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ABSTRACT: Abstract The generation of disease-specific induced pluripotent stem cell (iPS cell) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and for drug screening. Such innovation enables us to obtain autologous cell sources for regenerative medicine. Herein, we report the generation and characterization of iPS cells from the fibroblasts of patients with a family history of Duchenne muscular dystrophy (DMD); these fibroblasts were obtained from patients at 22 gestational weeks of age and exhibit exon duplication from exons 16 to 42. The DMD-iPS cells were generated by the ectopic expression of four transcription factors: OCT4, SOX2, KLF4 and c-MYC; the DMD-iPS cells expressed several pluripotency markers and could be differentiated into various somatic cell types both in vitro and in vivo. Furthermore, DMD-iPSCs showed the differentiation potential to neuronal lineage. Thus, DMD-iPS cells are expected to serve as an in vitro disease model system, which will lay a foundation for the production of autologous cell therapies that avoid immune rejection and enable the correction of gene defects prior to tissue reconstitution.
The International journal of neuroscience 03/2013; · 0.86 Impact Factor
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ABSTRACT: To reprogram amniotic fluid cells into pluripotent stem cells in order to create an optimal internal control model for directed cell differentiation.
Human amniotic fluid-derived cells (hAFDCs) from heterozygotic twin fetuses were induced by retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4. In vivo pluripotency, differentiation capacity and karyotype of hAFDCs induced pluripotent stem cells (hAFDCs-iPSCs) were determined.
hAFDC-iPSCs derived from heterozygotic twins have maintained self renewal, with expression of high pluripotency marker gene detected at both mRNA and protein levels. The cells have maintained their differentiation capacity both in vitro and vivo, and showed normal karyotypes after long-term culturing in vitro.
hAFDCs-iPSCs derived from heterozygotic twins have good consistency in terms of genetic background, and can provide a good internal control for directed differentiation of iPSCs, and may be used an ideal source for autologous cell replacement therapy in the later life of the fetus.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 08/2012; 29(4):398-403.
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ABSTRACT: Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.
Journal of Reproduction and Development 05/2012; · 1.46 Impact Factor
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ABSTRACT: Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may have a wide range of applications in cell and gene therapy. However, the safety issues and the low efficiency associated with generating human iPSCs have limited their usage in clinical settings. The cell type used to create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid cells from the prenatal diagnosis of a β-thalassemia patient can be efficiently reprogrammed using a doxycycline (DOX)-inducible humanized version of the single lentiviral "stem cell cassette" vector flanked by loxP sites, which can be excised with Cre recombinase. We also demonstrated that the patient-derived iPSCs can be characterized based on the expression of pluripotency markers, and they can be differentiated into various somatic cell types in vitro and in vivo. Moreover, microarray analysis demonstrates a high correlation coefficient between human β-thalassemia iPS cells and human embryonic stem (hES) cells but a low correlation coefficient between human β-thalassemia amniotic fluid cells and human β-thalassemia iPS cells. Our data suggest that amniotic fluid cells may be an ideal human somatic cell resource for rapid and efficient generation of patient-specific iPS cells.
Journal of Reproduction and Development 04/2012; 58(4):404-9. · 1.46 Impact Factor
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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.
Journal of Assisted Reproduction and Genetics 04/2012; 29(8):713-21. · 1.84 Impact Factor
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Yong Fan,
Yonghua Jiang, Xinjie Chen,
Zhanhui Ou,
Yifei Yin,
Shengchang Huang,
Zhaohui Kou,
Qing Li,
Xiaolin Long,
Jianqiao Liu,
Yuemei Luo,
Baoping Liao,
Shaorong Gao,
Xiaofang Sun
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ABSTRACT: Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5 nM TSA for 10 h following activation incubation increased the developmental competence of human SCNT embryos constructed from β-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.
