• 主题
高级搜索  >
历史搜索 清空历史

您现在的位置：首页>美国卫生研究院文献>Cellular Reprogramming

期刊信息

  • 期刊名称：

    -

  • 刊频： Bimonthly
  • NLM标题：
  • iso缩写： -
  • ISSN： -

年度选择

更多>>

  • 排序：
  • 显示：
  • 每页：
全选（0）
<2/8>
共145条结果
  • 1.

    【2hr】Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

    包量

    机译 重组人骨形态发生蛋白6增强卵母细胞重编程的潜力和克隆Development牛胚胎的后续发展。
    摘要：This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus–oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.
  • 2.

    【2hr】Correction: Cellular Reprogramming: 2015; 17/2:95—105

    包量

    机译 更正：Cellular Reprogramming：2015； 17/2：95—105
    • 作者：
    • 刊名：Cellular Reprogramming
    • -1年第4期
    摘要：
  • 3.

    【2hr】Development Characterization and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized Hand-Guided Cloned and Parthenogenetic Embryos

    包量

    机译 体外受精手工克隆和孤雌生殖胚胎的水牛（Bubalus bubalis）胚胎干细胞系的发育鉴定和多能性分析
    摘要：We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium.
  • 4.

    【2hr】Transcriptome Analysis of Pig In Vivo In Vitro–Fertilized and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway

    包量

    机译 用组蛋白脱乙酰基酶抑制剂治疗后的猪体内体外受精和核转移的胚泡胚期胚胎的转录组分析揭示了溶酶体途径的变化
    摘要：Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro–fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 μM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 μM ISAHA had the same percentage of blastocyst development as Scriptaid (p<0.05). Two treatments, 1.0 μM ISAHA and 1.0 μM SAHA, had higher mean cell number than No HDACi treatment (p<0.021). Embryo transfers performed with 10 μM SAHA- and 1 μM ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos (p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT.
  • 5.

    【2hr】Transdifferentiation of Fibroblasts by Defined Factors

    包量

    机译 成纤维细胞转分化的确定因素
    摘要：Cellular differentiation is usually considered to be an irreversible process during development due to robust lineage commitment. Feedback and feed-forward loops play a significant role in maintaining lineage-specific gene expression processes in various cell types, and, in turn, factors secreted by cells may regulate the homeostatic balance of these cycles during development and differentiation. The output of biological responses is controlled by such mechanisms in many regulatory pathways through gene networks involved in transcription, RNA metabolism, signal transduction, micromolecular synthesis, and degradation. The pluripotent stage during cellular conversion may be avoided through ectopic expression of lineage-specific factors. Lineage-specific transcription factors produced during development may strengthen cell type–specific gene expression patterns. Cellular phenotypes are further stabilized by epigenetic modifications. This reprogramming approach could have important implications for disease modeling and regenerative and personalized medicine.
  • 6.

    【2hr】Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine

    包量

    机译 从尿液分离的体细胞生产克隆的水牛小牛
    摘要：This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine- and tail skin–derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic index was lower (p<0.05) than that of control blastocysts. The global level of histone H3 acetylated at lysine 9 (H3K9ac) was similar in the three types of donor cells and in urine- and tail skin–derived HMC blastocysts and in vitro–fertilized (IVF) blastocysts (controls). The global level of histone H3 trimethylated at lysine 27 (H3K27me3) in the cells was in the order (p<0.05) urine≥tail skin>ear skin–derived cells, whereas in blastocysts, it was higher (p<0.05) in urine- and tail skin–derived HMC blastocysts than that in IVF blastocysts. The expression level of CASPASE3, CASPASE9, P53, DNMT1, DNMT3a, OCT4, and NANOG, which was similar in HMC blastocysts of three the groups, was lower (p<0.05) than that in IVF blastocysts, whereas that of HDAC1 was similar among the four groups. Following transfer of urine-derived embryos (n=10) to five recipients (two embryos/recipient), one of the recipients delivered a normal calf that is now 5 weeks old.
  • 7.

