Key Laboratory of Stem Cells and Reproductive Engineering

Changsha, China

Key Laboratory of Stem Cells and Reproductive Engineering

Changsha, China
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Yang Q.,Central South University | Zhou H.,Central South University | Li L.,Central South University | Du J.,Central South University | And 9 more authors.
Stem Cell Research | Year: 2015

Human embryonic stem cell (hESC) line was derived from abnormal blastocyst donated by Marfan syndrome patient after preimpantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that the hESC line carried the heterozygous deletion mutation, c.3536delA, of FBN1 gene. Characteristic tests proved that the hESC line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. © 2015 Elsevier B.V.

Tan Y.-Q.,Central South University | Tan Y.-Q.,Reproductive and Genetic Hospital of Citic Xiangya | Tan Y.-Q.,Key Laboratory of Stem Cells and Reproductive Engineering | Tan K.,Central South University | And 18 more authors.
Human Reproduction | Year: 2013

STUDY QUESTIONIs preimplantation genetic diagnosis (PGD) for translocation carriers more effective when done with a single-nucleotide polymorphism (SNP) array using trophectoderm (TE) biopsy and frozen embryo transfer (FET) compared with traditional PGD based on fluorescence in situ hybridization (FISH-PGD) using blastomere biopsy and fresh embryo transfer?SUMMARY ANSWERThe procedure using the SNP array combined with TE biopsy and FET significantly improves the clinical pregnancy rate for translocation carriers. The miscarriage rate also slightly decreases.WHAT IS KNOWN ALREADYFISH-PGD has been widely used in translocation carriers but the clinical outcomes have not been ideal. SNP arrays can detect both chromosome segmental imbalances and aneuploidy, and may overcome the limitations of FISH in PGD for translocation carriers.STUDY DESIGN, SIZE AND DURATIONThis was a retrospective study of 575 couples with chromosomal translocations, including 169 couples treated by SNP-PGD between October 2011 and August 2012, and 406 couples treated by FISH-PGD between January 2005 and October 2011.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study was set in an IVF center at the Reproductive and Genetic Hospital of CITIC-Xiangya, China. In total, 169 couples underwent SNP analysis, including 52 Robertsonian translocation carriers and 117 carriers of reciprocal translocations. Blastocysts (n = 773) were biopsied and FET was carried out on the balanced embryos. Four hundred and six couples underwent FISH-PGD, including 149 Robertsonian translocation carriers and 257 reciprocal translocation carriers. In total, 3968 embryos were biopsied and balanced embryos were transferred fresh. The SNP-PGD results and clinical outcomes were compared with those of FISH-PGD.MAIN RESULTS AND THE ROLE OF CHANCEReliable SNP-PGD results were obtained for 717 out of 773 (92.8%) biopsied blastocysts. The proportions of normal/balanced embryos, embryos with translocation-related and translocation-unrelated abnormalities, the median number of embryos per patient, the ongoing pregnancy rate per embryo transfer and the miscarriage rate were 58, 23, 19, 2, 69 and 12%, respectively, for Robertsonian translocation carriers and 36, 52, 12, 1, 74 and 11%, respectively, in reciprocal translocation carriers. Reliable FISH-PGD results were obtained for 3452 out of 3968 (87.0%) biopsied embryos. The proportions of normal/balanced embryos, unbalanced embryos, the median number of embryos per patient, the ongoing pregnancy rate per transfer and the miscarriage were 36, 64, 3, 38 and 17%, respectively, for Robertsonian translocation carriers and 20, 80, 1, 39 and 16%, respectively, for reciprocal translocation carriers. Thus, SNP-PGD achieved a higher pregnancy rate but a lower miscarriage rate than FISH-PGD. There were no significant differences in maternal age, basal endocrine level and the average number of retrieved oocytes and good-quality D3 embryos in the SNP-PGD group compared with the FISH-PGD group.LIMITATIONS, REASONS FOR CAUTIONThis was a retrospective study with the two groups treated in different periods; therefore, there is a chance of sample bias and a possibility that the results were influenced by other factors that changed over time. Furthermore, the two treatment protocols differ in several respects and we cannot say which makes the greatest contribution to the difference in success. Complete pregnancy outcomes of SNP-PGD have not been obtained as some embryos have not been transferred yet. We cannot exclude differences between the final data and the data in the present manuscript.WIDER IMPLICATIONS OF THE FINDINGSThe adoption of SNP-PGD combined with TE biopsy and FET may significantly improve the clinical pregnancy rate, and decrease the miscarriage rate after PGD for translocation carriers. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Leng L.,Central South University | Leng L.,Key Laboratory of Stem Cells and Reproductive Engineering | Tan Y.,Central South University | Tan Y.,Key Laboratory of Stem Cells and Reproductive Engineering | And 9 more authors.
Human Reproduction | Year: 2015