Stem cells and development 02/2011; 20(11):1951-9. · 4.15 Impact Factor
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ABSTRACT: Human embryonic stem cells (HESCs) are defined as self-renewing cells that retain their ability to differentiate into all cell types of the body. They have enormous potential in medical applications and as a model for early human development. There is a need for derivation of new HESC lines to meet emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. Here, we describe a modified culture medium containing human recombinant leukemia inhibitory factor and human basic fibroblast growth factor that significantly increases the number of human blastocysts formed and their quality, as well as the efficiency of HESC derivation from poor-quality embryos. Culturing poor-quality embryos in modified medium resulted in a two-fold increase in the blastocyst formation rate and a seven-fold increase over the derivation efficiency in conventional medium. We derived 15 HESC lines from poor-quality embryos cultured in modified culture medium and two HESC lines from quality embryos cultured in conventional culture medium. All cell lines shared typical human pluripotent stem cell features including similar morphology, normal karyotypes, expression of alkaline phosphatase, pluripotency genes, such as Oct4, and cell surface markers (SSEA-4, TRA-1-60, TRA-1-81), the ability to form teratomas in SCID mice, and the ability to differentiate into cells of three embryonic germ layers in vitro. Our data suggest that poor-quality embryos that have reached the blastocyst stage in our modified culture medium are a robust source for normal HESC line derivation.
Journal of Reproduction and Development 10/2010; 56(5):533-9. · 1.46 Impact Factor
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ABSTRACT: After fertilization, male and female gametes undergo extensive reprogramming to restore totipotency. Both DNA methylation and histone modification are important epigenetic reprogramming events. Previous studies have reported that the paternal pronucleus of the human zygote is actively demethylated to some extent, while the maternal pronucleus remains methylated. However, to our knowledge, the relationship between DNA methylation and H3K9 dimethylation patterns in human embryos has not been reported. In this study, we examined the dynamic DNA methylation and H3K9 dimethylation patterns in triploid and bipronucleated zygotes and early developing embryos. We sought to gain further insight into the relationship between DNA methylation and H3K9 dimethylation and to investigate whether removing a pronucleus from triploid zygotes affects DNA methylation and H3K9 dimethylation patterns. We found that active DNA demethylation of the two male pronuclei occurred in tripronuclear human zygotes while the female pronucleus remained methylated at 20 h post-insemination. In tripronuclear human zygotes, H3K9 was hypomethylated in the two paternal pronuclei relative to the maternal pronucleus. Our data show that there are no differences in the DNA methylation and H3K9 dimethylation patterns between tripronuclear and corrected bipronuclear human zygotes. However, correction of 3PN human zygotes dispermic in origin could not improve subsequent embryo development. In conclusion, DNA methylation and H3K9 dimethylation patterns are well correlated in tripronuclear zygotes and embryos; early embryo development is not affected by removal of a male pronucleus. Our results imply that limited developmental potential of either 3PN or corrected 2PN embryos may not be caused by the abnormalities in DNA methylation or H3K9 dimethylation modification.
Journal of Reproduction and Development 03/2010; 56(3):324-9. · 1.46 Impact Factor
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Xinjie Chen,
Tianqing Li,
Xuemei Li,
Yunhua Xie,
Xiangyu Guo,
Shaohui Ji,
Yiyu Niu,
Yang Yu,
Chenhui Ding,
Ruqiang Yao,
Shihua Yang,
Weizhi Ji,
Qi Zhou
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ABSTRACT: A simple monoculture system, combined with a chemically defined medium containing hepatocyte growth factor (HGF) and G5 supplement, was used to induce rhesus monkey embryonic stem cells (rESC) directly into neuroepithelial (NE) cells. Under these conditions, the generation of NE cells did not require the formation of embryoid bodies or co-culture with other cell types. The NE cells could further develop to generate neurons, astrocytes and oligodendrocytes. These results demonstrate a simple approach to obtain enriched and expandable populations of neural progenitors. Importantly, unlike other systems, the neural progenitors obtained using this approach may possess the potential to differentiate into various regional neural cells. Finally, the results suggest that the time-dependent shift in the differentiation potential of the rESC-derived neural progenitors in vitro reflects the developmental events that occur during neurogenesis in vivo. Thus, this system can be used to study the mechanisms of cell fate specification during non-human primate neurogenesis.
Reproductive biomedicine online 09/2009; 19(3):426-33. · 2.04 Impact Factor
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ABSTRACT: The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.