    【2hr】Stem Cell–Derived Bioactive Materials Accelerate Development of Porcine In Vitro–Fertilized Embryos

    包量

    机译 干细胞衍生的生物活性物质促进猪体外受精胚胎的发育
    摘要：Stem cells show the capability to proliferate in an undifferentiated state with long-term self-renewal, which gives the cells advantages for use as bioactive material (BM) for embryo culture in vitro. The objective of this experiment was to investigate the effect of two BMs—human adipose tissue–derived mesenchymal stem cell BM (hAT-MSC-BM) and human embryonic stem cell–derived BM (hESC-BM)—on porcine embryo development compared to commonly used bovine serum albumin (BSA) or serum treatment groups. In vitro–fertilized (IVF) embryos were cultured in PZM-5 with 4 mg/mL BSA until day 4 and equally divided into four groups. Starting from day 4 (until day 6), each group was treated with the following protein additives: 4 mg/mL BSA (control), 10% fetal bovine serum (FBS), 10% hAT-MSC-BM, or 10% hESC-BM. Our results show FBS- and two other BM-treated groups showed significant increases in blastocyst formation rate, hatching rate, and total cell number compared with the control group (p<0.05). The hAT-MSC-BM and hESC-BM treatment groups presented better-quality embryo development, especially from the middle expanding stage to hatching. In particular, the hAT-MSC-BM–treated group showed the highest developmental potential of all groups and formed the most expanding-stage blastocysts. The relative expression of reprogramming-related transcription factor (POU5F1, SOX2, DPPA5, and CDH1), antioxidant (PRDX5), and apoptosis (BCL2L1 and BIRC5) genes also increased in two types of BMs compared to the control. In addition, we investigated the protein synthesis of the tight junction– and gap junction–related genes, connexin 43 and zonula occludens-1 (ZO-1); these increased more than in the control. These results demonstrate that stem cell–derived BMs accelerate porcine preimplantation embryo development and that the BMs would be helpful in the development of preimplantation embryos.
  • 8.

    【2hr】Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer

    包量

    机译 牛体细胞核转移的有效卵母细胞玻璃化和存活技术
    摘要：Bovine somatic cell nuclear transfer (SCNT) using vitrified–thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (p<0.05). Spindle assessment via the Oosight Microscopy Imaging System and microtubule staining revealed that vitrified metaphase II oocytes (VT group) were not suitable for SCNT. However, enucleating and/or activating oocytes prior to freezing enhanced their developmental potential and suitability for SCNT. The cloning efficiency of the enucleated–activated–vitrified–thawed (EAVT) group (21.6%) was better than that of the other vitrification groups [enucleated–vitrified–thawed (EVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential–related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen–thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques.
  • 9.

    【2hr】Runx1 and Runx3 Are Downstream Effectors of Nanog in Promoting Osteogenic Differentiation of the Mouse Mesenchymal Cell Line C3H10T1/2

    包量

    机译 Runx1和Runx3是Nanog在促进小鼠间充质细胞系C3H10T1 / 2成骨分化中的下游效应器
    摘要：Previously, we reported that the transcription factor Nanog, which maintains the self-renewal of embryonic stem cells (ESCs), promotes the osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 through a genome reprogramming process. In the present study, to clarify the mechanism underlying the multipotency of mesenchymal stem cells (MSCs) and to develop a novel approach to bone regenerative medicine, we attempted to identify the downstream effectors of Nanog in promoting osteogenic differentiation of mouse mesenchymal cells. We demonstrated that Runx1 and Runx3 are the downstream effectors of Nanog, especially in the early and intermediate osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2.
  • 10.

