Hong-Qing Liao1, Qi OuYang2,3, Shuo-Ping Zhang2,4, De-Hua Cheng4, Guang-Xiu Lu2,3,4, Ge Lin5,6,7. 1. The Second Affiliated Hospital of South China University, 30th Jie-Fang Road, Hengyang, Hunan, 421001, China. potlhq@gmail.com. 2. Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha, Hunan, 410078, China. 3. National Engineering and Research Center of Human Stem Cell, Changsha, Hunan, 410078, China. 4. CITIC-Xiangya Reproductive and Genetic Hospital, Changsha, Hunan, 410078, China. 5. Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha, Hunan, 410078, China. linge_csu@yahoo.com. 6. National Engineering and Research Center of Human Stem Cell, Changsha, Hunan, 410078, China. linge_csu@yahoo.com. 7. CITIC-Xiangya Reproductive and Genetic Hospital, Changsha, Hunan, 410078, China. linge_csu@yahoo.com.
Abstract
PURPOSE: This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes. METHODS: After sequential culture for 5-6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis. RESULTS: The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P < 0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P < 0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P < 0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes. CONCLUSIONS: Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.
PURPOSE: This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes. METHODS: After sequential culture for 5-6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis. RESULTS: The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P < 0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P < 0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P < 0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes. CONCLUSIONS: Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.
Authors: Richard Porter; Taer Han; Michael J Tucker; James Graham; Juergen Liebermann; E Scott Sills Journal: J Assist Reprod Genet Date: 2003-09 Impact factor: 3.412
Authors: Maisam Mitalipova; John Calhoun; Soojung Shin; David Wininger; Thomas Schulz; Scott Noggle; Alison Venable; Ian Lyons; Allan Robins; Steven Stice Journal: Stem Cells Date: 2003 Impact factor: 6.277