study question: Can the induced pluripotent stem cells (iPSCs) derived from women with primary ovarian insufficiency (POI) differentiate into germ cells for potential disease modeling in vitro? summaryanswer: The iPSC lines derived from POI patients with 46, X, del(X)(q26) or 46, X, del(X)(q26)9qh+ could differentiate into germ cells and expressed lower levels of genes in the deletion region of the X chromosome. what is known already: iPSC technology has been envisioned as an approach for generating patient-specific stem cells for disease modeling and for developing novel therapies. It has also been confirmed that iPSCs differentiate into germ cells. study design, size, duration:We compared the differentiation ability of germcells and the gene expression level of germcell-related genes in theXchromosome deletion regionof iPSClines derived fromPOIpatients (n = 2)with an iPSCline derived fromnormal fibroblasts (n = 1). participants/materials, setting, methods: We established three iPSC lines from two patients with partial Xq deletioninduced POI and normal fibroblasts by overexpressing four factors: octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Nanog homeobox (NANOG), and lin-28 homolog (LIN28), using lentiviral vectors.We then generated stable-transfected fluorescent reporter cell lines under the control of the Asp-Glu-Ala-Asp box polypeptide 4 (DDX4, also called VASA) promoter, and selected clonal derived sublines.We induced subline differentiation into germ cells by adding Wnt3a (30 ng/ml) and bone morphogenetic protein 4 (100 ng/ml). After 12 days of differentiation, green fluorescent protein (GFP)-positive and GFP-negative cells were isolated via fluorescence-activated cell sorting and analyzed for endogenous VASA protein (immunostaining) and for germ cell markers and genes expressed in the deleted region of the X chromosome (quantitative RT-PCR). main results and the role of chance: The POI-and normal fibroblast-derived iPSCs had typical self-renewal and pluripotency characteristics. After stable transfection with the VASA-GFP construct, the sublines POI1-iPS-V.1, POI2-iPS-V.1 and hEF-iPS-V.1 produced green fluorescent cells in the differentiated cultures, and the percentage of GFP-positive cells increased over the 12 days of differentiation to a maximum of 6.9±0.33%, 5.3±0.57% and 8.5±0.29%, respectively, of the total cell population. Immunohistochemical analysis confirmed that endogenous VASA was enriched in the GFP-positive cells. Quantitative reverse transcription-PCR revealed significantly higher expression of germ cell markers [PR domain containing 1, with ZNF domain (PRDM1, BLIMP1), developmental pluripotency-associated 3 (DPPA3, STELLA), deleted in azoospermia-like (DAZL), and VASA (DDX4)] in GFP-positive cells than in GFP-negative cells. Moreover, the GFP-positive cells from POIiPSCs had reduced expression of the family with sequence similarity 122C (FAM122C), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), and RNA binding motif protein, X-linked (RBMX), genes located in the deleted region of the X chromosome and that are highly expressed in differentiated germ cells, compared with cells from normal iPSCs. limitations, reasons for caution: Gene expression profiling indicated that the germ cells differentiated from POI-iPSCs were pre-meiotic. Therefore,howthe differentiated primordial germ cells could progress further to meiosis and form follicles remains to be determined in the study of POI. wider implications of the findings: Our results might provide an in vitro model for studying germ cell development in patients with POI. study funding/competing interest(s): This work was supported by grants fromthe Major State Basic ResearchDevelopment Program of China (No. 2012CB944901), the National Science Foundation of China (No. 81222007 and 81471432), the Program for New Century Excellent Talents in University and the Fundamental Research Funds for Central Universities (No. 721500003). The authors have no competing interests to declare. © 2015 The Author.

Zhang S.,Central South University | Zhang S.,Reproductive and Genetic Hospital of Citic Xiangya | Luo K.,Central South University | Luo K.,Reproductive and Genetic Hospital of Citic Xiangya | And 23 more authors.
Fertility and Sterility | Year: 2016

Objective To evaluate whether the developmental potential of the blastocyst is affected by the number of trophectoderm (TE) cells biopsied in preimplantation genetic diagnosis (PGD) cycles. Design Retrospective study. Setting University-affiliated center. Patient(s) Women underwent PGD cycles of blastocyst biopsy and fluorescence in situ hybridization analysis. Intervention(s) Not applicable. Main Outcome Measure(s) Biopsied TE cell number of blastocysts, survival, and implantation rates. Result(s) The biopsied TE cell number was affected by the TE quality and experience of different embryologists. The diagnostic efficiency increased when from one to five cells were biopsied (86.7%, 91.7%%, 96.0%, 96.8%, to 98.7%) and was maximized when more than six cells were biopsied. To compare the clinical efficiencies, blastocysts were divided into four groups according to biopsied TE cell number: 1-5, 6-10, 11-15, and 16-41. For the blastocysts with grade A TE score, no significant difference was observed in the survival and implantation rates among the four groups. For the blastocysts with grades B and C TE scores, the survival rates showed no significant differences among the four groups, but a significant decreasing trend in implantation rates was observed with increasing biopsied TE cell number. Conclusion(s) The implantation potential is negatively affected by the biopsied TE cell number in blastocysts with poor TE morphological score. © 2016 American Society for Reproductive Medicine, Published by Elsevier Inc.