    【2hr】Conversion of Adipose Tissue-Derived Mesenchymal Stem Cells to Neural Stem Cell-Like Cells by a Single Transcription Factor Sox2

    包量

    机译 通过单个转录因子Sox2将脂肪组织来源的间充质干细胞转化为神经干细胞样细胞。
    摘要：Adipose tissue is an attractive source of easily accessible adult candidate cells for regenerative medicine. Adipose tissue–derived mesenchymal stem cells (ADSCs) have multipotency and strong proliferation and differentiation capabilities in vitro. However, as mesodermal multipotent stem cells, whether the ADSCs can convert into induced neural stem cells (NSCs) has so far not been demonstrated. In this study, we found that normally the naïve ADSCs cultured as either monolayer or spheres in NSC medium did not express Sox2 and Pax6 genes and proteins, and could not differentiate to neuron-like cells. However, when we introduced the Sox2 gene into ADSCs by retrovirus, they exhibited a typical NSC-like morphology, and could be passaged continuously, and expressed NSC specific markers Sox2 and Pax6. In addition, the ADSC-derived NSC-like cells displayed the ability to differentiate into neuron-like cells when switched to the differentiation culture medium, expressing neuronal markers, including Tuj1 and MAP2 genes and proteins. Our results suggest the ADSCs can be converted into induced NSC-like cells with a single transcription factor Sox2. This finding could provide another alternative cell source for cell therapy of neurological disorders.
  • 11.

    【2hr】Epigenetic Modification of Cloned Embryos Improves Nanog Reprogramming in Pigs

    包量

    机译 克隆胚胎的表观遗传修饰改善猪的Nanog重编程。
    摘要：Incomplete reprogramming of pluripotent genes in cloned embryos is associated with low cloning efficiency. Epigenetic modification agents have been shown to enhance the developmental competence of cloned embryos; however, the effect of the epigenetic modification agents on pluripotent gene reprogramming remains unclear. Here, we investigated Nanog reprogramming and the expression patterns of pluripotent transcription factors during early embryo development in pigs. We found that compared with fertilized embryos, cloned embryos displayed higher methylation in the promoter and 5′-untranslated region and lower methylation in the first exon of Nanog. When 5-aza-2′-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of porcine cloned embryos, Nanog methylation reprogramming was also improved, similar to that detected in fertilized counterparts. Furthermore, our results showed that the epigenetic modification agents improved the expression levels of Oct4 and Sox2 and effectively promoted Nanog transcription in cloned embryos. In conclusion, our results demonstrated that the epigenetic modification agent 5-aza-dC or TSA improved Nanog methylation reprogramming and the expression patterns of pluripotent transcription factors, thereby resulting in the enhanced expression of Nanog and high development of porcine cloned embryos. This work has important implications in the improvement of cloning efficiency.
  • 12.

    【2hr】Application Of Small Molecules Favoring Naïve Pluripotency during Human Embryonic Stem Cell Derivation

    包量

    机译 小分子天真性在人类胚胎干细胞衍生中的应用
    摘要：In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3β (GSK3β) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these “progenitor cells” did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs.
  • 13.

    【2hr】Buffalo Embryos Produced by Handmade Cloning from Oocytes Selected Using Brilliant Cresyl Blue Staining Have Better Developmental Competence and Quality and Are Closer to Embryos Produced by In Vitro Fertilization in Terms of Their Epigenetic Status and Gene Expression Pattern

    包量

    机译 通过精制甲酚蓝染色从卵母细胞中进行手工克隆生产的水牛胚具有更好的发育能力和质量并且在表观遗传状态和基因表达方式方面更接近于体外受精产生的胚
    摘要：We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB−). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB− oocytes (43.41±2.54 vs. 22.74±1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB− blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB− blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB− blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB− blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.
  • 14.

