-
Tianda Li,
Ziwei Wang,
Wei Li,
Fei Teng,
Zhonghua Liu,
Wanwan Zhu,
Fenglin Cao,
Lisi Sang,
Zhenkun Wang, Liu Wang,
Chao Sheng,
Qi Zhou,
Xiaoyang Zhao
-
Zhenkun Wang,
Chao Sheng,
Tianda Li,
Fei Teng,
Lisi Sang,
Fenglin Cao,
Ziwei Wang,
Wanwan Zhu,
Wei Li, Xiaoyang Zhao,
Zhonghua Liu,
Liu Wang,
Qi Zhou
[show abstract]
[hide abstract]
ABSTRACT: Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem (rES) cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem cells give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) inhibitors (two inhibitors, "2i") with feeder-conditioned medium, we successfully obtained high-quality rat embryoid bodies (rEBs) from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells (rNPCs) were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligodendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for γ-aminobutyric acid (GABA) and tyrosine hydroxylase (TH). In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson's disease and the development of rat nervous system.
Journal of Genetics and Genomics 12/2012; 39(12):643-51. · 1.88 Impact Factor
-
Zichuan Liu,
Haifeng Wan,
Eryao Wang, Xiaoyang Zhao,
Chenhui Ding,
Shuya Zhou,
Tianda Li,
Ling Shuai,
Chunjing Feng,
Yang Yu,
Qi Zhou,
Nathalie Beaujean
[show abstract]
[hide abstract]
ABSTRACT: Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.
Stem cells and development 06/2012; 21(16):3001-9. · 4.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos.
In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed
with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA
methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells
(J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem
cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not
improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either.
Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor
nuclear reprogramming.
KeywordsDNA methylation-nuclear transfer-DNA methyltransferase 3a-DNA methyltransferase 3b
04/2012; 5(5):439-444.
-
Nan Cao,
Zumei Liu,
Zhongyan Chen,
Jia Wang,
Taotao Chen, Xiaoyang Zhao,
Yu Ma,
Lianju Qin,
Jiuhong Kang,
Bin Wei,
Liu Wang,
Ying Jin,
Huang-Tian Yang
[show abstract]
[hide abstract]
ABSTRACT: Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.
Cell Research 12/2011; 22(1):219-36. · 8.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It has been established that exogenous expression of four transcription factors (Oct4, Klf4, Sox2, and c-Myc) can reprogram mammalian somatic cells to pluripotent states. Further studies demonstrated that such induced pluripotent stem cells (iPSCs) could be generated with fewer exogenous transcription factors, facilitated by endogenous expression of reprogramming factors and/or synthetic small molecules. Here, we reported identification of a new small molecule, a protein arginine methyltransferase inhibitor AMI-5, which enabled Oct4-induced reprogramming of mouse embryonic fibroblasts in combination with transforming growth factor (TGF)-β inhibitor A-83-01. The Oct4-induced iPSCs were shown similar to mouse embryonic stem cells with respect to typical pluripotency criteria. More importantly, they were shown to give rise to liveborn pups through tetraploid complementation assays, demonstrating the high quality of full reprogramming induced by this condition. Furthermore, this study suggests that regulation of protein arginine methylation might be involved in the reprogramming process.
Stem Cells 03/2011; 29(3):549-53. · 7.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It has been established that exogenous expression of four transcription factors (Oct4, Klf4, Sox2 and c-Myc) can reprogram mammalian somatic cells to pluripotent states. Further studies demonstrated that such induced pluripotent stem cells (iPSCs) could be generated with fewer exogenous transcription factors, facilitated by endogenous expression of reprogramming factors and/or synthetic small molecules. Here, we reported identification of a new small molecule, a protein arginine methyltransferase (PRMT) inhibitor AMI-5, which enabled Oct4-induced reprogramming of MEFs in combination with TGF-β inhibitor A-83-01. The Oct4-induced iPSCs were shown similar to mouse embryonic stem cells (mESCs) with respect to typical pluripotency criteria. More importantly, they were shown to give rise to live-born pups through tetraploid complementation assays, demonstrating the high quality of full reprogramming induced by this condition. Furthermore, this study suggests that regulation of protein arginine methylation might be involved in the reprogramming process.
Stem Cells 01/2011; · 7.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.
Cell Research 12/2010; 21(5):770-8. · 8.19 Impact Factor
-
Yingying Wang,
Tang Hai,
Zichuan Liu,
Shuya Zhou,
Zhuo Lv,
Chenhui Ding,
Lei Liu,
Yuyu Niu, Xiaoyang Zhao,
Man Tong,
Liu Wang,
Alice Jouneau,
Xun Zhang,
Weizhi Ji,
Qi Zhou
[show abstract]
[hide abstract]
ABSTRACT: Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.
Biochemical and Biophysical Research Communications 07/2010; 397(3):407-12. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Knockout Brown Norway (BN) rat could be a useful disease model for human disorders, however, a failure to derive embryonic stem (ES) cells disturbs the further development of the model. In this study, we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley (SD) rats. The BN rat ES cells expressed the key transcription factors, and were able to form embryonic bodies (EBs) when being differentiated in vitro. After injecting the BN rat ES cells into the SD rat blastocysts, high-contribution chimeric rats were generated and could survive to their adulthood. Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.
Journal of Genetics and Genomics 07/2010; 37(7):467-73. · 1.88 Impact Factor
-
Shuya Zhou,
Chenhui Ding, Xiaoyang Zhao,
Eryao Wang,
Xiangpeng Dai,
Lei Liu,
Wei Li,
Zichuan Liu,
Haifeng Wan,
Chunjing Feng,
Tang Hai,
Liu Wang,
Qi Zhou
Cell Research 07/2010; 20(7):850-3. · 8.19 Impact Factor
-
Lei Liu,
Guan-Zheng Luo,
Wei Yang, Xiaoyang Zhao,
Qinyuan Zheng,
Zhuo Lv,
Wei Li,
Hua-Jun Wu,
Liu Wang,
Xiu-Jie Wang,
Qi Zhou
[show abstract]
[hide abstract]
ABSTRACT: Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.
Journal of Biological Chemistry 04/2010; 285(25):19483-90. · 4.77 Impact Factor