Tan K.,Central South University | Tan K.,Key laboratory of Stem Cells and Reproductive Engineering | Tan Y.Q.,Central South University | Tan Y.Q.,Key laboratory of Stem Cells and Reproductive Engineering | And 19 more authors.
Genomics Data | Year: 2014

Translocation is one of the more common structural rearrangements of chromosomes, with a prevalence of 0.2%. The two most common types of chromosomal translocations, Robertsonian and reciprocal, usually result in no obvious phenotypic abnormalities when balanced. However, these are still associated with reproductive risks, such as infertility, spontaneous abortion and the delivery of babies with mental retardation or developmental delay. In recent years, array-based whole-genome amplification (WGA) technologies, including microarray comparative genomic hybridization (array CGH; aCGH) and single-nucleotide polymorphism (SNP) micro-arrays, have enabled the screening of every chromosome for whole-chromosome aneuploidy and segmental imbalance. These techniques have been shown to have clinical application for translocation carriers. Promising studies have indicated that array-based PGD of translocation carriers can lead to transfer pregnancy rates of 45-70% [2]. In addition to genetic testing techniques, the embryo biopsy stage (polar body, cleavage embryo or blastocyst) and the mode of embryo transfer (fresh or frozen embryos) can affect the outcome of PGD. It is now generally recommended that blastomere biopsy should be replaced by blastocyst biopsy to avoid a high mosaic rate and biopsy-related damage to cleavage-stage embryos, which might affect embryo development. However, more clinical data are required to confirm that the technique of SNP array-based PGD (SNP-PGD) combined with trophectoderm (TE) biopsy and frozen embryo transfer (FET) is superior to traditional FISH-PGD combined with Day 3 (D3) blastomere biopsy and fresh embryo transfer. © 2014.

Zhou H.,Central South University | Yang Q.,Central South University | Li L.,Central South University | Du J.,Central South University | And 9 more authors.
Stem Cell Research | Year: 2016

A human embryonic stem cell (hESC) line was derived from abnormal embryo donated by Glucose-6-phosphate dehydrogenase (G6PD) deficiency patient. Sequencing analysis confirmed that the hESC line possessed the mutant contributing to abnormal expression of G6PD. Further characteristic analysis demonstrated that the favism hESC line maintained stable and normal karyotype, expressed pluripotent markers and had the capacity of generating the derivatives from all three germ layers. © 2015 The Authors.

Zeng S.,Central South University | Zeng S.,Key Laboratory of Stem Cells and Reproductive Engineering | Liu L.,Central South University | Liu L.,Key Laboratory of Stem Cells and Reproductive Engineering | And 19 more authors.
Journal of Cell Science | Year: 2014

High telomerase activity is a characteristic of human embryonic stem cells (hESCs), however, the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion, but stabilized at later passages. We report that the core unit of telomerase, hTERT, was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore, this was regulated in a Wnt-β-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation, demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation, suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines, we were able to establish that telomere length remained relatively stable at 12.02 ± 61.01 kb during later passages (15-95). In contrast, telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary, we propose that correct, stable telomere length may serve as a potential biomarker for genetically stable hESCs. © 2014. Published by The Company of Biologists Ltd.

Sun Y.,Central South University | Sun Y.,Key Laboratory of Stem Cells and Reproductive Engineering | Yang Y.,Chongqing Medical University | Zeng S.,Central South University | And 5 more authors.
PLoS ONE | Year: 2014

Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. © 2014 Sun et al.

Xie P.,Central South University | Zhou H.,Central South University | Zhao X.,Reproductive and oGenetic Hospital of CITIC Xiangya | Du J.,Central South University | And 9 more authors.
Stem Cell Research | Year: 2016

We established a human embryonic stem cell (hESC) line chHES-427 from the abnormal embryo carrying homozygous deletion of exon 7 of survival motor neuron gene (SMN). This cell line maintained a normal karyotype 46, XX during long-term culture. Further characteristic analysis suggested that the cells expressed the pluripotency-related markers and had the capacity to differentiate into the derivatives from the three germ layers in vitro. © 2016 The Authors.

Xie P.,Central South University | Xie P.,Key Laboratory of Stem Cells and Reproductive Engineering | Sun Y.,Central South University | Sun Y.,Key Laboratory of Stem Cells and Reproductive Engineering | And 14 more authors.
Stem Cells | Year: 2014

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. © AlphaMed Press 2013.

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