    【2hr】Early Development of Cloned Bovine Embryos Produced from Oocytes Enucleated by Fluorescence Metaphase II Imaging Using a Conventional Halogen-Lamp Microscope

    包量

    机译 使用常规卤素灯显微镜从通过荧光中期II成像去核的卵母细胞产生的克隆牛胚胎的早期发育
    摘要：Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used a new experimental system that enables fluorescent observation of chromosomes in living oocytes without any damage. We succeeded in visualizing and removing the MII chromosome in matured bovine oocytes. This experimental system consists of injecting fluorescence-labeled antibody conjugates that bind to chromosomes and fluorescent observation using a conventional halogen-lamp microscope. The cleavage rates and blastocyst rates of bovine embryos following in vitro fertilization (IVF) decreased as the concentration of the antibody increased (p<0.05). The enucleation rate of the conventional method (blind enucleation) was 86%, whereas all oocytes injected with the antibody conjugates were enucleated successfully. Fusion rates and developmental rates of SCNT embryos produced with the enucleated oocytes were the same as those of the blind enucleation group (p>0.05). For the production of SCNT embryos, the new system can be used as a reliable predictor of the location of metaphase plates in opaque oocytes, such as those in ruminant animals.
  • 15.

    【2hr】Downregulation of DNA Methyltransferase 1 in Zona-Free Cloned Buffalo (Bubalus bubalis) Embryos by Small Interefering RNA Improves In Vitro Development But Does Not Alter DNA Methylation Level

    包量

    机译 DNA甲基转移酶1下调的小Intereferrefer RNA在无Zona克隆的水牛（Bubalus bubalis）胚胎中改善了体外发育但不会改变DNA甲基化水平
    摘要：Aberrant epigenetic reprogramming, especially genomic hypermethylation, is implicated as the primary reason behind the failure of the cloning process during somatic cell nuclear transfer (SCNT). We transfected one-cell-stage zona-free buffalo embryos produced by handmade cloning with 50 nM DNMT1 small interfering RNA (siRNA), using lipofectamine, to knockdown the DNA methyltransferase 1 (DNMT1) gene. siRNA treatment decreased (p<0.001) the expression level of DNMT1 mRNA and DNMT1 protein in the one-cell-stage embryos and increased (p<0.05) the blastocyst rate (52.3±1.3% vs. 45.3±2.5%) compared to that in the controls, but did not reduce the DNA methylation level similar to the in vitro–fertilized (IVF) embryos. It also increased (p<0.05) the relative mRNA abundance of P53 and CASPASE 3, but not that of HDAC1, DNMT1, and DNMT3a, in the blastocysts of the siRNA group compared to the controls. The global level of H3K18ac was higher (p<0.05) in the blastocysts of the siRNA group than in the controls, whereas that of H3K9ac and H3K27me3 was not significantly different between the two groups. In conclusion, lipofection can be successfully used for transfection of DNMT1 siRNA into one-cell-stage zona-free cloned buffalo embryos. It results in a concomitant decrease in the DNMT1 mRNA and protein levels in the one-cell-stage embryos. siRNA-mediated knockdown increases the blastocyst rate but does not alter the DNA methylation level.
  • 16.

    【2hr】Deficiency of Genomic Reprogramming in Trophoblast Stem Cells Following Nuclear Transfer

    包量

    机译 核移植后滋养层干细胞中基因组重编程的缺陷。
    摘要：To examine the genomic reprogrammability of trophoblast stem (TS) cells using a nuclear transfer technique, we produced TS cloned embryos using five TS cell lines from three strains of mice (ICR, B6D2F1, and B6CBF1) as donors and observed developmental ability during preimplantation development. The developmental rates of the TS cloned embryos that developed to the two-cell, four- to eight-cell, morula, and blastocyst stages were 58–83%, 0–38.6%, 0–21.3%, and 0–15.9%, respectively, indicating that more than 50% of TS cloned embryos arrested at the two-cell stage. These TS cloned two-cell embryos were expressed low level of Dappa3 (also known as PGC7/Stella), indicating that zygotic gene activation (ZGA) was disrupted in these embryos. However, a small portion of the TS cloned embryos (0–15.9%) reached the blastocyst stage. In these TS cloned blastocysts, the numbers of trophectoderm (TE) and inner cell mass (ICM) cells were 31.9±4.6 and 12.1±3.0, respectively, which were not significantly different from those in the fertilized embryos. In addition, the gene expression analysis showed that Oct3/4, and Cdx2, which are ICM- and TE-specific marker genes, respectively, and Dppa3, and Hdac1, which are zygotic gene activation-related genes, were expressed in TS cloned blastocysts at the same levels as in the fertilized blastocysts. These results indicate that although TS cloned embryos are able to differentiate into ICM cells, the genomic reprogrammability of TS cells is very low following nuclear transfer.
  • 17.

