF-1, the frequency ratio of chromosome abnormality was reduced. Our present study will facilitate the development of hESC lines derivation in subsequent studies and also provide an additional choice for infertile couples.Stem cells and development 11/2013; · 4.15 Impact Factor
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ABSTRACT: Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.Cell cycle (Georgetown, Tex.) 01/2012; 12(2). · 5.24 Impact Factor
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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. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.Journal of Cellular Biochemistry 04/2013; 114(9). · 3.37 Impact Factor