Biochemical and Biophysical Research Communications 05/2009; 382(1):119-23. · 2.48 Impact Factor
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Weiqiang Liu,
Yifei Yin,
Xiaolin Long,
Yumei Luo,
Yonghua Jiang,
Wenhong Zhang,
Hongzi Du,
Shaoying Li,
Yuhong Zheng,
Qing Li, Xinjie Chen,
Baoping Liao,
Guohong Xiao,
Weihua Wang,
Xiaofang Sun
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ABSTRACT: Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.
Journal of Genetics and Genomics 04/2009; 36(4):229-39. · 1.88 Impact Factor
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ABSTRACT: The oocyte plays a key role in reprogramming the epigenetic status of donor cell nuclei, and the absence of reprogramming elements in the cytoplasm or aberrant accumulation of proteins can trigger the abnormal development of nuclear transfer (NT) embryos. Previous studies have demonstrated the relationship between oocyte morphology and both embryo development and pregnancy outcome. In the present study, we compared the morphology of oocytes with subsequent development of human somatic cell NT (SCNT) embryos.
Piezo-assisted SCNT technology was used to produce reconstructed embryos, with almost 92% of oocytes reconstructed successfully. Depending on their morphologies, we separated metaphase II oocytes into four grades according to criteria which assess oocyte morphology, first polar body and perivitelline space, and especially, cytoplasm granula distribution.
Embryos from oocytes of Grades A and B could develop to the blastocyst stage with similar development efficiency for every developmental stage. However, embryos from Grade C oocytes arrested at or before the 8-cell stage then degraded, and the donor cell genome could not be activated and reprogrammed in such oocytes. For Grade D oocytes, cleavage was not observed in the reconstructed embryos, suggesting that the oocytes themselves have no developmental potential.
Our study revealed that different levels of developmental competence of SCNT embryos resulting from different oocyte reprogramming potentials associated with different morphologies. The results suggest that effective methods for improving oocyte quality should be studied, and that human SCNT efficiency would be increased following simple assessment of established oocyte morphology criterion.
Human Reproduction 01/2009; 24(3):649-57. · 4.47 Impact Factor
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Xiaofang Sun,
Xiaolin Long,
Yifei Yin,
Yonghua Jiang, Xinjie Chen,
Weiqiang Liu,
Wenhong Zhang,
Hongzi Du,
Shaoying Li,
Yuhong Zheng,
Shu Kong,
Qianying Pang,
Yu Shi,
Yulin Huang,
Shengchan Huang,
Baoping Liao,
Guohong Xiao,
Weihua Wang
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ABSTRACT: Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar.
Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines.
All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines.
The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.
Human Reproduction 08/2008; 23(10):2185-93. · 4.47 Impact Factor
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Cell Research 07/2008; · 8.19 Impact Factor
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Cell Research 07/2008; · 8.19 Impact Factor
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Yang Yu,
Chenhui Ding,
Eryao Wang, Xinjie Chen,
Xuemei Li,
Chunli Zhao,
Yong Fan,
Liu Wang,
Nathalie Beaujean,
Qi Zhou,
Alice Jouneau,
Weizhi Ji
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ABSTRACT: Even though it generates healthy adults, nuclear transfer in mammals remains an inefficient process. Mainly attributed to abnormal reprograming of the donor chromatin, this inefficiency may also be caused at least partly by a specific effect of the cloning technique which has not yet been well investigated. There are two main procedures for transferring nuclei into enucleated oocytes: fusion and piezoelectric microinjection, the latter being used mostly in mice. We have, therefore, decided to compare the quality and the developmental ability, both in vivo and in vitro, of embryos reconstructed with electrofusion or piezoelectric injection. In addition, the effect of piezo setups of differing electric strengths was investigated. Along with the record of the rate of development, we compared the nuclear integrity in the blastomeres during the first cleavages as well as the morphological and cellular quality of the blastocysts. Our results show that the piezo-assisted micromanipulation can induce DNA damage in the reconstructed embryos, apoptosis, and reduced cell numbers in blastocysts as well as a lower rate of development to term. Even if piezo-driven injection facilitates a faster and more efficient rate of reconstruction, it should be used with precaution and with as low parameters as possible.
Reproduction (Cambridge, England) 06/2007; 133(5):947-54. · 3.09 Impact Factor