    【2hr】Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression but Reduces Induced Pluripotent Stem Cell Colony Formation

    包量

    机译 蛋白酶体的长期抑制循环上调Oct3 / 4和Nanog基因表达但减少诱导的多能干细胞集落形成。
    摘要：There is ample evidence that the ubiquitin–proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells.
  • 18.

    【2hr】Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

    包量

    机译 转座子介导的重编程牛诱导多能干细胞的衍生和表征。
    摘要：Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line–competent bovine iPSCs and will facilitate genetic modification of the bovine genome.
  • 19.

    【2hr】The Effects of ROCK Inhibitor Y-27632 on Injectable Spheroids of Bovine Corneal Endothelial Cells

    包量

    机译 ROCK抑制剂Y-27632对牛角膜内皮细胞可注射球体的影响
    摘要：The spheroids of 3-dimensional culture and Rho-associated kinase (ROCK) inhibitor Y-27632 have shown many advantages for the promotion of cellular viability and proliferation. The objective of this study was to investigate the effect of Y-27632 on the growth and injectability of bovine corneal endothelial cells (B-CECs) maintained in vitro as spheroid cultures. Immunofluorescence staining showed that Y-27632 did not alter the cell type specificity of B-CECs, but it significantly enhanced B-CEC spherical viability and proliferation by a Live/Dead assay, 5-ethynyl-2′-deoxyuridine (EdU) labeling assay, and Cell Counting Kit-8 (CCK-8) assay. The uniform B-CEC spheroids could easily form in multiwall agarose micromolds and had a higher stemness potential than single B-CECs. Injectable B-CEC spheroids were able to form monolayer growth, and polygonal B-CECs completely covered culture plates or Descemet's membrane of decellularized corneas under inverted microscopy and scanning electron microscopy (SEM). B-CEC spheroids were generated from agarose microwells on day 1 and then adherent culture with Y-27632 for day 5. However, small B-CEC spheroids still existed on culture plates or decellularized corneas when B-CEC spheroids were cultured in the same condition except for absence of Y-27632. Our findings that CEC spheroids with Y-27632 are injectable in vitro have important implications for the favorable treatment of CEC deficiency.
  • 20.

    【2hr】Assessment of Difference in Gene Expression Profile Between Embryos of Different Derivations

    包量

    机译 评估不同衍生胚胎之间基因表达谱的差异
    摘要：Researchers have exerted sustained efforts to improve the viability of somatic cell nuclear transfer (SCNT) embryos, testing their experimental designs and probing the resultant embryos. However, the lack of a reliable method to estimate the efficacy of these experimental attempts is a chief hindrance to tackling the low-viability problem in SCNT. Here, we introduce a procedure that assesses the degree of difference in gene expression profiles (GEPs) of blastocysts from each other as a representative control of good quality. We first adapted a multiplex reverse transcription-polymerase chain reaction strategy to obtain GEPs for 15 reprogramming-related genes from single mouse blastocysts. GEPs of individual blastocysts displayed a broad range of variations, the extent of which was calculated using a weighted root mean square deviation (wRMSD). wRMSD-based quantitation of GEP difference (qGEP) found that GEP difference between in vivo–derived blastocysts (in vivo) and SCNT blastocysts was greater than the difference between in vivo blastocysts and in vitro–produced (IVP) blastocysts, demonstrating that the SCNT group was more distantly related to the in vivo group than the IVP group. Our qGEP approach for grading individual blastocysts would be useful for selecting a better protocol to derive embryos of better quality prior to field applications.
题名 作者 来源 发表时间

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号